TW202144575A

TW202144575A - Treatment of phenylketonuria with aav and therapeutic formulations

Info

Publication number: TW202144575A
Application number: TW110111976A
Authority: TW
Inventors: 彼得克羅希; 哈西卜拉阿基夫; 傑佛瑞博桂格; 拉傑夫瑪西卡; 皓鈴翁; 顧仲華; 維亮黃; 琪迪絲蒂艾拉雅; 天惠周
Original assignee: 美商拜奧馬林製藥公司
Priority date: 2020-04-03
Filing date: 2021-03-31
Publication date: 2021-12-01
Also published as: AR122409A1; WO2021202943A1

Abstract

Provided herein are pharmaceutical compositions and methods for treating phenylketonuria in a human subject.

Description

使用AAV及治療調配物之苯酮尿症治療Phenylketonuria Treatment Using AAV and Therapeutic Formulations

本發明係關於藉由向患有苯酮尿症之個體投與AAV病毒顆粒使胺基酸、神經傳遞質及/或神經傳遞質代謝物之含量正常化而治療苯酮尿症之方法，以及包含其之治療調配物。The present invention pertains to methods of treating phenylketonuria by administering AAV viral particles to an individual suffering from phenylketonuria to normalize levels of amino acids, neurotransmitters and/or neurotransmitter metabolites, and Therapeutic formulations containing the same.

苯酮尿症(PKU)為由肝***酸羥化酶(PAH)之活性受損造成的先天性胺基酸代謝異常，該酶負責代謝***酸(Phe)。患有導致PKU及高***酸血症(HPA)之PAH突變之患者呈現Phe含量升高、神經生理功能受損及認知發展降低。對於患有PKU之患者而言，除非Phe含量使用可以與藥物療法組合使用之膳食限制而維持在低含量下，否則可能存在不可逆之精神遲鈍。當前指南推薦患者為了生命對血漿Phe維持嚴格控制，此係因為PKU之失控血液Phe含量與顯著神經心理及神經認知問題相關，影響超過兒童期之患者進入青年期及成人期。PKU之神經症狀由許多神經傳遞質之異常產生而引起，該異常產生由PAH損失引起，需要PAH損失將Phe轉化為合成各種神經傳遞質所需之前驅體代謝物。Phenylketonuria (PKU) is an inborn error of amino acid metabolism caused by impaired activity of hepatic phenylalanine hydroxylase (PAH), the enzyme responsible for metabolizing phenylalanine (Phe). Patients with PAH mutations that cause PKU and hyperphenylalaninemia (HPA) exhibit elevated Phe levels, impaired neurophysiological function, and reduced cognitive development. For patients with PKU, unless Phe levels are kept low using dietary restrictions that can be used in combination with drug therapy, there may be irreversible mental retardation. Current guidelines recommend that patients maintain strict control of plasma Phe for life. This is because uncontrolled blood Phe levels in PKU are associated with significant neuropsychological and neurocognitive problems, affecting patients beyond childhood into adolescence and adulthood. The neurological symptoms of PKU result from the abnormal production of many neurotransmitters resulting from the loss of PAH, which is required to convert Phe into precursor metabolites required for the synthesis of various neurotransmitters.

當前針對PKU之治療包括以後生活中一直遵守低Phe之膳食。此膳食療法難以維持且始終無法消除可能由***酸含量升高引起之損害神經影響。妊娠期間小於理想的膳食控制可導致出生缺陷。另外，PKU/HPA患者在遵循限制膳食的同時很難過正常生活，且膳食療法可以與幾種營養素缺乏相關，其中一些對大腦發展有害。Current treatment for PKU includes adhering to a low Phe diet throughout life. This dietary regimen is difficult to maintain and does not always eliminate the damaging neurological effects that may be caused by elevated levels of phenylalanine. Less than ideal dietary control during pregnancy can lead to birth defects. Additionally, PKU/HPA patients have difficulty leading a normal life while following a restricted diet, and dietary therapy can be associated with several nutrient deficiencies, some of which are detrimental to brain development.

當前批准用於苯酮尿症之治療包括：PALYNZIQ® (培格瓦力-pqpz (pegvaliase-pqpz))；每日一次投與之酶***酸氨解離酶之聚乙二醇化形式；及KUVAN® (沙丙蝶呤(sapropterin))或四氫生物喋呤(tetrahydrobiopterin；BH4)，其由於BH4-反應性而每日一次向患有HPA之患者投與，通常與限制Phe之膳食一起。BH4係PAH酶之天然輔因子，且可以增加殘餘PAH酶活性以將Phe代謝成酪胺酸。KUVAN®描述於美國專利第7,732,599號、第8,003,126號、第7,566,462號及第8,178,670號中，其各者以全文引用之方式併入本文中。PALYNZIQ® (培格瓦力-pqpz)描述於美國專利第7,531,341號、第7,534,595號、第7,537,923號、第7,790,433號、第7,560,263號、第9,557,34號中，其各者以全文引用之方式併入本文中。Current approved treatments for phenylketonuria include: PALYNZIQ® (pegvaliase-pqpz (pegvaliase-pqpz)); a pegylated form of the enzyme phenylalanine ammonia lyase administered once daily; and KUVAN® (sapropterin) or tetrahydrobiopterin (BH4), which are administered once daily to patients with HPA due to BH4-reactivity, usually with a Phe-restricted diet. BH4 is a natural cofactor for PAH enzymes and can increase residual PAH enzyme activity to metabolize Phe to tyrosine. KUVAN® is described in US Pat. Nos. 7,732,599, 8,003,126, 7,566,462, and 8,178,670, each of which is incorporated herein by reference in its entirety. PALYNZIQ® (Pegvari-pqpz) is described in US Pat. Nos. 7,531,341, 7,534,595, 7,537,923, 7,790,433, 7,560,263, 9,557,34, each of which is incorporated by reference in its entirety into this article.

已在動物模型中嘗試多種基因療法方法，包括使用腺相關病毒(AAV)載體之方法。舉例而言，Ahmed等人在美國基因與細胞療法協會(American Society of Gene & Cell Therapy；ASGCT)年度會議(2018年5月17日)上報告：在PAHenu2小鼠模型中投與包裝由廣泛表達啟動子(非組織特異性)驅動之人類PAH轉基因的AAVHSC15將Phe含量正常化至小於150 µM，同時包括肝特異性啟動子(HMI-102)之經優化載體將血清Phe正常化為低十倍的劑量。三名人類患者似乎已在臨床試驗NCT03952156中使用基於含有人類PAH基因之功能複本之AAVHSC15載體的基因療法治療，其中低劑量之兩個參與者無效果(Phe含量無降低)，而中間劑量之第三個患者據報導儘管不經歷治療含量，但經歷Phe含量之一定下降。Various approaches to gene therapy have been attempted in animal models, including those using adeno-associated virus (AAV) vectors. For example, Ahmed et al. report at the American Society of Gene & Cell Therapy (ASGCT) annual meeting (May 17, 2018): Administered packaging in the PAHenu2 mouse model is widely expressed AAVHSC15 of a promoter (non-tissue-specific) driven human PAH transgene normalizes Phe content to less than 150 µM, while an optimized vector including a liver-specific promoter (HMI-102) normalizes serum Phe to ten-fold lower dose. Three human patients appear to have been treated with gene therapy based on the AAVHSC15 vector containing a functional copy of the human PAH gene in clinical trial NCT03952156, with two participants at the low dose having no effect (no reduction in Phe levels) and the third at the intermediate dose Three patients were reported to experience some reduction in Phe levels despite not experiencing therapeutic levels.

國際專利公開案WO 2018/126112 (PCT/US2017/068897)報告在TBG(一種基於人類甲狀腺激素結合球蛋白啟動子及微球蛋白/比庫寧(bikunin)增強子的雜合啟動子)控制下，向PAH基因剔除小鼠投與帶有密碼子優化之人類PAH cDNA的AAV8載體。仍需要用呈現有效以臨床上顯著方式降低Phe含量之安全、持久且穩定的長期表現的一次性基因療法治療人類個體。International Patent Publication WO 2018/126112 (PCT/US2017/068897) reports under the control of TBG, a hybrid promoter based on human thyroid hormone binding globulin promoter and microglobulin/bikunin enhancer , the AAV8 vector with codon-optimized human PAH cDNA was administered to PAH knockout mice. There remains a need to treat human subjects with a one-time gene therapy that exhibits a safe, durable and stable long-term performance of effectively reducing Phe levels in a clinically significant manner.

本發明提供藉由向患有苯酮尿症之個體投與複製缺陷型重組AAV (rAAV)顆粒以及包含其之醫藥組合物使胺基酸、神經傳遞質及/或神經傳遞質代謝物之含量正常化而治療苯酮尿症之方法。The present invention provides an increase in the levels of amino acids, neurotransmitters and/or neurotransmitter metabolites by administering replication-deficient recombinant AAV (rAAV) particles and pharmaceutical compositions comprising the same to individuals with phenylketonuria Methods of normalizing and treating phenylketonuria.

在一個態樣中，本發明提供一種降低有需要之人類個體中之血漿***酸(Phe)含量的方法，其包含向該個體投與治療有效量之重組腺相關病毒(rAAV)顆粒，該重組腺相關病毒(rAAV)顆粒包含AAV衣殼及重組載體建構體，該重組載體建構體包含編碼功能性***酸羥化酶(PAH)之核酸及視情況選用之異源肝特異性轉錄調節區。In one aspect, the invention provides a method of reducing plasma phenylalanine (Phe) levels in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of recombinant adeno-associated virus (rAAV) particles, the recombinant Adeno-associated virus (rAAV) particles comprise an AAV capsid and a recombinant vector construct comprising a nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcriptional regulatory region.

在一相關態樣中，本發明提供一種治療患有苯酮尿症(PKU)之人類個體之方法，其包含向該個體投與治療有效量之重組腺相關病毒(rAAV)顆粒，該顆粒包含AAV衣殼及重組載體建構體，該重組載體建構體包含編碼功能性***酸羥化酶(PAH)之核酸及視情況選用之異源肝特異性轉錄調節區。In a related aspect, the present invention provides a method of treating a human subject suffering from phenylketonuria (PKU), comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) particle comprising AAV capsids and recombinant vector constructs comprising nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcriptional regulatory region.

在另一相關態樣中，本發明提供根據所揭示之方法使用之如本文所描述之重組載體建構體或AAV顆粒的組合物。本發明亦提供如本文所描述之重組載體建構體或AAV顆粒之用途，其用於製備用以根據本文所描述之方法治療之藥劑。In another related aspect, the present invention provides compositions of recombinant vector constructs or AAV particles as described herein for use according to the disclosed methods. The present invention also provides the use of a recombinant vector construct or AAV particle as described herein for the manufacture of a medicament for treatment according to the methods described herein.

rAAV顆粒較佳地為複製缺陷型的。rAAV顆粒可包含重組載體建構體，或rAAV顆粒可藉由包含向細胞提供重組載體建構體之方法產生，該重組載體建構體包含(a)以下中之一或兩者：(i) AAV 5'反向末端重複序列(ITR)及(ii) AAV 3' ITR；(b)異源肝特異性轉錄調節區；及(c)編碼功能性人類***酸羥化酶(hPAH)之核酸序列，視情況其中該等AAV ITR為AAV2 ITR。較佳地，編碼功能性hPAH之核酸可操作地連接至肝特異性表現控制元件。載體建構體可包括額外表現控制元件，例如：啟動子及/或增強子；內含子；視情況，來自與內含子相同之基因的外顯子；及多腺苷酸化(polyA)信號。本文中進一步描述此類元件。較佳地，rAAV顆粒亦包含具有肝趨向性之AAV衣殼，視情況為AAV5型衣殼。The rAAV particles are preferably replication defective. The rAAV particle may comprise a recombinant vector construct, or the rAAV particle may be produced by a method comprising providing a cell with a recombinant vector construct comprising (a) one or both of the following: (i) AAV 5' Inverted terminal repeats (ITR) and (ii) AAV 3' ITR; (b) heterologous liver-specific transcriptional regulatory regions; and (c) nucleic acid sequences encoding functional human phenylalanine hydroxylase (hPAH), depending on Situations wherein such AAV ITRs are AAV2 ITRs. Preferably, the nucleic acid encoding a functional hPAH is operably linked to a liver-specific expression control element. The vector construct may include additional expression control elements such as: promoters and/or enhancers; introns; optionally, exons from the same gene as the intron; and polyadenylation (polyA) signals. Such elements are further described herein. Preferably, the rAAV particles also comprise an AAV capsid with liver tropism, optionally an AAV5 type capsid.

國際專利公開案第WO 2019/217513 (PCT/US2019/031252)號，揭示編碼密碼子優化之PAH之核酸序列、肝特異性轉錄調節區、增強子、啟動子、內含子、多腺苷酸化信號及其他載體元件、AAV載體及病毒顆粒，及藉由正常化患有苯酮尿症之個體中之胺基酸、神經傳遞質及神經傳遞質代謝物之含量來治療苯酮尿症之方法，該公開案以全文引用之方式併入本文中。International Patent Publication No. WO 2019/217513 (PCT/US2019/031252), discloses nucleic acid sequence encoding codon-optimized PAH, liver-specific transcriptional regulatory region, enhancer, promoter, intron, polyadenylation Signaling and other vector elements, AAV vectors and viral particles, and methods of treating phenylketonuria by normalizing levels of amino acids, neurotransmitters, and neurotransmitter metabolites in individuals with phenylketonuria , which is incorporated herein by reference in its entirety.

在一些實施例中，編碼hPAH之核酸編碼與SEQ ID NO: 2至少95%一致之功能性hPAH胺基酸序列。此類編碼功能性hPAH之核酸可包含與SEQ ID NO: 1中之任一者至少80%一致的核苷酸序列。可替代地，此類編碼功能性hPAH之核酸可包含與SEQ ID NO: 7至13中之任一者至少80%一致的核苷酸序列。舉例而言，編碼功能性hPAH之核酸與SEQ ID NO: 7至少80%一致。In some embodiments, the hPAH-encoding nucleic acid encodes a functional hPAH amino acid sequence that is at least 95% identical to SEQ ID NO:2. Such nucleic acid encoding a functional hPAH may comprise a nucleotide sequence that is at least 80% identical to any one of SEQ ID NO: 1. Alternatively, such nucleic acid encoding a functional hPAH may comprise a nucleotide sequence that is at least 80% identical to any one of SEQ ID NOs: 7-13. For example, the nucleic acid encoding a functional hPAH is at least 80% identical to SEQ ID NO:7.

在一些實施例中，肝特異性轉錄調節區包含hAAT啟動子之片段及HCR增強子/ApoE增強子之片段。在此類實施例中，編碼PAH之核酸可操作地連接至與HCR增強子/ApoE增強子之片段連接的hAAT啟動子之片段。在一些實施例中，肝特異性轉錄調節區包含與SEQ ID NO: 3、4或24中之任一者至少90%一致的核苷酸序列。在一些實施例中，重組載體建構體進一步包含與SEQ ID NO: 6至少90%一致之核苷酸序列。可替代地，在一些實施例中，肝特異性轉錄調節區包含不同類型之啟動子，且包含與SEQ ID NO: 25或26中之任一者至少90%一致的核苷酸序列。In some embodiments, the liver-specific transcriptional regulatory region comprises a fragment of the hAAT promoter and a fragment of the HCR enhancer/ApoE enhancer. In such embodiments, the nucleic acid encoding the PAH is operably linked to a fragment of the hAAT promoter linked to a fragment of the HCR enhancer/ApoE enhancer. In some embodiments, the liver-specific transcriptional regulatory region comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 3, 4, or 24. In some embodiments, the recombinant vector construct further comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:6. Alternatively, in some embodiments, the liver-specific transcriptional regulatory region comprises a different type of promoter and comprises a nucleotide sequence that is at least 90% identical to any of SEQ ID NOs: 25 or 26.

重組載體建構體可進一步包含內含子，例如天然PAH內含子或其片段、β球蛋白內含子或其片段或hAAT內含子或其片段，或其組合。可能需要包括毗鄰外顯子之部分以確保適當剪接。在一些實施例中，內含子包含與SEQ ID NO: 14、SEQ ID NO: 27、SEQ ID NO: 29或SEQ ID NO: 34至少90%一致之核苷酸序列。The recombinant vector construct may further comprise introns, such as native PAH introns or fragments thereof, beta globin introns or fragments thereof, or hAAT introns or fragments thereof, or combinations thereof. It may be necessary to include portions of adjacent exons to ensure proper splicing. In some embodiments, the intron comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 34.

重組載體建構體可進一步包含多腺苷酸化信號，例如牛生長激素(bGH)或人類生長激素(hGH)多腺苷酸化信號。在一些實施例中，重組載體建構體包含bGH多腺苷酸化信號。The recombinant vector construct may further comprise a polyadenylation signal, such as a bovine growth hormone (bGH) or human growth hormone (hGH) polyadenylation signal. In some embodiments, the recombinant vector construct comprises a bGH polyadenylation signal.

在一些實施例中，重組載體建構體包含與SEQ ID NO: 18至少90%一致之核苷酸序列，且AAV衣殼為AAV5型衣殼。在某些實施例中，重組載體建構體包含與SEQ ID NO: 52至少90%一致之核苷酸序列，且AAV衣殼為AAV5型衣殼。在一些實施例中，重組載體建構體包含與SEQ ID NO: 15至23或52中之任一者至少90%一致的核苷酸序列，且AAV衣殼為AAV5型衣殼。In some embodiments, the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 18, and the AAV capsid is an AAV5 type capsid. In certain embodiments, the recombinant vector construct comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 52, and the AAV capsid is an AAV5 type capsid. In some embodiments, the recombinant vector construct comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 15 to 23 or 52, and the AAV capsid is an AAV5 type capsid.

在一些實施例中，重組載體建構體包含與SEQ ID NO: 18至少95%一致之核苷酸序列，且AAV衣殼為AAV5型衣殼。在某些實施例中，重組載體建構體包含與SEQ ID NO: 52至少95%一致之核苷酸序列，且AAV衣殼為AAV5型衣殼。在一些實施例中，重組載體建構體包含與SEQ ID NO: 15至23中之任一者至少95%一致的核苷酸序列，且AAV衣殼為AAV5型衣殼。In some embodiments, the recombinant vector construct comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 18, and the AAV capsid is an AAV5 type capsid. In certain embodiments, the recombinant vector construct comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 52, and the AAV capsid is an AAV5 capsid. In some embodiments, the recombinant vector construct comprises a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 15-23, and the AAV capsid is an AAV5 type capsid.

在具體實施例中，重組載體建構體包含SEQ ID NO: 18之核苷酸序列，且AAV衣殼為AAV5型衣殼。在某些實施例中，重組載體建構體包含SEQ ID NO: 52之核苷酸序列，且AAV衣殼為AAV5型衣殼。在一些實施例中，重組載體建構體包含SEQ ID NO: 15至23中之任一者的核苷酸序列，且AAV衣殼為AAV5型衣殼。In a specific embodiment, the recombinant vector construct comprises the nucleotide sequence of SEQ ID NO: 18 and the AAV capsid is an AAV5 type capsid. In certain embodiments, the recombinant vector construct comprises the nucleotide sequence of SEQ ID NO: 52 and the AAV capsid is an AAV5 type capsid. In some embodiments, the recombinant vector construct comprises the nucleotide sequence of any one of SEQ ID NOs: 15-23, and the AAV capsid is an AAV5 type capsid.

在實施例中之任一者中，AAV衣殼可包含與SEQ ID NO: 35至51中之任一者至少85%一致，視情況與SEQ ID NO: 44至少85%、90%或95%一致的胺基酸序列(AAV5)。在一些實施例中，AAV衣殼為具有肝趨向性之AAV衣殼。在實施例中之任一者中，具有肝趨向性之AAV衣殼可為不包括AAV8及/或AAVHSC15之AAV衣殼。較佳地，具有肝趨向性之AAV衣殼為AAV5型衣殼。In any of the embodiments, the AAV capsid may comprise at least 85% identity to any one of SEQ ID NOs: 35 to 51, optionally at least 85%, 90% or 95% to SEQ ID NO: 44 Consensus amino acid sequence (AAV5). In some embodiments, the AAV capsid is an AAV capsid with liver tropism. In any of the embodiments, the AAV capsid with liver tropism can be an AAV capsid that does not include AAV8 and/or AAVHSC15. Preferably, the AAV capsid with liver tropism is AAV5 type capsid.

在實施例中之任一者中，個體患有苯酮尿症(PKU)，視情況為典型PKU或重度PKU。在一些實施例中，在該投與之前該個體之血漿Phe含量為600 μmol/L或更高。在一些實施例中，在該投與之前該個體之血漿Phe含量為1200 μmol/L或更高。在一些實施例中，個體為15歲或更大。在一些實施例中，個體為成人。在一些實施例中，個體為女性，例如非妊娠女性。In any of the embodiments, the individual suffers from phenylketonuria (PKU), classic PKU or severe PKU, as the case may be. In some embodiments, the subject has a plasma Phe level of 600 μmol/L or higher prior to the administration. In some embodiments, the subject has plasma Phe levels of 1200 μmol/L or greater prior to the administration. In some embodiments, the individual is 15 years old or older. In some embodiments, the individual is an adult. In some embodiments, the individual is female, eg, a non-pregnant female.

在一些實施例中，個體未接受治療PKU之藥物療法。舉例而言，該個體在該投與之前至少30天未接受培格瓦力，及/或該個體在該投與之前至少7天未接受沙丙蝶呤。在一些實施例中，個體在該投與之前至少30天未接受類固醇。在一些實施例中，個體在該投與之前未患有臨床上顯著之肝病。在一些實施例中，個體在該投與之前在血液中不具有可檢測之抗AAV衣殼抗體(例如並非AAV5血清反應呈陽性)。In some embodiments, the individual is not receiving drug therapy to treat PKU. For example, the individual has not received pegvar for at least 30 days prior to the administration, and/or the individual has not received sapropterin for at least 7 days prior to the administration. In some embodiments, the individual has not received steroids for at least 30 days prior to the administration. In some embodiments, the subject does not have clinically significant liver disease prior to the administration. In some embodiments, the individual does not have detectable anti-AAV capsid antibodies in blood prior to the administration (eg, is not seropositive for AAV5).

在本發明之方法中，rAAV顆粒以單次給藥經靜脈內投與。在一些實施例中，rAAV顆粒以在每公斤個體體重約1E13至約5E14載體基因體(vg/kg)範圍內之劑量，約2E13至約2E14 (vg/kg)之劑量，例如約1E13 vg/kg之劑量、或約2E13 vg/kg之劑量、或約6E13 vg/kg之劑量、或約2E14 vg/kg之劑量投與。在一個實施例中，rAAV顆粒以每公斤個體體重2E13載體基因體之劑量投與。在另一實施例中，rAAV顆粒以每公斤個體體重3E13載體基因體之劑量投與。在另一實施例中，rAAV顆粒以每公斤個體體重4E13載體基因體之劑量投與。在另一實施例中，rAAV顆粒以每公斤個體體重5E13載體基因體之劑量投與。在另一實施例中，rAAV顆粒以每公斤個體體重6E13載體基因體之劑量投與。在另一實施例中，rAAV顆粒以每公斤個體體重7E13載體基因體之劑量投與。在另一實施例中，rAAV顆粒以每公斤個體體重8E13載體基因體之劑量投與。在另一實施例中，rAAV顆粒以每公斤個體體重9E13載體基因體之劑量投與。In the methods of the present invention, the rAAV particles are administered intravenously in a single dose. In some embodiments, the rAAV particles are at a dose ranging from about 1E13 to about 5E14 vector genome per kilogram of body weight (vg/kg), a dose of about 2E13 to about 2E14 (vg/kg), eg, about 1E13 vg/kg A dose of kg, or a dose of about 2E13 vg/kg, or a dose of about 6E13 vg/kg, or a dose of about 2E14 vg/kg was administered. In one embodiment, the rAAV particles are administered at a dose of 2E13 vector gene bodies per kilogram of body weight of the subject. In another embodiment, the rAAV particles are administered at a dose of 3E13 vector gene bodies per kilogram of body weight of the subject. In another embodiment, the rAAV particles are administered at a dose of 4E13 vector gene bodies per kilogram of body weight of the subject. In another embodiment, the rAAV particles are administered at a dose of 5E13 vector gene bodies per kilogram of body weight of the subject. In another embodiment, the rAAV particles are administered at a dose of 6E13 vector gene bodies per kilogram of body weight of the subject. In another embodiment, the rAAV particles are administered at a dose of 7E13 vector gene bodies per kilogram of body weight of the subject. In another embodiment, the rAAV particles are administered at a dose of 8E13 vector gene bodies per kilogram of body weight of the subject. In another embodiment, the rAAV particles are administered at a dose of 9E13 vector gene bodies per kilogram of body weight of the subject.

本發明之方法可進一步包含向個體投與預防有效量之皮質類固醇或其他全身性免疫抑制劑以預防肝毒性，隨後檢測肝毒性(例如，如藉由高於正常值上限(ULN)或至少2倍基線ALT之ALT升高所檢測)。此在本文中亦稱為「預防性免疫抑制治療」。在一些實施例中，與投與本發明之rAAV顆粒同時投與預防有效量之皮質類固醇或免疫抑制劑。在其他實施例中，在投與rAAV顆粒之後開始投與預防有效量之皮質類固醇或免疫抑制劑，例如在投與rAAV顆粒之後3至10週但在檢測肝毒性之前開始。可投與皮質類固醇或免疫抑制劑持續預防性治療時段，例如持續至少約3至13週之時段，且較佳地隨後投與遞減量之皮質類固醇或免疫抑制劑持續遞減時段，，例如持續約2、3或4週之時段。舉例而言，皮質類固醇之預防有效量為10毫克/天至40毫克/天之普賴松等效物劑量持續至少約3至13週之時段，接著遞減量之該皮質類固醇持續約2、3或4週之時段。在一些實施例中，投與預防有效量之皮質類固醇持續約13週之時段，接著遞減量之該皮質類固醇持續約3週之時段。舉例而言，與rAAV顆粒之該投與同時投與40毫克/天之普賴松等效物劑量持續約13週之時段，接著遞減量之普賴松等效物持續約3週之時段(例如30毫克/天之普賴松等效物劑量持續一週，20毫克/天持續一週，及10毫克/天持續一週)。其他普賴松等效物皮質類固醇可以適當劑量使用，例如***(dexamethasone)、普賴松(prednisone)、普賴蘇穠(prednisolone)、氟可體松(fludrocortisone)、氫化可體松(hydrocortisone)或布***(budesonide)。參見Liu等人，Allergy, Asthma & Clinical Immunology 9:30 (2013)中之普賴松等效物劑量的描述，其以全文引用之方式併入本文中。在一些實施例中，以3毫克/天之劑量投與布***持續約14週之時段，接著遞減持續約3週之時段。可以預防有效劑量投與來預防肝毒性之其他全身性免疫抑制劑包括(1)鈣調神經磷酸酶抑制劑，例如他克莫司(tacrolimus)或環孢素(cyclosporine)；(2)抗增殖劑或IMDH抑制劑，例如黴酚酸酯、來氟米特(leflunomide)或硫唑嘌呤；(3) mTOR抑制劑，例如西羅莫司(sirolimus)或依維莫司(everolimus)；(4)詹納斯(Janus)激酶抑制劑，例如托法替尼(tofacitinib)；或(5)免疫抑制劑抗體。舉例而言，免疫抑制劑為他克莫司或黴酚酸酯。The methods of the invention may further comprise administering to the individual a prophylactically effective amount of a corticosteroid or other systemic immunosuppressive agent to prevent liver toxicity, followed by detection of liver toxicity (eg, as by a higher than upper limit of normal (ULN) or at least 2 Measured by an increase in ALT that was twice the baseline ALT). This is also referred to herein as "prophylactic immunosuppressive therapy." In some embodiments, a prophylactically effective amount of a corticosteroid or immunosuppressant is administered concurrently with the administration of the rAAV particles of the invention. In other embodiments, administration of a prophylactically effective amount of a corticosteroid or immunosuppressive agent is initiated after administration of the rAAV particles, eg, 3 to 10 weeks after administration of the rAAV particles but before detection of hepatotoxicity. The corticosteroid or immunosuppressant may be administered for a period of prophylactic treatment, such as for a period of at least about 3 to 13 weeks, and preferably followed by administration of a decreasing amount of the corticosteroid or immunosuppressant for a period of decreasing, such as for about 2, 3 or 4 week period. For example, a prophylactically effective amount of a corticosteroid is a 10 mg/day to 40 mg/day prisone equivalent dose for a period of at least about 3 to 13 weeks, followed by a decreasing amount of the corticosteroid for about 2, 3 weeks or a 4-week period. In some embodiments, a prophylactically effective amount of a corticosteroid is administered for a period of about 13 weeks, followed by a decreasing amount of the corticosteroid for a period of about 3 weeks. For example, a dose of 40 mg/day of prisone equivalent is administered concurrently with this administration of rAAV particles for a period of about 13 weeks, followed by a decreasing amount of prisone equivalent for a period of about 3 weeks ( For example, 30 mg/day of prisone equivalent doses for one week, 20 mg/day for one week, and 10 mg/day for one week). Other prednisone equivalent corticosteroids may be used in appropriate doses, such as dexamethasone, prednisone, prednisolone, fludrocortisone, hydrocortisone ( hydrocortisone) or budesonide. See Liu et al., Allergy, Asthma & Clinical Immunology 9:30 (2013) for a description of prisone equivalent dosages, which is incorporated herein by reference in its entirety. In some embodiments, budesonide is administered at a dose of 3 mg/day for a period of about 14 weeks, followed by tapering for a period of about 3 weeks. Other systemic immunosuppressants that can be administered in prophylactically effective doses to prevent liver toxicity include (1) calcineurin inhibitors such as tacrolimus or cyclosporine; (2) antiproliferative or IMDH inhibitors such as mycophenolate mofetil, leflunomide or azathioprine; (3) mTOR inhibitors such as sirolimus or everolimus; (4) ) Janus kinase inhibitors, such as tofacitinib; or (5) immunosuppressive antibodies. For example, the immunosuppressant is tacrolimus or mycophenolate mofetil.

本發明之方法可包含在檢測肝毒性(例如，如藉由高於正常值上限(ULN)或至少2倍基線ALT之ALT升高所檢測)之後，向個體投與預防有效量之皮質類固醇或其他全身性免疫抑制劑以治療肝毒性。此在本文中亦稱為「治療性免疫抑制治療」。本發明之方法可進一步包含以下步驟：(a)在該投與之前，視情況在該投與之前約一個月測定該個體之血液中的肝毒性標記之基線含量，及(b)在該投與之後，視情況每週或更頻繁地測定該個體之血液中的投與後該肝毒性標記之含量。此類方法可進一步包含以下步驟：(c)在藉由生物化學或臨床症狀檢測肝毒性之後，向個體投與治療有效量之皮質類固醇或其他全身性免疫抑制劑持續治療性治療時段，例如持續至少約5至約8週或更長(例如5、6、7或8週或更長)之時段，且較佳地接著投與遞減量之皮質類固醇或其他免疫抑制劑持續遞減時段，例如持續約2、3或4週之時段。舉例而言，步驟(c)包含在藉由(i)大於正常值上限(ULN)之投與後該肝毒性標記之含量，或(ii)大於或等於該肝毒性標記的基線含量兩倍之投與後該肝毒性標記之含量來檢測肝毒性之後，向該個體投與治療有效量之皮質類固醇持續至少約5至約8週之時段，接著遞減量之皮質類固醇持續約2、3或4週之時段。在此類實施例中之任一者中，肝毒性標記為ALT及/或AST，較佳地ALT。在一些實施例中，在該檢測之後，投與40毫克/天之普賴松等效物劑量持續約5週之時段，接著遞減量之普賴松等效物持續約3週之時段(例如30毫克/天之普賴松等效物劑量持續一週、20毫克/天持續一週及10毫克/天持續一週)。可在檢測肝毒性之後以有效劑量投與之其他全身性免疫抑制劑包括(1)鈣調神經磷酸酶抑制劑，例如他克莫司或環孢素；(2)抗增殖劑或IMDH抑制劑，例如黴酚酸酯、來氟米特或硫唑嘌呤；(3) mTOR抑制劑，例如西羅莫司或依維莫司； (4)詹納斯激酶抑制劑，例如托法替尼；或(5)免疫抑制劑抗體。舉例而言，免疫抑制劑為他克莫司或黴酚酸酯。The methods of the invention can comprise administering to the subject a prophylactically effective amount of a corticosteroid or a Other systemic immunosuppressants to treat liver toxicity. This is also referred to herein as "therapeutic immunosuppressive therapy." The methods of the invention may further comprise the steps of: (a) prior to the administration, optionally about one month prior to the administration, determining a baseline level of a marker of hepatotoxicity in the blood of the individual, and (b) at the time of the administration and thereafter, the level of the hepatotoxic marker post-administration in the subject's blood is determined weekly or more frequently as appropriate. Such methods may further comprise the step of: (c) administering to the individual a therapeutically effective amount of a corticosteroid or other systemic immunosuppressant for a therapeutic treatment period, e.g., after detection of hepatotoxicity by biochemical or clinical symptoms a period of at least about 5 to about 8 weeks or longer (eg, 5, 6, 7 or 8 weeks or longer), and preferably followed by administration of a decreasing amount of corticosteroid or other immunosuppressant for a period of decreasing, such as continuously Periods of about 2, 3 or 4 weeks. For example, step (c) comprises following administration by (i) greater than the upper limit of normal (ULN) level of the hepatotoxic marker, or (ii) greater than or equal to twice the baseline level of the hepatotoxic marker Following administration of the level of the hepatotoxic marker to detect hepatotoxicity, the subject is administered a therapeutically effective amount of corticosteroid for a period of at least about 5 to about 8 weeks, followed by a decreasing amount of corticosteroid for about 2, 3 or 4 weeks time of week. In any of such embodiments, the hepatotoxicity marker is ALT and/or AST, preferably ALT. In some embodiments, following the detection, a dose of 40 mg/day of the prisone equivalent is administered for a period of about 5 weeks, followed by a decreasing amount of the prisone equivalent for a period of about 3 weeks (eg, Prisone equivalent doses of 30 mg/day for one week, 20 mg/day for one week and 10 mg/day for one week). Other systemic immunosuppressants that can be administered at effective doses after detection of hepatotoxicity include (1) calcineurin inhibitors such as tacrolimus or cyclosporine; (2) antiproliferative agents or IMDH inhibitors , such as mycophenolate mofetil, leflunomide, or azathioprine; (3) mTOR inhibitors, such as sirolimus or everolimus; (4) Janus kinase inhibitors, such as tofacitinib; or (5) immunosuppressant antibodies. For example, the immunosuppressant is tacrolimus or mycophenolate mofetil.

在某些相關實施例中，本發明提供如本文所描述之重組載體建構體或AAV顆粒之組合物，其用於與預防性投與免疫抑制劑(例如皮質類固醇)及/或治療性投與本文所描述之免疫抑制劑(例如皮質類固醇)一起共投與。本發明亦提供如本文所描述之重組載體建構體或AAV顆粒之用途，其係用於製備用以與預防性投與免疫抑制劑及/或治療性投與本文所描述之免疫抑制劑一起共投與的藥劑。類似地，本發明提供一種免疫抑制劑組合物，其用於根據本文所描述之免疫抑制劑預防性投與及/或免疫抑制劑治療性投與來預防及/或治療與投與AAV顆粒相關之任何肝毒性。本發明亦提供免疫抑制劑之用途，其用於製備用於根據本文所描述之免疫抑制劑預防性投與及/或免疫抑制劑治療性投與來預防及/或治療與投與AAV顆粒相關之任何肝毒性的藥劑。In certain related embodiments, the present invention provides compositions of recombinant vector constructs or AAV particles as described herein for use in conjunction with prophylactic administration of immunosuppressants (eg, corticosteroids) and/or therapeutic administration The immunosuppressive agents described herein (eg, corticosteroids) are co-administered together. The present invention also provides the use of a recombinant vector construct or AAV particle as described herein for the preparation of co-administration with prophylactic administration of an immunosuppressant and/or therapeutic administration of an immunosuppressant as described herein administered drug. Similarly, the present invention provides an immunosuppressive composition for use in the prophylactic and/or therapeutic administration of AAV particles in accordance with the immunosuppressive prophylactic and/or immunosuppressive therapeutic administration described herein any hepatotoxicity. The present invention also provides the use of an immunosuppressant for the preparation of an immunosuppressive agent for prophylactic and/or therapeutic administration in accordance with the immunosuppressive prophylactic and/or immunosuppressive therapeutic administration described herein in connection with the administration of AAV particles any hepatotoxic agent.

該等方法亦可進一步包含監測游離基因體形成之步驟，該等步驟包含視情況藉由PCR或南方墨點法自個體之肝細胞提取DNA及檢測環形載體基因體。該等方法亦可進一步包含監測AAV整合之步驟，該等步驟包含自個體之肝細胞提取染色體或DNA及檢測AAV載體基因體，例如藉由PCR，其描述於Schnepp等人,J . Virol . , 79(23):14793-14803 (2005)中，或藉由目標富集定序(TES)，其描述於Gnirke等人,Nat Biotechnol . 27(2): 182-189 (2009)中。The methods may also further comprise the steps of monitoring the formation of episomal bodies comprising, as appropriate, extracting DNA from individual hepatocytes by PCR or Southern blotting and detecting circular vector gene bodies. The methods may further comprise the step of monitoring the integrated AAV, such step comprising extracts from hepatocytes of an individual and the detection of chromosomal DNA or AAV vector genome, for example by PCR, which is described in Schnepp et al., J. Virol., 79(23):14793-14803 (2005), or by target enrichment sequencing (TES), which is described in Gnirke et al., Nat Biotechnol . 27(2): 182-189 (2009).

該等方法亦可進一步包含每週量測個體之血漿Phe含量，及視情況對個體進行Phe挑戰測試或Phe呼吸測試(其量測Phe氧化)之步驟。The methods may also further comprise the steps of measuring the subject's plasma Phe levels on a weekly basis, and optionally subjecting the subject to a Phe challenge test or a Phe breath test, which measures Phe oxidation.

該等方法亦可進一步包含每週量測個體之一或多種神經傳遞質或神經傳遞質代謝物之血漿含量的步驟。舉例而言，該一或多種神經傳遞質或神經傳遞質代謝物為苯乙胺、苯乙醇胺、酪胺、多巴胺、正腎上腺素、腎上腺素、色胺、羥基色胺、苯乙酸、苯乙醯基麩醯胺酸、杏仁酸、羥基苯乙酸、DOPAC、高香草酸、DOMA、MOPEG、香草基杏仁酸、吲哚乙酸及5-羥基吲哚乙酸。The methods may also further comprise the step of measuring weekly plasma levels of one or more neurotransmitters or neurotransmitter metabolites in the subject. For example, the one or more neurotransmitters or neurotransmitter metabolites are phenylethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid, phenylacetylene glutamic acid, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillyl mandelic acid, indoleacetic acid and 5-hydroxyindoleacetic acid.

在不存在同時藥物療法之情況下，本發明之方法可導致血漿Phe含量(例如平均血漿Phe含量，或兩個連續血漿Phe含量之平均值)臨床上顯著降低。舉例而言，在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與之後8週降低至360 μmol/L或更低，或在該投與之後2、3或4年降低至360 μmol/L或更低。舉例而言，在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與之後8週在120與360 μmol/L之間。在一些實施例中，在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與之後8週降低至120 μmol/L或更低，或在未同時藥物療法之情況下，在該投與之後2、3或4年降低至120 μmol/L或更低。In the absence of concurrent drug therapy, the methods of the present invention can result in a clinically significant reduction in plasma Phe levels (eg, mean plasma Phe levels, or the average of two consecutive plasma Phe levels). For example, in the absence of concomitant drug therapy, the subject's plasma Phe levels decrease to 360 μmol/L or less 8 weeks after the administration, or to 2, 3 or 4 years after the administration 360 μmol/L or less. For example, without concomitant drug therapy, the subject's plasma Phe levels were between 120 and 360 μmol/L 8 weeks after the administration. In some embodiments, the subject's plasma Phe levels decrease to 120 μmol/L or less 8 weeks after the administration without concomitant drug therapy, or, without concomitant drug therapy, at the Reduce to 120 μmol/L or less 2, 3 or 4 years after administration.

本發明之方法可允許個體經受來自完整食物來源之Phe攝入增加。舉例而言，在該投與之後該個體之血漿Phe含量在120與360 μmol/L之間，或在30與360 μmol/L之間，且與基線處之Phe受限膳食相比，個體耐受Phe攝入增加。The methods of the present invention may allow individuals to experience increased intake of Phe from whole food sources. For example, the subject's plasma Phe level is between 120 and 360 μmol/L, or between 30 and 360 μmol/L following the administration, and the subject is resistant to a Phe-restricted diet at baseline compared to a Phe-restricted diet. Increased intake of Phe.

本發明之方法可降低在該投與之後個體之神經傳遞質或神經傳遞質代謝物之血漿含量。舉例而言，該一或多種神經傳遞質或神經傳遞質代謝物為苯乙胺、苯乙醇胺、酪胺、多巴胺、正腎上腺素、腎上腺素、色胺、羥基色胺、苯乙酸、苯乙醯基麩醯胺酸、杏仁酸、羥基苯乙酸、DOPAC、高香草酸、DOMA、MOPEG、香草基杏仁酸、吲哚乙酸及5-羥基吲哚乙酸。The methods of the present invention can reduce plasma levels of neurotransmitters or neurotransmitter metabolites in an individual following such administration. For example, the one or more neurotransmitters or neurotransmitter metabolites are phenylethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid, phenylacetylene glutamic acid, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillyl mandelic acid, indoleacetic acid and 5-hydroxyindoleacetic acid.

本發明之方法可使得該個體之生活品質在該投與之後改善，視情況如藉由PKU-QOL或Q-LES-Q-SF調查表所量測。本發明之方法可使得該個體之神經認知症狀或量測在該投與之後改善，視情況如藉由CANTAB所量測。較佳地，該個體在該投與之後未罹患低***酸血症。The methods of the present invention may result in an improvement in the subject's quality of life following the administration, as measured by the PKU-QOL or Q-LES-Q-SF questionnaire, as appropriate. The methods of the present invention may result in an improvement in the individual's neurocognitive symptoms or measures following the administration, as measured by CANTAB, as appropriate. Preferably, the individual does not suffer from hypophenylalaninemia following the administration.

在另一態樣中，本發明提供一種醫藥組合物，其包含濃度為至少1E13 vg/ml，例如約1E13 vg/ml至約5E14 vg/ml、約2E13 vg/ml至約2E14 vg/ml、約1E13 vg/ml、約2E13 vg/ml、約3E13 vg/ml、約4E13 vg/ml、5E13 vg/ml、約6E13 vg/ml、約7E13 vg/ml、約8E13 vg/ml、約9E13 vg/ml或約2E14 vg/ml之rAAV顆粒；緩衝劑；等張劑；冷凍保存劑；及界面活性劑，該醫藥組合物在約-60℃或更低溫度下儲存期間穩定持續至少約1年、1.5年或2年。在一些實施例中，界面活性劑為濃度小於0.2% w/v，或小於0.15% w/v，例如約0.1% w/v之泊洛沙姆。在一些實施例中，冷凍保存劑為糖，例如海藻糖。In another aspect, the present invention provides a pharmaceutical composition comprising a concentration of at least 1E13 vg/ml, such as about 1E13 vg/ml to about 5E14 vg/ml, about 2E13 vg/ml to about 2E14 vg/ml, About 1E13 vg/ml, about 2E13 vg/ml, about 3E13 vg/ml, about 4E13 vg/ml, 5E13 vg/ml, about 6E13 vg/ml, about 7E13 vg/ml, about 8E13 vg/ml, about 9E13 vg rAAV particles per ml or about 2E14 vg/ml; buffers; isotonic agents; cryopreservatives; and surfactants, the pharmaceutical composition is stable for at least about 1 year during storage at about -60°C or less , 1.5 years or 2 years. In some embodiments, the surfactant is a poloxamer at a concentration of less than 0.2% w/v, or less than 0.15% w/v, eg, about 0.1% w/v. In some embodiments, the cryopreservative is a sugar, such as trehalose.

在一些實施例中，醫藥組合物為水性的且包含濃度為至少1E13 vg/ml之rAAV顆粒；濃度為約5至約15 mM之磷酸鈉；濃度為約100 mM至約165 mM之氯化鈉；為糖，視情況為海藻糖之冷凍保存劑；及濃度為小於0.2% w/v之泊洛沙姆。磷酸鈉可包含磷酸氫二鈉及磷酸二氫鈉。視情況，糖為濃度為約60 mM至約80 mM之海藻糖。視情況，泊洛沙姆為濃度為約0.05% w/v至0.15% w/v之泊洛沙姆188。In some embodiments, the pharmaceutical composition is aqueous and comprises rAAV particles at a concentration of at least 1E13 vg/ml; sodium phosphate at a concentration of about 5 to about 15 mM; sodium chloride at a concentration of about 100 mM to about 165 mM ; is a sugar, as the case may be, a cryopreservative for trehalose; and a poloxamer at a concentration of less than 0.2% w/v. The sodium phosphate can include disodium hydrogen phosphate and sodium dihydrogen phosphate. Optionally, the sugar is trehalose at a concentration of about 60 mM to about 80 mM. A Poloxamer is Poloxamer 188 at a concentration of about 0.05% w/v to 0.15% w/v, as appropriate.

在一些實施例中，磷酸二氫鈉之濃度為大於0.1 mg/mL且小於0.5 mg/mL，視情況約0.3 mg/mL，且該磷酸氫二鈉之濃度為大於2.5 mg/ml且小於3 mg/ml，視情況約2.7 mg/mL。在一些實施例中，氯化鈉之濃度為大於5 mg/ml且小於8 mg/ml，視情況約7 mg/ml。在一些實施例中，糖為濃度大於20 mg/ml至小於40 mg/ml，或約25 mg/ml至約35 mg/ml，或約28 mg/ml之二水合海藻糖。在一些實施例中，泊洛沙姆188之濃度為小於1.5 mg/ml或約1 mg/ml。在一些實施例中，醫藥組合物包含濃度為約2E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM二水合海藻糖及0.1% w/v泊洛沙姆188。在某些實施例中，醫藥組合物包含濃度為約3E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM二水合海藻糖及0.1% w/v泊洛沙姆188。在一些實施例中，醫藥組合物包含濃度為約4E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM二水合海藻糖及0.1% w/v泊洛沙姆188。在某些實施例中，醫藥組合物包含濃度為約5E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM二水合海藻糖及0.1% w/v泊洛沙姆188。在一些實施例中，醫藥組合物包含濃度為約6E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM二水合海藻糖及0.1% w/v泊洛沙姆188。在某些實施例中，醫藥組合物包含濃度為約7E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM二水合海藻糖及0.1% w/v泊洛沙姆188。在一些實施例中，醫藥組合物包含濃度為約8E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM二水合海藻糖及0.1% w/v泊洛沙姆188。在某些實施例中，醫藥組合物包含濃度為約9E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM二水合海藻糖及0.1% w/v泊洛沙姆188。在一些實施例中，醫藥組合物包含濃度為約2E14 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM二水合海藻糖及0.1% w/v泊洛沙姆188。In some embodiments, the concentration of sodium dihydrogen phosphate is greater than 0.1 mg/mL and less than 0.5 mg/mL, optionally about 0.3 mg/mL, and the concentration of disodium hydrogen phosphate is greater than 2.5 mg/ml and less than 3 mg/mL mg/ml, about 2.7 mg/mL as the case may be. In some embodiments, the concentration of sodium chloride is greater than 5 mg/ml and less than 8 mg/ml, optionally about 7 mg/ml. In some embodiments, the sugar is trehalose dihydrate at a concentration of greater than 20 mg/ml to less than 40 mg/ml, or about 25 mg/ml to about 35 mg/ml, or about 28 mg/ml. In some embodiments, the concentration of poloxamer 188 is less than 1.5 mg/ml or about 1 mg/ml. In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 2E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. In certain embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 3E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188 . In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 4E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. In certain embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 5E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188 . In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 6E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. In certain embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 7E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188 . In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 8E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. In certain embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 9E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188 . In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 2E14 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188.

在具體實施例中，醫藥組合物包含本文所描述之重組AAV顆粒。在具體實施例中，重組rAAV顆粒包含重組載體建構體，其中該重組載體建構體包含(a)以下中之一或兩者：(i) AAV 5'反向末端重複序列(ITR)及(ii) AAV 3' ITR；(b)異源肝特異性轉錄調節區；及(c)編碼功能性人類***酸羥化酶(hPAH)之核酸序列，視情況其中該等AAV ITR為AAV2 ITR。較佳地，編碼功能性hPAH之核酸可操作地連接至肝特異性表現控制元件。載體建構體可包括額外表現控制元件，例如：啟動子及/或增強子；內含子；視情況，來自與內含子相同之基因的外顯子；及多腺苷酸化(polyA)信號。本文中進一步描述此類元件。較佳地，rAAV顆粒亦包含具有肝趨向性之AAV衣殼，視情況為AAV5型衣殼。In specific embodiments, the pharmaceutical composition comprises the recombinant AAV particles described herein. In specific embodiments, the recombinant rAAV particle comprises a recombinant vector construct, wherein the recombinant vector construct comprises (a) one or both of: (i) AAV 5' inverted terminal repeats (ITR) and (ii) ) AAV 3' ITR; (b) a heterologous liver-specific transcriptional regulatory region; and (c) a nucleic acid sequence encoding a functional human phenylalanine hydroxylase (hPAH), optionally wherein the AAV ITRs are AAV2 ITRs. Preferably, the nucleic acid encoding a functional hPAH is operably linked to a liver-specific expression control element. The vector construct may include additional expression control elements such as: promoters and/or enhancers; introns; optionally, exons from the same gene as the intron; and polyadenylation (polyA) signals. Such elements are further described herein. Preferably, the rAAV particles also comprise an AAV capsid with liver tropism, optionally an AAV5 type capsid.

在一些實施例中，重組載體建構體包含與SEQ ID NO: 18至少90%一致之核苷酸序列，且AAV衣殼為AAV5型衣殼。在某些實施例中，重組載體建構體包含與SEQ ID NO: 52至少90%一致之核苷酸序列，且AAV衣殼為AAV5型衣殼。在一些實施例中，重組載體建構體包含與SEQ ID NO: 15至23中之任一者至少90%一致的核苷酸序列，且AAV衣殼為AAV5型衣殼。In some embodiments, the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 18, and the AAV capsid is an AAV5 type capsid. In certain embodiments, the recombinant vector construct comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 52, and the AAV capsid is an AAV5 type capsid. In some embodiments, the recombinant vector construct comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 15-23, and the AAV capsid is an AAV5 type capsid.

較佳地，醫藥組合物為液體水溶液，或經凍乾，且用於在冷凍溫度下儲存。在此等實施例中之任一者中，該組合物用於向患有苯酮尿症之患者靜脈內投與rAAV顆粒。Preferably, the pharmaceutical composition is a liquid aqueous solution, or lyophilized, and used for storage at refrigerated temperatures. In any of these embodiments, the composition is used to administer rAAV particles intravenously to a patient with phenylketonuria.

熟習此項技術者在閱讀本說明書時將顯而易知其他實施例。Other embodiments will be apparent to those skilled in the art upon reading this specification.

定義：definition:

除非另外定義，否則本文所用之技術及科學術語具有與本發明所屬領域之一般熟習此項技術者通常所理解相同之含義。參見例如Singleton等人, Dictionary of Microbiology and Molecular Biology第2版, J. Wiley & Sons (New York, N.Y. 1994)；Sambrook等人, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989)。出於本發明之目的，以下術語定義如下。Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd Edition, J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). For the purposes of the present invention, the following terms are defined as follows.

如本文所用，在基因遞送之上下文中，術語「載體」或「基因遞送載體」可指充當基因遞送媒劑且包含封裝於例如包膜或衣殼內之核酸(亦即，包含本文所描述之載體建構體中之任一者的載體基因體)的顆粒。基因遞送載體可為病毒基因遞送載體或非病毒基因遞送載體。可替代地，在一些上下文中，術語「載體」可用於僅指代載體基因體或載體建構體。適用於本文中之病毒載體可為小病毒、腺病毒、反轉錄病毒、慢病毒或單純疱疹病毒。小病毒可為腺病毒相關病毒(AAV)。As used herein, in the context of gene delivery, the term "vector" or "gene delivery vector" can refer to a nucleic acid that acts as a gene delivery vehicle and comprises a nucleic acid encapsulated, eg, within an envelope or capsid (ie, comprising the particle of any of the vector constructs). The gene delivery vector can be a viral gene delivery vector or a non-viral gene delivery vector. Alternatively, in some contexts, the term "vector" may be used to refer only to a vector gene body or a vector construct. Viral vectors suitable for use herein can be parvoviruses, adenoviruses, retroviruses, lentiviruses or herpes simplex virus. The small virus may be an adeno-associated virus (AAV).

如本文所用，術語「AAV」為腺相關病毒之標準縮寫。腺相關病毒為僅在其中某些功能由共感染輔助病毒提供之細胞中生長之單股DNA小病毒。存在多種已表徵之AAV之血清型。AAV之一般資訊及綜述可見於以下：例如Carter, 1989, Handbook of Parvoviruses, 第1卷, 第169頁至第228頁；及Berns, 1990, Virology, 第1743頁至第1764頁, Raven Press, (New York)；Gao等人, 2011, Methods Mol. Biol. 807: 93-118；Ojala等人, 2018, Mol. Ther. 26(1): 304-19。然而，完全預期此等相同原理將適用於其他AAV血清型，因為熟知的是各種血清型在結構上及功能上相當緊密相關，甚至在基因層面上亦如此。(參見例如，Blacklowe, 1988, Parvoviruses and Human Disease之第165-174頁, J. R. Pattison,編；及Rose, Comprehensive Virology 3:1-61 (1974))。舉例而言，所有AAV血清型明顯展現由同源rep基因介導之極類似複製特性；並且所有皆帶有三種相關衣殼蛋白質。相關性程度藉由沿基因體長度在血清型之間展現廣泛交叉雜交之異雙螺旋分析；及對應於「反向末端重複序列(ITR)」之末端處之類似自結合片段之存在進一步表明。As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated viruses are small single-stranded DNA viruses that grow only in cells in which some functions are provided by co-infecting helper viruses. There are various serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228; and Berns, 1990, Virology, pp. 1743-1764, Raven Press, ( New York); Gao et al., 2011, Methods Mol. Biol. 807: 93-118; Ojala et al., 2018, Mol. Ther. 26(1): 304-19. However, it is fully expected that these same principles will apply to other AAV serotypes, as it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, eg, Blacklowe, 1988, Parvoviruses and Human Disease, pp. 165-174, J. R. Pattison, eds.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes; and all carry three related capsid proteins. The degree of relatedness is further indicated by heteroduplex analysis showing extensive cross-hybridization between serotypes along the length of the genome; and the presence of similar self-associating fragments at the ends corresponding to "inverted terminal repeats (ITRs)".

如本文所用，「AAV載體建構體」係指單股或雙股核酸，其具有可操作地連接至轉錄調節元件(亦稱為「表現控制元件」)之側接編碼蛋白之序列(在一個實施例中，編碼功能性治療蛋白之序列，例如，編碼hPAH之序列)的AAV 5'反向末端重複序列(ITR)序列或AAV 3' ITR，該等轉錄調節元件與編碼蛋白之序列為異源的及/或與AAV病毒基因體為異源的，亦即一或多種啟動子及/或增強子，且視情況多腺苷酸化序列及/或一或多種***在編碼蛋白之序列的外顯子之間的內含子。單股AAV載體係指存在於AAV病毒顆粒基因體中的核酸，並且可為本文所揭示之核酸序列的有義股或反義股。此類單股核酸之尺寸以鹼基提供。雙鏈AAV載體係指用於表現或轉移AAV載體核酸的存在於質體(例如pUC19)之DNA或雙股病毒(例如桿狀病毒)之基因體中之核酸。此類雙股核酸之尺寸以鹼基對(bp)提供。As used herein, an "AAV vector construct" refers to a single- or double-stranded nucleic acid having sequences (in one implementation) flanked by protein-encoding sequences operably linked to transcriptional regulatory elements (also known as "expression control elements") For example, a sequence encoding a functional therapeutic protein, e.g., a sequence encoding hPAH, an AAV 5' inverted terminal repeat (ITR) sequence or an AAV 3' ITR sequence that is heterologous to the protein-encoding sequence and/or heterologous to the AAV viral genome, i.e., one or more promoters and/or enhancers, and optionally polyadenylation sequences and/or one or more insertions in the extremity of the protein-encoding sequence introns between sons. A single-stranded AAV vector refers to a nucleic acid present in the genome of an AAV virion, and can be either the sense or antisense strand of the nucleic acid sequences disclosed herein. The dimensions of such single-stranded nucleic acids are provided in bases. A double-stranded AAV vector system refers to nucleic acid present in the DNA of a plastid (eg, pUC19) or the gene body of a double-stranded virus (eg, baculovirus) used for expression or transfer of AAV vector nucleic acid. The dimensions of such double-stranded nucleic acids are provided in base pairs (bp).

儘管已在文獻中報導AAV顆粒具有＞5.0 kb之AAV基因體，在許多此等情況下，編碼基因之5'或3'末端似乎經截短(參見Hirsch等人,Molec . Ther . 18:6-8, 2010，及Ghosh等人,Biotech . Genet . Engin . Rev . 24:165- 178, 2007)。然而，已顯示重疊同源重組發生於具有5'端截短與3'端截短之核酸之間的AAV感染細胞中以產生編碼大型蛋白之「完整」核酸，進而重構功能性全長基因。Although AAV particles have been reported in the literature to have an AAV gene body >5.0 kb, in many of these cases the 5' or 3' end of the coding gene appears to be truncated (see Hirsch et al., Molec . Ther . 18:6 ... -8, 2010 and Ghosh et al., Biotech Genet Engin Rev 24:. 165- 178, 2007). However, overlapping homologous recombination has been shown to occur in AAV-infected cells between nucleic acids with 5' and 3' truncations to generate "whole" nucleic acids encoding large proteins, thereby reconstituting functional full-length genes.

過大AAV載體在5'端處隨機截斷且缺少5' AAV ITR。因為AAV為單股DNA病毒，且包裝有義或反義股，所以過大AAV載體中之有義股缺少5' AAV ITR及編碼目標蛋白之基因之5'端的可能部分，且過大AAV載體中之反義股缺少3' ITR及編碼目標蛋白之基因之3'端的可能部分。功能性轉基因藉由目標細胞內之有義及反義截短基因體之黏接產生於過大AAV載體感染細胞中。因此，在某些實施例中，AAV PAH載體及/或病毒顆粒包含至少一個ITR。Oversized AAV vectors are randomly truncated at the 5' end and lack the 5' AAV ITR. Because AAV is a single-stranded DNA virus and packages sense or antisense strands, the sense strand in an oversized AAV vector lacks the 5' AAV ITR and possibly a portion of the 5' end of the gene encoding the protein of interest, and the oversized AAV vector lacks the sense strand. The antisense strand lacks the 3' ITR and a possible portion of the 3' end of the gene encoding the protein of interest. Functional transgenes are produced in oversized AAV vector-infected cells by cohesion of sense and antisense truncated gene bodies within the target cells. Thus, in certain embodiments, AAV PAH vectors and/or viral particles comprise at least one ITR.

如本文所用之術語「反向末端重複序列(ITR)」係指順式充當DNA複製起點及病毒基因體之封裝信號的在AAV基因體之5'及3'端發現的本領域中公認的區。AAV ITR與AAV rep編碼區一起實現***兩個側接ITR之間之核苷酸序列的有效切除及救援以及整合至宿主細胞基因體中。某些AAV相關ITR之序列揭示於Yan等人,J . Virol . (2005)第79卷, 第364頁至第379頁中，該文獻以全文引用之方式併入本文中。可用於本文中之ITR序列可為保留功能性能力之全長、野生型ITR或其片段，或可為能夠以順式其複製來源作用之全長、野生型AAV ITR之序列變異體。適用於本文所提供之實施例之重組AAV PAH載體中之AAV ITR可來源於任何已知AAV血清型，且在某些較佳實施例中，來源於AAV2血清型。The term "inverted terminal repeat (ITR)" as used herein refers to an art-recognized region found at the 5' and 3' ends of the AAV gene body that serves in cis as an origin of DNA replication and an encapsulation signal for the viral genome . The AAV ITR together with the AAV rep coding region enables efficient excision and rescue of the nucleotide sequence inserted between the two flanking ITRs and integration into the host cell genome. Certain sequences of AAV ITR correlation disclosed in Yan et al., J. Virol. (2005) Vol. 79, page 364 to page 379, incorporated by reference in this document it is incorporated herein by reference. The ITR sequences useful herein can be full-length, wild-type ITRs or fragments thereof that retain functional capability, or can be sequence variants of full-length, wild-type AAV ITRs capable of functioning in cis for their source of replication. AAV ITRs suitable for use in the recombinant AAV PAH vectors of the embodiments provided herein can be derived from any known AAV serotype, and in certain preferred embodiments, from the AAV2 serotype.

術語「控制序列」係指在特定宿主生物體中表現可操作地連接之編碼序列所需之DNA序列。適用於原核生物之控制序列例如包括啟動子、視情況選用之操縱序列及核糖體結合位點。已知真核細胞利用啟動子、多腺苷酸化信號及增強子。The term "control sequences" refers to DNA sequences required for expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for use in prokaryotes include, for example, promoters, optional operator sequences and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.

「轉錄調節元件」係指涉及基因轉錄調節之基因的核苷酸序列，包括啟動子，加上反應元件、活化因子及增強子序列，用於結合轉錄因子以幫助RNA聚合酶結合且促進表現；以及抑制蛋白所結合的操縱或靜默序列，以阻斷RNA聚合酶附接且阻止表現。術語「肝特異性轉錄調節元件」或「肝特異性轉錄調節區」係指在肝組織中特異性產生較佳基因表現之調節元件或區。"Transcriptional regulatory element" means the nucleotide sequence of a gene involved in the regulation of gene transcription, including promoters, plus response elements, activators and enhancer sequences for binding transcription factors to aid RNA polymerase binding and promote expression; and manipulation or silencing sequences to which arrestin binds to block RNA polymerase attachment and prevent expression. The term "liver-specific transcriptional regulatory element" or "liver-specific transcriptional regulatory region" refers to a regulatory element or region that specifically results in better gene expression in liver tissue.

如本文所用，術語「可操作地連接」用於描述調節元件與基因或其編碼區之間的連接。典型地，基因表現處於一或多個調節元件控制下，例如(不限於)組成性或誘導性啟動子、組織特異性調節元件及增強子。基因或編碼區稱為「可操作地連接至」或「以操作方式連接至」調節元件或與調節元件「可操作地相關」，意謂基因或編碼區受調節元件的控制或影響。舉例而言，若啟動子影響編碼序列之轉錄或表現，則啟動子可操作地連接至編碼序列。As used herein, the term "operably linked" is used to describe the linkage between a regulatory element and a gene or its coding region. Typically, gene expression is under the control of one or more regulatory elements, such as, without limitation, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. A gene or coding region is referred to as being "operably linked to" or "operably linked to" or "operably associated with" a regulatory element, meaning that the gene or coding region is under the control or influence of the regulatory element. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

術語「分離」在關於本發明之核酸分子使用時通常係指自至少一種與其天然來源通常相關的污染核酸鑑別且分離的核酸序列。經分離之核酸可以與自然界中發現的形式或環境不同的形式或環境存在。因此，經分離之核酸分子區別於在天然細胞中存在的核酸分子。The term "isolated" as used in relation to the nucleic acid molecules of the invention generally refers to a nucleic acid sequence identified and isolated from at least one contaminating nucleic acid that is normally associated with its natural source. An isolated nucleic acid may exist in a form or environment different from that found in nature. Thus, isolated nucleic acid molecules are distinguished from nucleic acid molecules present in natural cells.

如本文所用，術語「變異體」係指具有與參考聚核苷酸(或多肽)實質上類似的序列之聚核苷酸(或多肽)。熟習此項技術者已知在聚核苷酸、蛋白質或多肽中引入核苷酸及胺基酸變化之程序(參見例如Sambrook等人(1989))。在聚核苷酸之情況下，相較於參考聚核苷酸，變異體可在5'端、3'端及/或一或多個內部位點處具有一或多個核苷酸之缺失、取代、添加。變異體與參考聚核苷酸之間的序列類似性及/或差異可使用此項技術中已知之習知技術(例如聚合酶鏈反應(PCR)及雜合技術)檢測。變異體聚核苷酸亦包括合成衍生之聚核苷酸，諸如例如藉由使用定點突變誘發產生之聚核苷酸。一般而言，如藉由熟練的技術人員已知之序列比對程式所測定，包括但不限於DNA之聚核苷酸之變異體可以與參考聚核苷酸具有至少約50%、約55%、約60%、約65%、約70%、約75%、約80%、約85%、約90%、約91%、約92%、約93%、約94%、約95%、約96%、約97%、約98%、約99%或更大之序列一致性。在多肽之情況下，與參考多肽相比，變異體可以具有一或多個胺基酸之缺失、取代、添加。變異體與參考多肽之間之序列類似性及/或差異可以使用此項技術中已知之習知技術(例如西方墨點法(Western blot))檢測。一般而言，如藉由熟練的技術人員已知之序列比對程式所測定，多肽之變異體可以與參考多肽具有至少約60%、約65%、約70%、約75%、約80%、約85%、約90%、約91%、約92%、約93%、約94%、約95%、約96%、約97%、約98%、約99%或更大之序列一致性。As used herein, the term "variant" refers to a polynucleotide (or polypeptide) having a substantially similar sequence to a reference polynucleotide (or polypeptide). Procedures for introducing nucleotide and amino acid changes in polynucleotides, proteins or polypeptides are known to those skilled in the art (see, eg, Sambrook et al. (1989)). In the case of polynucleotides, variants may have deletions of one or more nucleotides at the 5' end, the 3' end and/or at one or more internal sites compared to the reference polynucleotide , replace, add. Sequence similarity and/or differences between a variant and a reference polynucleotide can be detected using conventional techniques known in the art, such as polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as, for example, polynucleotides produced by the use of site-directed mutagenesis. Generally, variants of polynucleotides, including but not limited to DNA, can be at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96% %, about 97%, about 98%, about 99% or greater sequence identity. In the case of polypeptides, variants may have deletions, substitutions, additions of one or more amino acids compared to the reference polypeptide. Sequence similarity and/or differences between the variant and the reference polypeptide can be detected using conventional techniques known in the art (eg, Western blot). In general, a variant of a polypeptide may be at least about 60%, about 65%, about 70%, about 75%, about 80%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or greater sequence identity .

術語「一致性」、「同源性」及其語法變化形式意謂兩個或更多個參考實體在其為「比對」序列時相同。因此，藉助於實例，在兩個多肽序列相同時，其至少在參考區或部分內具有相同胺基酸序列。在兩個聚核苷酸序列相同時，其至少在參考區或部分內具有相同聚核苷酸序列。一致性可在序列之界定區域(區或結構域)內。一致性之「區域」或「區」係指相同的兩個或更多個參考實體之部分。因此，在兩個蛋白質或核酸序列在一或多個序列區域或區內相同時，其在該區內共用一致性。「比對」序列係指多個聚核苷酸或蛋白質(胺基酸)序列，其與參考序列相比通常含有對缺失或額外鹼基或胺基酸(間隙)的校正。「實質同源性」意謂分子在結構上或在功能上保守，以使得其具有或經預測具有至少部分的參考分子或與其共用同源性之參考分子之相關/對應區或部分之一或多種結構或功能(例如，生物功能或活性)的結構或功能。The terms "identity", "homology" and grammatical variations thereof mean that two or more referenced entities are the same when they are "aligned" sequences. Thus, by way of example, when two polypeptide sequences are identical, they have the same amino acid sequence at least within the reference region or portion. When two polynucleotide sequences are identical, they have the same polynucleotide sequence at least within the reference region or portion. The identity can be within a defined region (region or domain) of the sequence. A "region" or "region" of identity refers to the same part of two or more reference entities. Thus, when two protein or nucleic acid sequences are identical in one or more sequence regions or regions, they share identity within that region. An "aligned" sequence refers to a plurality of polynucleotide or protein (amino acid) sequences that typically contain corrections for deletions or additional bases or amino acids (gaps) compared to a reference sequence. "Substantially homologous" means that a molecule is structurally or functionally conserved such that it has or is predicted to have at least part of the reference molecule or one of the related/corresponding regions or parts of the reference molecule with which it shares homology or The structure or function of various structures or functions (eg, biological functions or activities).

「核酸序列一致性或同源性百分比(%)」定義為在比對各別序列且必要時引入間隙以實現最大序列一致性百分比後與參考序列一致之候選序列中之核苷酸的百分比。出於測定核酸序列一致性百分比之目的之比對可以此項技術內之各種方式實現，例如使用公開可獲得之電腦軟體，諸如ALIGN或Megalign (DNASTAR)軟體。熟習此項技術者可以測定用於量測比對之適當參數，包括用於達成所比較序列之全長內之最大比對所需的任何演算法。"Percent (%) nucleic acid sequence identity or homology" is defined as the percentage of nucleotides in a candidate sequence that are identical to the reference sequence after aligning the respective sequences and introducing gaps where necessary to achieve a maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be accomplished in various ways within the art, eg, using publicly available computer software, such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

相對於本文中所鑑別之PAH胺基酸序列，「胺基酸序列一致性或同源性百分比(%)」定義為候選序列中之胺基酸殘基的百分比，該等候選序列中之胺基酸殘基在比對序列且必要時引入間隙以實現最大序列一致性百分比後與PAH多肽序列中之胺基酸殘基一致，且不將任何保守取代視為序列一致性之一部分。出於測定胺基酸序列一致性百分比之目的之比對可以此項技術內之各種方式實現，例如使用公開可獲得之電腦軟體，諸如ALIGN或Megalign (DNASTAR)軟體。熟習此項技術者可以測定用於量測比對之適當參數，包括用於達成所比較序列之全長內之最大比對所需的任何演算法。"Percent amino acid sequence identity or homology (%)" is defined as the percentage of amino acid residues in candidate sequences relative to the PAH amino acid sequences identified herein The amino acid residues are identical to the amino acid residues in the PAH polypeptide sequence after aligning the sequences and introducing gaps where necessary to achieve maximum percent sequence identity, and no conservative substitutions are considered part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in various ways within the art, eg, using publicly available computer software, such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

「密碼子優化(Codon optimization/codon optimized)」係指核苷酸序列中所產生之變化，以使得其與非密碼子優化序列相比更可能以相對較高含量表現。其不改變各密碼子編碼之胺基酸。"Codon optimization/codon optimized" refers to changes made in a nucleotide sequence such that it is more likely to be expressed at relatively high levels compared to non-codon optimized sequences. It does not change the amino acid encoded by each codon.

如本文所用，「內含子」廣泛定義為可藉由RNA剪接移除之核苷酸序列。「RNA剪接」意謂自前mRNA切除內含子以形成成熟mRNA。內含子可位於基因之編碼區上游、下游或內部。將內含子***核苷酸序列中可以藉由此項技術中已知之任何方法完成。***內含子之位置之唯一限制為考慮AAV病毒顆粒(約5 kbp)之封裝限制。As used herein, an "intron" is broadly defined as a nucleotide sequence that can be removed by RNA splicing. "RNA splicing" means excision of introns from pre-mRNA to form mature mRNA. Introns can be located upstream, downstream or within the coding region of a gene. Insertion of an intron into a nucleotide sequence can be accomplished by any method known in the art. The only restriction on the location of the intron insertion is to take into account the packaging restriction of AAV virions (approximately 5 kbp).

在某些實施例中，重組AAV載體建構體包含(a)核酸，其包含AAV2 5'反向末端重複序列(ITR) (其如此項技術中已知可或可不經修飾)；(b)肝特異性轉錄調節區；(c)一或多個內含子；(d)功能性hPAH蛋白編碼區；(e)多腺苷酸化序列；及(f) AAV2 3' ITR(其如此項技術中已知可或可不經修飾)。In certain embodiments, the recombinant AAV vector construct comprises (a) a nucleic acid comprising an AAV2 5' inverted terminal repeat (ITR) (which may or may not be modified as known in the art); (b) a liver (c) one or more introns; (d) a functional hPAH protein coding region; (e) a polyadenylation sequence; and (f) the AAV2 3' ITR as described in the art known with or without modification).

本文所提供之其他實施例係針對編碼功能性PAH多肽之載體建構體，其中該等建構體在一或多個不同定向上包含一或多個上述建構體之個別元件及其組合。本文所提供之另一實施例係針對呈相反定向之上述建構體。在另一實施例中，提供包含本文所描述之AAV載體建構體之重組AAV病毒顆粒及其用於治療個體之PKU之用途。Additional embodiments provided herein are directed to vector constructs encoding functional PAH polypeptides, wherein the constructs comprise one or more of the individual elements of the above-described constructs, and combinations thereof, in one or more different orientations. Another embodiment provided herein is directed to the above-described constructs in the opposite orientation. In another embodiment, recombinant AAV viral particles comprising the AAV vector constructs described herein and their use for treating PKU in an individual are provided.

「AAV病毒粒子(AAV virion)」或「AAV病毒顆粒(AAV viral particle)」或「AAV載體顆粒」係指由如本文所描述之至少一種AAV衣殼蛋白及衣殼化AAV載體建構體構成之病毒顆粒。若該顆粒包含異源聚核苷酸(亦即除野生型AAV基因體(諸如待遞送至哺乳動物細胞之轉基因)以外的聚核苷酸)，則其通常稱為「重組AAV載體顆粒」、「AAV顆粒」、「重組AAV顆粒」、「rAAV顆粒」或簡單稱為「AAV載體」。AAV載體顆粒之產生必定包括AAV載體基因體之產生，因此載體基因體含於AAV載體顆粒內。應理解，提及囊封於載體顆粒內之聚核苷酸AAV載體建構體及其複製係指AAV載體基因體。"AAV virion" or "AAV viral particle" or "AAV vector particle" refers to a structure consisting of at least one AAV capsid protein as described herein and an encapsidated AAV vector construct virus particles. If the particle comprises a heterologous polynucleotide (ie, a polynucleotide other than the wild-type AAV gene body (such as a transgene to be delivered to mammalian cells), it is commonly referred to as a "recombinant AAV vector particle", "AAV particle", "recombinant AAV particle", "rAAV particle" or simply "AAV vector". The production of AAV vector particles necessarily involves the production of AAV vector gene bodies, and thus the vector gene bodies are contained within the AAV vector particles. It will be understood that reference to a polynucleotide AAV vector construct encapsulated within a vector particle and its replication refers to the AAV vector genome.

如本文所用，「治療性AAV病毒」係指AAV病毒粒子、AAV病毒顆粒、AAV載體顆粒或AAV病毒，其包含編碼治療蛋白(諸如本文所描述之hPAH)之異源聚核苷酸。如本文所用之「AAV載體建構體」或「AAV載體基因體」係指包含編碼所關注蛋白質(亦稱為轉基因)之聚核苷酸的載體建構體，該等所關注蛋白質由AAV末端重複序列(ITR)側接且可操作地連接至一或多個表現控制元件。此類AAV載體建構體可以複製及包裝至已經編碼及表現rep及cap基因產物之載體轉染的宿主細胞中之感染性病毒顆粒(若存在)中。As used herein, "therapeutic AAV virus" refers to an AAV virion, AAV virion, AAV vector particle or AAV virus comprising a heterologous polynucleotide encoding a therapeutic protein, such as hPAH described herein. "AAV vector construct" or "AAV vector gene body" as used herein refers to a vector construct comprising polynucleotides encoding proteins of interest (also known as transgenes) consisting of AAV terminal repeats (ITR) flanked and operably connected to one or more presentation control elements. Such AAV vector constructs can replicate and package into infectious viral particles (if present) in host cells that have been transfected with vectors encoding and expressing the rep and cap gene products.

如本文所用，「治療性蛋白質」係指具有置換或補償內源蛋白質活性損失或降低之生物活性的多肽。舉例而言，功能性PAH為PKU之治療蛋白。As used herein, a "therapeutic protein" refers to a polypeptide having a biological activity that replaces or compensates for the loss or reduction of endogenous protein activity. For example, functional PAHs are therapeutic proteins for PKU.

如本文所用之「神經傳遞質」係指自神經細胞釋放之化學物質，其從而將脈衝自神經細胞傳遞至另一神經、肌肉、器官或其他組織。神經傳遞質為神經資訊自一個細胞至另一細胞之信使。在某些實施例中，神經傳遞質包括苯乙胺、酪胺、多巴胺、正腎上腺素、腎上腺素、色胺及血清素。如本文所用之「神經傳遞質代謝物」係指在一個或兩個酶促步驟下游的神經傳遞質降解之後的產物。神經傳遞質代謝物之非限制性實例包括苯乙酸、苯乙醯基甘胺酸、苯乙醯基麩醯胺酸、DOPAC、高香草酸、二羥基苯基乙二醇(DOPEG)、3-甲氧基-4-羥基苯基二醇(MHPG、MOPEG)吲哚乙酸及5-羥基吲哚乙酸。"Neurotransmitter," as used herein, refers to a chemical released from a nerve cell that transmits impulses from the nerve cell to another nerve, muscle, organ, or other tissue. Neurotransmitters are messengers of neural information from one cell to another. In certain embodiments, neurotransmitters include phenethylamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, and serotonin. "Neurotransmitter metabolite" as used herein refers to the product following neurotransmitter degradation one or two enzymatic steps downstream. Non-limiting examples of neurotransmitter metabolites include phenylacetic acid, phenethylglycine, phenethylglutamic acid, DOPAC, homovanillic acid, dihydroxyphenylethylene glycol (DOPEG), 3- Methoxy-4-hydroxyphenyldiol (MHPG, MOPEG) indoleacetic acid and 5-hydroxyindoleacetic acid.

如本文所用之「苯酮尿症(PKU)」係指由酶***酸羥化酶(PAH)之缺乏引起之遺傳性代謝疾病。此導致***酸(Phe)之含量升高及神經傳遞質及神經傳遞質代謝物之含量降低，這可能會影響大腦功能，引起嚴重智力殘疾、行為異常、認知損傷、精神症狀及言語遲緩及癲癇。"Phenylketonuria (PKU)" as used herein refers to an inherited metabolic disorder caused by deficiency of the enzyme phenylalanine hydroxylase (PAH). This results in increased levels of phenylalanine (Phe) and decreased levels of neurotransmitters and neurotransmitter metabolites, which may affect brain function, causing severe intellectual disability, behavioral abnormalities, cognitive impairment, psychiatric symptoms and speech delay and epilepsy .

如本文所用之「治療(treat或treatment)」係指治療性治療，其係指出於減少或消除彼等病徵或症狀或改善其進展、嚴重程度或持續時間之目的，向展現病理學病徵或症狀(亦即PKU)之個體投與治療。病徵及症狀可以為生物化學、細胞、組織學、功能、主觀或客觀的。PKU之病徵包括血液或血漿Phe含量升高、血液或血漿神經傳遞質含量降低(例如Tyr含量)及神經認知症狀。"Treat or treatment" as used herein refers to therapeutic treatment, which refers to the reduction or elimination of such signs or symptoms, or the improvement of their progression, severity, or duration, towards the development of pathological signs or symptoms. (i.e., PKU) is administered therapy. Signs and symptoms can be biochemical, cellular, histological, functional, subjective or objective. Symptoms of PKU include elevated blood or plasma Phe levels, decreased blood or plasma neurotransmitter levels (eg, Tyr levels), and neurocognitive symptoms.

如本文所用之「神經認知症狀」係指與患有苯酮尿症之個體相關之特定神經、行為及認知症狀。特定言之，***酸羥化酶活性損失導致患有苯酮尿症之個體不能產生足夠的神經傳遞質含量。不能產生足夠的神經傳遞質直接導致多種神經、認知及行為症狀。在一個實施例中，神經認知症狀包括IQ降低、注意力缺乏及包括策略規劃、抑制控制、工作記憶及認知靈活性之執行功能缺乏。"Neurocognitive symptoms" as used herein refers to the specific neurological, behavioral and cognitive symptoms associated with individuals with phenylketonuria. Specifically, the loss of phenylalanine hydroxylase activity results in an inability to produce adequate neurotransmitter levels in individuals with phenylketonuria. The inability to produce enough neurotransmitters directly leads to a variety of neurological, cognitive, and behavioral symptoms. In one embodiment, neurocognitive symptoms include decreased IQ, inattention, and deficits in executive function including strategic planning, inhibitory control, working memory, and cognitive flexibility.

如本文所用之「治療有效」意謂能有效地減少病理學之病徵或症狀的量。在短暫肝毒性之情況下，其可係指有效減少肝毒性標記之皮質類固醇治療的量。在PKU之情況下，其可以指有效使得血液或血漿Phe含量臨床上顯著降低之量。儘管此將會觀測到初始效應，但也會觀測到其他效應，其指示病理學之病徵或症狀的有效減少，其包括Phe活性增加(例如，如藉由Phe呼吸測試上的Phe氧化的增加之比率所量測)；神經傳遞質或神經傳遞質代謝物之血液或血漿含量增加(例如，增加之酪胺酸(Tyr)含量)；耐受膳食蛋白攝入之增加及/或醫療食品攝入之降低(對於Phe降低或無Phe之食物的需求降低)的增加之能力；神經認知症狀減少；注意力不集中症狀降低及測量執行功能之改進(例如，ADHD-RS IV或CANTAB得分增加)；健康相關生活品質之改進(例如，PKU-QOL或Q-LES-Q-SF得分之改進)；營養標記之改進及/或空腹血脂之正常化。As used herein, "therapeutically effective" means an amount effective to reduce the signs or symptoms of a pathology. In the case of transient hepatotoxicity, it may refer to an amount of corticosteroid therapy effective to reduce markers of hepatotoxicity. In the context of PKU, it may refer to an amount effective to cause a clinically significant reduction in blood or plasma Phe levels. While this will be the primary effect observed, other effects will also be observed that are indicative of an effective reduction in signs or symptoms of pathology, including increased Phe activity (eg, as by increased Phe oxidation on the Phe breath test). ratios); increased blood or plasma levels of neurotransmitters or neurotransmitter metabolites (eg, increased tyrosine (Tyr) levels); increased dietary protein intake and/or medical food intake Increased ability to decrease (reduced need for Phe-reduced or Phe-free foods); decrease in neurocognitive symptoms; decrease in inattention symptoms and improvement in measures of executive function (eg, increase in ADHD-RS IV or CANTAB score); Improvement in health-related quality of life (eg, improvement in PKU-QOL or Q-LES-Q-SF score); improvement in nutritional markers and/or normalization of fasting lipids.

如本文所用之「改善」係指減少疾病之症狀之嚴重程度、進展或持續時間之作用。"Ameliorating" as used herein refers to the effect of reducing the severity, progression or duration of symptoms of a disease.

如本文所用之「穩定治療(stably treating或stable treatment)」係指使用向個體投與之治療性載體建構體、AAV顆粒或細胞，其中該個體穩定表現由載體建構體、AAV顆粒或細胞表現的治療蛋白。穩定表現之治療蛋白意謂蛋白質在臨床上顯著的時間長度內表現。As used herein, "stably treating or stable treatment" refers to the use of a therapeutic vector construct, AAV particle or cell administered to an individual, wherein the individual stably expresses the expression expressed by the vector construct, AAV particle or cell therapeutic protein. Stable expression of the therapeutic protein means that the protein is expressed for a clinically significant length of time.

如本文關於PKU所用之「臨床上顯著之時間長度」或「耐久性」意謂以治療有效含量表現持續一定時間長度，該時間長度對血漿Phe含量及/或對病理學之其他病徵或症狀具有有意義的影響。在某些實施例中，臨床上顯著的時間長度為表現至少六個月、至少八個月、至少一年、至少兩年、至少三年、至少四年、至少五年、至少六年、至少七年、至少八年、至少九年、至少十年或個體一生。"Clinically significant length of time" or "durability" as used herein with respect to PKU means manifested at therapeutically effective levels for a length of time that has a significant effect on plasma Phe levels and/or on other signs or symptoms of pathology meaningful impact. In certain embodiments, the clinically significant length of time is at least six months, at least eight months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least Seven years, at least eight years, at least nine years, at least ten years, or an individual's lifetime.

如本文所用，術語「有效量」係指足以實現有益或理想生物及/或臨床結果之量。As used herein, the term "effective amount" refers to an amount sufficient to achieve beneficial or desirable biological and/or clinical results.

如本文所用，「個體」係指為治療、觀測或實驗之目標之動物。「動物」包括冷血及溫血脊椎動物及無脊椎動物，諸如魚、甲殼類動物、爬行動物及尤其哺乳動物。如本文所用之術語「禽類」包括但不限於雞、鴨、鵝、鵪鶉、火雞及雉雞。如本文所用之「哺乳動物」係指屬於哺乳綱之個體，且包括但不限於人類、家養動物及農耕動物、動物園動物、競技用動物及寵物動物。哺乳動物之非限制性實例包括小鼠；大鼠；兔；天竺鼠；狗；貓；綿羊；山羊；奶牛；馬；靈長類動物，諸如猴、黑猩猩及猿及尤其人類。在一些實施例中，哺乳動物為人類，包括嬰兒、幼兒、青少年或成人，例如年齡低於2歲、年齡低於5歲、年齡為2至低於5歲、年齡為5至9歲、年齡為9至低於12歲、年齡為5至低於12歲、年齡為12至低於15歲或年齡為15至18歲、或15歲或更大、或18歲或更大、或年齡為低於3歲、年齡為3至低於6歲、年齡為低於6歲、年齡為6至低於9歲、年齡為9至低於12歲、年齡為6至低於12歲或年齡為12至低於18歲、或15歲或更大、或18歲或更大。As used herein, "individual" refers to an animal that is the subject of treatment, observation, or experimentation. "Animal" includes cold-blooded and warm-blooded vertebrates and invertebrates, such as fish, crustaceans, reptiles and especially mammals. The term "avian" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, and pheasants. As used herein, "mammal" refers to an individual belonging to the class Mammalia, and includes, but is not limited to, humans, domestic and agricultural animals, zoo animals, sports animals, and pet animals. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; In some embodiments, the mammal is a human, including an infant, toddler, juvenile, or adult, eg, less than 2 years old, less than 5 years old, 2 to less than 5 years old, 5 to 9 years old, be 9 to under 12 years old, 5 to under 12 years old, 12 to under 15 years old, or 15 to 18 years old, or 15 years old or older, or 18 years old or older, or Under 3 years old, 3 to under 6 years old, under 6 years old, 6 to under 9 years old, 9 to under 12 years old, 6 to under 12 years old, or 12 to under 18 years old, or 15 years old or older, or 18 years old or older.

一般而言，「醫藥學上可接受之載劑」為對細胞無毒性或過度有害的載劑。例示性醫藥學上可接受之載劑包括無菌、無熱原質水及無菌、無熱原質磷酸鹽緩衝鹽水。醫藥學上可接受之載劑包括生理學上可接受之載劑。術語「醫藥學上可接受之載劑」包括生理學上相容之任何及所有溶劑、分散介質、包衣、抗細菌劑及抗真菌劑、等張劑及吸收延遲劑及其類似物。 5.1 重組AAV載體建構體In general, a "pharmaceutically acceptable carrier" is one that is not toxic or unduly harmful to cells. Exemplary pharmaceutically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free phosphate buffered saline. Pharmaceutically acceptable carriers include physiologically acceptable carriers. The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. 5.1 Recombinant AAV vector constructs

本發明之重組載體建構體可用於藉由本文所描述之方法產生rAAV顆粒，其包含向適合之宿主細胞提供重組載體建構體以及Rep及Cap基因。重組載體建構體可包含(a)以下中之一或兩者：(i) AAV 5'反向末端重複序列(ITR)序列及(ii) AAV 3' ITR；(b)異源肝特異性轉錄調節區；及(c)編碼功能性人類***酸羥化酶(hPAH)之核酸，視情況其中該等AAV ITR為AAV2 ITR。較佳地，編碼功能性hPAH之核酸可操作地連接至肝特異性表現控制元件。載體建構體可包括額外表現控制元件，例如：啟動子及/或增強子；內含子；視情況，來自與內含子相同之基因的外顯子；及多腺苷酸化(polyA)信號。本文中進一步描述此類元件。較佳地，rAAV顆粒亦包含具有肝趨向性之AAV衣殼，視情況為AAV5型衣殼。The recombinant vector constructs of the present invention can be used to generate rAAV particles by the methods described herein, which comprise providing the recombinant vector construct and the Rep and Cap genes to a suitable host cell. The recombinant vector construct may comprise (a) one or both of: (i) AAV 5' inverted terminal repeat (ITR) sequences and (ii) AAV 3' ITR; (b) heterologous liver-specific transcripts and (c) a nucleic acid encoding a functional human phenylalanine hydroxylase (hPAH), optionally wherein the AAV ITRs are AAV2 ITRs. Preferably, the nucleic acid encoding a functional hPAH is operably linked to a liver-specific expression control element. The vector construct may include additional expression control elements such as: promoters and/or enhancers; introns; optionally, exons from the same gene as the intron; and polyadenylation (polyA) signals. Such elements are further described herein. Preferably, the rAAV particles also comprise an AAV capsid with liver tropism, optionally an AAV5 type capsid.

在一個實施例中，載體建構體包含編碼功能性活性(PAH)蛋白質之核酸。hPAH編碼序列可為野生型、密碼子優化或變異體。一種野生型hPAH基因具有SEQ ID NO: 1之核苷酸序列。一種野生型hPAH具有SEQ ID NO: 2之胺基酸序列。In one embodiment, the vector construct comprises a nucleic acid encoding a functionally active (PAH) protein. The hPAH coding sequence can be wild type, codon optimized or a variant. A wild-type hPAH gene has the nucleotide sequence of SEQ ID NO:1. A wild-type hPAH has the amino acid sequence of SEQ ID NO:2.

本文所描述之載體建構體可包含與野生型核苷酸序列不同但仍編碼與SEQ ID NO: 2之胺基酸至少90%、95%或98%一致之功能性hPAH胺基酸序列的核苷酸序列。根據此態樣，核苷酸序列可與SEQ ID NO: 1具有實質同源性，例如至少70%、75%、80%、85%、90%、95%、98%或99%同源性，只要其編碼與SEQ ID NO: 2之胺基酸至少90%一致之功能性hPAH。若核酸編碼包含已改變至野生型胺基酸中之任一者之序列的蛋白質，則蛋白質仍應為功能蛋白。技術人員應瞭解，可對一些蛋白質之胺基酸做出少量變化而不會不利地影響蛋白質之功能。The vector constructs described herein can comprise a nucleus that differs from the wild-type nucleotide sequence but still encodes a functional hPAH amino acid sequence that is at least 90%, 95%, or 98% identical to the amino acids of SEQ ID NO: 2 nucleotide sequence. According to this aspect, the nucleotide sequence may have substantial homology to SEQ ID NO: 1, such as at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% homology , as long as it encodes a functional hPAH that is at least 90% identical to the amino acid of SEQ ID NO: 2. If the nucleic acid encodes a protein comprising a sequence that has been altered to any of the wild-type amino acids, the protein should still be a functional protein. The skilled artisan will appreciate that small changes to the amino acids of some proteins can be made without adversely affecting the function of the protein.

可替代地，核苷酸序列可與SEQ ID NO: 7、8、9、10、11、12或13中之任一者具有實質同源性，例如至少70%、75%、80%、85%、90%、95%、98%或99%同源性，只要核苷酸序列編碼與SEQ ID NO: 2之胺基酸至少90%、95%或98%一致之功能性hPAH，較佳地為與SEQ ID NO: 2之胺基酸至少95%一致之功能性hPAH。術語實質同源性可以參考同源性百分比(%)進一步定義。此在本文中其他處進一步詳細地討論。在一些實施例中，載體建構體包含編碼人類PAH之核苷酸序列，其中該核苷酸序列與SEQ ID NO: 52之核苷酸2894至4252至少90%一致。在某些實施例中，載體建構體包含編碼人類PAH之核苷酸序列，其中該核苷酸序列與SEQ ID NO: 52之核苷酸2894至4252至少95%一致。在一些實施例中，載體建構體包含編碼人類PAH之核苷酸序列，其中該核苷酸序列包含SEQ ID NO: 52之核苷酸2894至4252。Alternatively, the nucleotide sequence may have substantial homology, such as at least 70%, 75%, 80%, 85%, to any of SEQ ID NOs: 7, 8, 9, 10, 11, 12 or 13 %, 90%, 95%, 98% or 99% homology, as long as the nucleotide sequence encodes a functional hPAH that is at least 90%, 95% or 98% identical to the amino acid of SEQ ID NO: 2, preferably is a functional hPAH that is at least 95% identical to the amino acid of SEQ ID NO: 2. The term substantial homology can be further defined with reference to percent (%) homology. This is discussed in further detail elsewhere herein. In some embodiments, the vector construct comprises a nucleotide sequence encoding a human PAH, wherein the nucleotide sequence is at least 90% identical to nucleotides 2894 to 4252 of SEQ ID NO: 52. In certain embodiments, the vector construct comprises a nucleotide sequence encoding a human PAH, wherein the nucleotide sequence is at least 95% identical to nucleotides 2894 to 4252 of SEQ ID NO: 52. In some embodiments, the vector construct comprises a nucleotide sequence encoding a human PAH, wherein the nucleotide sequence comprises nucleotides 2894 to 4252 of SEQ ID NO:52.

如本文所描述，編碼hPAH蛋白質之核苷酸序列可以經修飾以改進蛋白質之表達效率。舉例而言，核苷酸序列可以經密碼子優化。此可結合人工減少CpG二核苷酸含量及移除有義及反義方向上之任何額外ORF來進行。已展示CpG二核苷酸含量在樹突狀細胞中活化TLR9，引起潛在的免疫活化及CTL反應。AAV-載體基因體中之產物可以ssDNA形式遞送，因此降低CpG含量。降低CpG含量可減少肝炎及ALT。作為另一實例，所關注蛋白質之核苷酸序列中之剪接供體及/或剪接受體中之一或多者經修飾以降低額外剪接之可能性。As described herein, the nucleotide sequence encoding the hPAH protein can be modified to improve the expression efficiency of the protein. For example, the nucleotide sequence can be codon optimized. This can be done in conjunction with artificially reducing the CpG dinucleotide content and removing any additional ORFs in the sense and antisense orientations. CpG dinucleotide content has been shown to activate TLR9 in dendritic cells, leading to potential immune activation and CTL responses. The product in the AAV-vector genome can be delivered as ssDNA, thus reducing the CpG content. Reducing CpG levels can reduce hepatitis and ALT. As another example, one or more of the splice donor and/or splice acceptor in the nucleotide sequence of the protein of interest is modified to reduce the likelihood of additional splicing.

適用於包括於異源肝特異性轉錄調節區中之肝特異性調節元件之實例包括(但不限於)小鼠甲狀腺素運載蛋白啟動子(mTTR)、內源性人類因子VIII啟動子(F8)、人類脂蛋白元E肝控制區及其活性片段、人類α-1-抗胰蛋白酶啟動子(hAAT)及其活性片段、人類α-1-微球蛋白啟動子及其片段、人類凝血酶原啟動子及其活性片段、人類白蛋白最小啟動子及小鼠白蛋白啟動子。亦涵蓋來源於肝特異性轉錄因子結合位點之增強子，諸如EBP、DBP、HNF1、HNF3、HNF4、HNF6及Enh1。編碼hPAH之核酸可操作地連接至此類肝特異性調節元件中之任一者。在具體實例中，肝特異性調節元件為包含hAAT啟動子之片段及HCR增強子/ApoE增強子之片段的啟動子。在一或多個實施例中，肝特異性轉錄調節區為包含人類α-1-抗胰蛋白酶(hAAT)啟動子、肝控制區(HCR)增強子及/或脂蛋白元E(ApoE)增強子之部分的合成啟動子序列。舉例而言，肝特異性調節元件包含與SEQ ID NO: 3、4或24中之任一者至少90%一致的核苷酸序列。在又其他實例中，肝特異性調節元件包含與SEQ ID NO: 25或26中之任一者至少90%一致的核苷酸序列。Examples of liver-specific regulatory elements suitable for inclusion in heterologous liver-specific transcriptional regulatory regions include, but are not limited to, mouse transthyretin promoter (mTTR), endogenous human factor VIII promoter (F8) , Human lipoprotein E liver control region and its active fragments, human alpha-1-antitrypsin promoter (hAAT) and its active fragments, human alpha-1-microglobulin promoter and its fragments, human prothrombin Promoter and its active fragments, human albumin minimal promoter and mouse albumin promoter. Also encompassed are enhancers derived from liver-specific transcription factor binding sites, such as EBP, DBP, HNFl, HNF3, HNF4, HNF6, and Enhl. The nucleic acid encoding hPAH is operably linked to any of such liver-specific regulatory elements. In a specific example, the liver-specific regulatory element is a promoter comprising a fragment of the hAAT promoter and a fragment of the HCR enhancer/ApoE enhancer. In one or more embodiments, the liver-specific transcriptional regulatory region comprises a human alpha-1-antitrypsin (hAAT) promoter, a liver control region (HCR) enhancer, and/or a lipoprotein E (ApoE) enhancer Synthetic promoter sequences of subparts. For example, the liver-specific regulatory element comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 3, 4, or 24. In yet other examples, the liver-specific regulatory element comprises a nucleotide sequence that is at least 90% identical to any of SEQ ID NOs: 25 or 26.

在一些實施例中，肝特異性轉錄調節區包含縮短之ApoE增強子序列(SEQ ID NO: 4)或與其至少80%、85%、90%、95%或98%一致的核苷酸序列；186鹼基人類α抗胰蛋白酶(hAAT)近端啟動子(SEQ ID NO: 3)或語氣至少80%、85%、90%、95%或98%一致的核苷酸序列，其包括5'非轉譯區(UTR)之42個鹼基；一或多種選自由以下組成之組之增強子：(i) 34鹼基人類ApoE/C1增強子、(ii) 32鹼基人類AAT啟動子末端X區及(iii)人類AAT近端啟動子之末端元件之80個額外鹼基；及編碼人類PAH之核酸。在另一實施例中，肝特異性轉錄調節區包含α-微球蛋白增強子序列及186鹼基人類α抗胰蛋白酶(AAT)近端啟動子。In some embodiments, the liver-specific transcriptional regulatory region comprises a shortened ApoE enhancer sequence (SEQ ID NO: 4) or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto; 186 base human alpha antitrypsin (hAAT) proximal promoter (SEQ ID NO: 3) or a nucleotide sequence that is at least 80%, 85%, 90%, 95% or 98% identical in tone, including the 5' 42 bases of untranslated region (UTR); one or more enhancers selected from the group consisting of: (i) 34 base human ApoE/C1 enhancer, (ii) 32 base human AAT promoter terminal X region and (iii) 80 additional bases of the terminal element of the proximal promoter of human AAT; and a nucleic acid encoding human PAH. In another embodiment, the liver-specific transcriptional regulatory region comprises an alpha-microglobulin enhancer sequence and a 186-base human alpha antitrypsin (AAT) proximal promoter.

在一些實施例中，重組載體建構體包含與SEQ ID NO: 6至少90%一致之核苷酸序列，其包括ApoE增強子或HCR、hAAT啟動子、LGI內含子/hAAT內含子、血紅素內含子及血紅素外顯子之小部分。In some embodiments, the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 6, which includes the ApoE enhancer or HCR, hAAT promoter, LGI intron/hAAT intron, hemoglobin Introns and small parts of heme exons.

肝特異性啟動子之具體實例包括LP1、HLP、HCR-hAAT、ApoE-hAAT、LSP、TBG及TTR。此等啟動子更詳細地描述於以下參考文獻中： LP1(具有人類AAT啟動子(255 bp)之人類ApoE HCR核心序列(192 bp))：Nathwani A等人，Blood. 2006年4月1日; 107(7): 2653-2661；雜交肝特異性啟動子(HLP) (具有經修飾之人類α-1-抗胰蛋白酶(αAT)啟動子(217 bp)之人類脂蛋白元E (ApoE)肝控制區(HCR)片段(34 bp))：McIntosh J.等人，Blood. 2013年4月25日; 121(17): 3335-3344；HCR-hAAT (具有具有人類AAT啟動子(408 bp)之ApoE增強子(1-4x154 bp)且包括內含子A (1.4 kbp)及3'UTR (1.7 kbp)的ApoE-HCR (319 bp))：Miao CH等人，Mol Ther. 2000;1: 522-532；ApoE-hAAT：Okuyama T等人，Human Gene Therapy, 7, 637-645 (1996)；LSP：Wang L等人，Proc Natl Acad Sci U S A. 1999年3月30日; 96(7): 3906-3910；甲狀腺素結合球蛋白(TBG)啟動子：Yan等人，Gene 506:289-294 (2012)；及甲狀腺素運載蛋白(TTR)啟動子：Costa等人，Mol. Cell. Biol. 8:81-90 (1988)。Specific examples of liver-specific promoters include LP1, HLP, HCR-hAAT, ApoE-hAAT, LSP, TBG, and TTR. These promoters are described in more detail in the following references: LP1 (Human ApoE HCR core sequence (192 bp) with human AAT promoter (255 bp)): Nathwani A et al. Blood. Apr 1, 2006 ; 107(7): 2653-2661; Hybrid liver-specific promoter (HLP) (human lipoprotein E (ApoE) with modified human alpha-1-antitrypsin (αAT) promoter (217 bp) Liver Control Region (HCR) fragment (34 bp)): McIntosh J. et al. Blood. 2013 Apr 25; 121(17): 3335-3344; HCR-hAAT (with human AAT promoter (408 bp) ) ApoE enhancer (1-4x154 bp) and ApoE-HCR (319 bp) including intron A (1.4 kbp) and 3' UTR (1.7 kbp): Miao CH et al., Mol Ther. 2000; 1 : 522-532; ApoE-hAAT: Okuyama T et al, Human Gene Therapy, 7, 637-645 (1996); LSP: Wang L et al, Proc Natl Acad Sci US A. 1999 Mar 30; 96( 7): 3906-3910; Thyroxine-binding globulin (TBG) promoter: Yan et al., Gene 506: 289-294 (2012); and Transthyretin (TTR) promoter: Costa et al., Mol. Cell . Biol. 8:81-90 (1988).

舉例而言，De Simone等人(EMBO Journal，第6卷，第9號，第2759頁至第2766頁，1987)描述多種來源於人類α-1-反胰蛋白酶啟動子之啟動子。舉例而言，其自-1200至+44表徵人類AAT啟動子內之肝特異性活性所需的順式作用及反式作用元件。HLP中之人類AAT啟動子由遠端X元件(32 bp)及近端A及B元件(185 bp)組成。Frain等人(MOL CELL BIO, 1990年3月, 第10卷, 第3號, 第991頁至第999頁)描述來源於人類白蛋白啟動子之啟動子數目。舉例而言，其自-1022至-1表徵人類白蛋白基因內之啟動子及增強子元件。For example, De Simone et al. (EMBO Journal, Vol. 6, No. 9, pp. 2759-2766, 1987) describe various promoters derived from the human alpha-1-antitrypsin promoter. For example, it characterizes the cis- and trans-acting elements required for liver-specific activity within the human AAT promoter from -1200 to +44. The human AAT promoter in HLP consists of a distal X element (32 bp) and proximal A and B elements (185 bp). Frain et al. (MOL CELL BIO, March 1990, Vol. 10, No. 3, pp. 991-999) describe the number of promoters derived from the human albumin promoter. For example, it characterizes promoter and enhancer elements within the human albumin gene from -1022 to -1.

Dang等人(J BIOL CHEM, 第270卷, 第38號, 9月22日之期刊, 第22577頁至第22585頁, 1995)描述人類脂蛋白元E基因(774 bp)之肝控制區(HCR)。Shachter等人(J. Lipid Res. 1993. 第34卷: 第1699頁至第1707頁)表徵ApoE HCR(154 bp)中之肝特異性增強子。此等HCR片段可以與諸如人類AAT啟動子或其片段之其他轉錄調節元件組合。Chow等人(J Biol Chem. 1991年10月5日;266(28):18927-33)自-940至-860(80 bp)表徵人類凝血酶原增強子。Rouet等人(第267卷, 第29號, 10月15日之期刊, 第20765頁至第20773頁,1992；Nucleic Acids Res. 1995年2月11日; 23(3): 395至404；及Biochemical Journal, 1998年九月15日, 334 (3) 577至584)表徵肝特異性人類α-1-微球蛋白/比庫甯增強子之序列。美國專利第7,323,324號亦描述人類AAT啟動子、人類α-微球蛋白/比庫甯增強子、人類白蛋白啟動子及人類凝血酶原增強子。Dang et al. (J BIOL CHEM, Vol. 270, No. 38, Journal of September 22, pp. 22577-22585, 1995) describe the hepatic control region (HCR) of the human lipoprotein E gene (774 bp) ). Shachter et al. (J. Lipid Res. 1993. Vol. 34: pp. 1699-1707) characterized a liver-specific enhancer in the ApoE HCR (154 bp). These HCR fragments can be combined with other transcriptional regulatory elements such as the human AAT promoter or fragments thereof. Chow et al. (J Biol Chem. 1991 Oct 5;266(28):18927-33) characterized the human prothrombin enhancer from -940 to -860 (80 bp). Rouet et al. (Vol. 267, No. 29, Journal of Oct. 15, pp. 20765-20773, 1992; Nucleic Acids Res. 1995 Feb. 11; 23(3): 395-404; and Biochemical Journal, Sept. 15, 1998, 334(3) 577-584) Characterization of the sequence of the liver-specific human alpha-1-microglobulin/bikunin enhancer. US Patent No. 7,323,324 also describes the human AAT promoter, the human alpha-microglobulin/bikunin enhancer, the human albumin promoter, and the human prothrombin enhancer.

在一些實施例中，啟動子包含上述經鑑別之增強子中之一或多者之多個複本。在一些實施例中，啟動子建構體在一或多個不同定向上包含一或多個上文所描述之個別增強子元件及其組合。In some embodiments, the promoter comprises multiple copies of one or more of the above-identified enhancers. In some embodiments, the promoter construct comprises one or more of the individual enhancer elements described above, and combinations thereof, in one or more different orientations.

在一些實施例中，啟動子與編碼一或多種所關注蛋白質之聚核苷酸可操作地連接。在一些實施例中，啟動子與編碼hPAH蛋白質之聚核苷酸可操作地連接。In some embodiments, a promoter is operably linked to a polynucleotide encoding one or more proteins of interest. In some embodiments, the promoter is operably linked to the polynucleotide encoding the hPAH protein.

啟動子之尺寸可以變化。由於AAV之有限封裝能力，因此較佳使用尺寸小但同時允許在宿主細胞中產生高含量之所關注蛋白質的啟動子。舉例而言，在一些實施例中，啟動子為至多約1.5 kb、至多約1.4 kb、至多約1.35 kb、至多約1.3 kb、至多約1.25 kb、至多約1.2 kb、至多約1.15 kb、至多約1.1 kb、至多約1.05 kb、至多約1 kb、至多約800個鹼基對、至多約600個鹼基對、至多約400個鹼基對、至多約200個鹼基對或至多約100個鹼基對。The size of the promoter can vary. Due to the limited encapsulation capabilities of AAVs, it is preferred to use promoters that are small in size but at the same time allow high levels of the protein of interest to be produced in the host cell. For example, in some embodiments, the promoter is at most about 1.5 kb, at most about 1.4 kb, at most about 1.35 kb, at most about 1.3 kb, at most about 1.25 kb, at most about 1.2 kb, at most about 1.15 kb, at most about 1.1 kb, up to about 1.05 kb, up to about 1 kb, up to about 800 base pairs, up to about 600 base pairs, up to about 400 base pairs, up to about 200 base pairs, or up to about 100 base pairs base pair.

在一些實施例中，載體建構體包含肝特異性轉錄調節區，其中該肝特異性調節區包含與SEQ ID NO: 52之核苷酸160至839至少90%一致的核苷酸序列。在某些實施例中，載體建構體包含肝特異性轉錄調節區，其中該肝特異性調節區包含與SEQ ID NO: 52之核苷酸160至839至少95%一致的核苷酸序列。在一些實施例中，載體建構體包含肝特異性轉錄調節區，其中該肝特異性調節區包含SEQ ID NO: 52之核苷酸160至839。In some embodiments, the vector construct comprises a liver-specific transcriptional regulatory region, wherein the liver-specific regulatory region comprises a nucleotide sequence that is at least 90% identical to nucleotides 160 to 839 of SEQ ID NO: 52. In certain embodiments, the vector construct comprises a liver-specific transcriptional regulatory region, wherein the liver-specific regulatory region comprises a nucleotide sequence that is at least 95% identical to nucleotides 160 to 839 of SEQ ID NO: 52. In some embodiments, the vector construct comprises a liver-specific transcriptional regulatory region, wherein the liver-specific regulatory region comprises nucleotides 160 to 839 of SEQ ID NO:52.

在一些實施例中，載體建構體包含編碼功能性hPAH之核酸序列，該核酸序列可操作地連接至異源肝特異性轉錄調節區。載體建構體可包含其他調節元件。在一些實施例中，在本文所描述之載體建構體中，表現控制元件包括以下中之一或多者：啟動子及/或增強子；內含子；及多腺苷酸化(polyA)信號。In some embodiments, the vector construct comprises a nucleic acid sequence encoding a functional hPAH operably linked to a heterologous liver-specific transcriptional regulatory region. The vector construct may contain other regulatory elements. In some embodiments, in the vector constructs described herein, the expression control elements include one or more of the following: promoters and/or enhancers; introns; and polyadenylation (polyA) signals.

在一些實施例中，載體建構體包含以下中之至少一或兩者：AAV之5'反向末端重複序列(ITR)及3' AAV ITR、啟動子、編碼功能性hPAH之核酸及視情況選用之轉錄後調節元件，其中啟動子、編碼功能性hPAH之核酸及轉錄後調節元件位於5' AAV ITR下游及3' AAV ITR上游。In some embodiments, the vector construct comprises at least one or both of the 5' inverted terminal repeat (ITR) and 3' AAV ITR of AAV, a promoter, a nucleic acid encoding a functional hPAH, and optionally The post-transcriptional regulatory element, wherein the promoter, the nucleic acid encoding a functional hPAH, and the post-transcriptional regulatory element are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR.

在某些實施例中，重組AAV載體建構體包含核酸，其包含(a) AAV2 5'反向末端重複序列(ITR) (其如此項技術中已知可經修飾或可不經修飾)；(b)肝特異性轉錄調節區；(c)功能性hPAH編碼區；(d)一或多個內含子，其包括較長內含子之片段；(e)視情況選用之外顯子或其片段；(f)多腺苷酸化序列；及(f) AAV2 3' ITR (其如此項技術中已知可經修飾或可不經修飾)。In certain embodiments, a recombinant AAV vector construct comprises a nucleic acid comprising (a) an AAV2 5' inverted terminal repeat (ITR) (which may or may not be modified as known in the art); (b) ) liver-specific transcriptional regulatory region; (c) functional hPAH coding region; (d) one or more introns, including fragments of longer introns; (e) optional exons or their fragment; (f) polyadenylation sequence; and (f) AAV2 3' ITR (which may or may not be modified as known in the art).

本文所提供之其他實施例係針對編碼功能性PAH多肽之載體建構體，其中該等建構體在一或多個不同定向上包含一或多個上述建構體之個別元件及其組合。Additional embodiments provided herein are directed to vector constructs encoding functional PAH polypeptides, wherein the constructs comprise one or more of the individual elements of the above-described constructs, and combinations thereof, in one or more different orientations.

各種額外調節元件可以用於載體建構體中，例如進一步增加所關注蛋白質在宿主細胞、多腺苷酸化信號、核糖體結合序列及/或共同剪接受體或剪接供體位點中之表現量的增強子。在一些實施例中，調節元件可以有助於將重組DNA分子維持在宿主細胞之染色體外及/或改進載體效能(例如，骨架/基質附接區(S/MAR))。此類調節元件為此項技術中所熟知。Various additional regulatory elements can be used in the vector construct, such as to further increase the level of expression of the protein of interest in the host cell, polyadenylation signals, ribosome binding sequences and/or common splice acceptor or splice donor sites. son. In some embodiments, regulatory elements can help maintain recombinant DNA molecules extrachromosomally in the host cell and/or improve vector performance (eg, backbone/matrix attachment regions (S/MARs)). Such regulatory elements are well known in the art.

本文所揭示之載體建構體可包括調節元件，諸如轉錄起始區及/或轉錄終止區。轉錄終止區之實例包括但不限於多腺苷酸化信號序列。多腺苷酸化信號序列之實例包括但不限於人類生長激素(hGH) poly(A)、牛生長激素(bGH) poly(A)、SV40後poly(A)、兔β-球蛋白(rBG) poly(A)、胸苷激酶(TK) poly(A)序列及其任何變異體。在一些實施例中，轉錄終止區位於轉錄後調節元件下游。在一些實施例中，轉錄終止區為多腺苷酸化信號序列。在一些實施例中，轉錄終止區為bGH poly(A)序列。The vector constructs disclosed herein can include regulatory elements, such as transcription initiation regions and/or transcription termination regions. Examples of transcription termination regions include, but are not limited to, polyadenylation signal sequences. Examples of polyadenylation signal sequences include, but are not limited to, human growth hormone (hGH) poly(A), bovine growth hormone (bGH) poly(A), post-SV40 poly(A), rabbit beta-globulin (rBG) poly (A), thymidine kinase (TK) poly(A) sequence and any variants thereof. In some embodiments, the transcription termination region is located downstream of the post-transcriptional regulatory element. In some embodiments, the transcription termination region is a polyadenylation signal sequence. In some embodiments, the transcription termination region is a bGH poly(A) sequence.

在一些實施例中，載體建構體包含poly(A)序列，其中該poly(A)序列包含與SEQ ID NO: 52之核苷酸4258至4484至少90%一致的核苷酸序列。在某些實施例中，載體建構體包含poly(A)序列，其中該poly(A)序列包含與SEQ ID NO: 52之核苷酸4258至4484至少95%一致的核苷酸序列。在一些實施例中，載體建構體包含poly(A)序列，其中該poly(A)序列包含SEQ ID NO: 52之核苷酸4258至4484。In some embodiments, the vector construct comprises a poly(A) sequence, wherein the poly(A) sequence comprises a nucleotide sequence that is at least 90% identical to nucleotides 4258 to 4484 of SEQ ID NO: 52. In certain embodiments, the vector construct comprises a poly(A) sequence, wherein the poly(A) sequence comprises a nucleotide sequence that is at least 95% identical to nucleotides 4258 to 4484 of SEQ ID NO: 52. In some embodiments, the vector construct comprises a poly(A) sequence, wherein the poly(A) sequence comprises nucleotides 4258 to 4484 of SEQ ID NO:52.

在一些實施例中，載體建構體可以包括額外轉錄及轉譯起始序列及/或額外轉錄及轉譯終止子，其為此項技術中已知的。In some embodiments, the vector construct may include additional transcriptional and translational initiation sequences and/or additional transcriptional and translational terminators, which are known in the art.

在一些實施例中，載體包含一或多個內含子。內含子可有助於處理哺乳動物宿主細胞中之RNA轉錄物，增加所關注蛋白質之表現及/或使載體至AAV顆粒中之封裝優化。與在不存在內含子元件之情況下的表現相比較，包括內含子元件可增強表現(參見例如，Kurachi等人, 1995, J Biol Chem. 1995年3月10日；270(10):5276-81)。AAV載體典型地接受具有限定之尺寸範圍(大體上為約4 kb至約5.2 kb，或略多)之DNA的***。然而，封裝不存在最小尺寸且小載體基因體封裝極有效。內含子及內含子片段滿足此要求，同時亦增強表現。因此，本發明涵蓋在AAV載體中包括hPAH內含子序列(例如PAH之內含子2之一部分)，或代替hPAH內含子之一部分的其他內含子或其他DNA序列。在一些實施例中，內含子為第二hPAH內含子或hPAH第二內含子之2116 bp截短形式。另外，可使用核酸之其他5'及3'非轉譯區代替針對hPAH所敍述之彼等。此類內含子之非限制性實例為血紅素(β-球蛋白)內含子及/或hAAT(人類α-1-抗胰蛋白酶)內含子。在其他實施例中，內含子序列為複合hAAT/β-球蛋白內含子。在一些實施例中，內含子為合成內含子。In some embodiments, the vector comprises one or more introns. Introns can aid in the processing of RNA transcripts in mammalian host cells, increase the expression of proteins of interest and/or optimize encapsulation of vectors into AAV particles. Inclusion of intronic elements can enhance performance compared to performance in the absence of intronic elements (see, e.g., Kurachi et al., 1995, J Biol Chem. 1995 Mar 10;270(10): 5276-81). AAV vectors typically accept insertion of DNA having a limited size range (generally from about 4 kb to about 5.2 kb, or slightly more). However, there is no minimum size for encapsulation and vectorlet genome encapsulation is extremely efficient. Introns and intron fragments meet this requirement while also enhancing performance. Accordingly, the present invention contemplates the inclusion of hPAH intron sequences (eg, a portion of intron 2 of PAH) in an AAV vector, or other introns or other DNA sequences in place of a portion of the hPAH intron. In some embodiments, the intron is the second hPAH intron or a 2116 bp truncated version of the hPAH second intron. In addition, other 5' and 3' untranslated regions of nucleic acids can be used in place of those described for hPAH. Non-limiting examples of such introns are the heme (beta-globin) intron and/or the hAAT (human alpha-1-antitrypsin) intron. In other embodiments, the intron sequence is a complex hAAT/beta-globin intron. In some embodiments, the intron is a synthetic intron.

在一些實施例中，內含子包含與SEQ ID NO: 14或SEQ ID NO: 27或29或34至少90%一致之核苷酸序列。In some embodiments, the intron comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14 or SEQ ID NO: 27 or 29 or 34.

在一些實施例中，載體建構體包含內含子區，其中該內含子包含與SEQ ID NO: 52之核苷酸885至2828至少90%一致的核苷酸序列。在某些實施例中，載體建構體包含內含子區，其中該內含子包含與SEQ ID NO: 52之核苷酸885至2828至少95%一致的核苷酸序列。在一些實施例中，載體建構體包含內含子區，其中該內含子包含SEQ ID NO: 52之核苷酸885至2828。In some embodiments, the vector construct comprises an intron region, wherein the intron comprises a nucleotide sequence that is at least 90% identical to nucleotides 885 to 2828 of SEQ ID NO: 52. In certain embodiments, the vector construct comprises an intron region, wherein the intron comprises a nucleotide sequence that is at least 95% identical to nucleotides 885 to 2828 of SEQ ID NO: 52. In some embodiments, the vector construct comprises an intron region, wherein the intron comprises nucleotides 885 to 2828 of SEQ ID NO:52.

載體中之內含子之位置及尺寸可以變化。在一些實施例中，內含子位於啟動子與編碼所關注蛋白質之序列之間。在一些實施例中，內含子位於編碼所關注蛋白質之序列的下游。在一些實施例中，內含子位於啟動子內。在一些實施例中，內含子包括增強子元件。在一些實施例中，內含子位於編碼所關注蛋白質之序列內，較佳地位於編碼所關注蛋白質之序列之外顯子之間。在一些實施例中，內含子可包含編碼所關注蛋白質之序列內之天然存在之內含子的全部或一部分。The location and size of the introns in the vector can vary. In some embodiments, the intron is located between the promoter and the sequence encoding the protein of interest. In some embodiments, the intron is located downstream of the sequence encoding the protein of interest. In some embodiments, the intron is located within a promoter. In some embodiments, the intron includes an enhancer element. In some embodiments, introns are located within the sequence encoding the protein of interest, preferably between exons of the sequence encoding the protein of interest. In some embodiments, an intron may comprise all or a portion of a naturally occurring intron within the sequence encoding the protein of interest.

載體建構體可合併來自任何已知生物體之基因體的序列。序列可以其天然形式併入或可以任何方式修飾以獲得所需活性。舉例而言，序列可包含***、刪除或取代。The vector construct can incorporate sequences from the genome of any known organism. Sequences can be incorporated in their native form or modified in any way to obtain the desired activity. For example, sequences may contain insertions, deletions or substitutions.

在本文之任何實施例中，rAAV顆粒包含重組載體建構體，其包含與SEQ NO: 15至23或52中之任一者至少90%一致的核苷酸序列。較佳地，重組載體建構體包含與SEQ ID NO: 18或SEQ ID NO: 52至少90%一致之核苷酸序列，且AAV衣殼為AAV5型衣殼。In any of the embodiments herein, the rAAV particle comprises a recombinant vector construct comprising a nucleotide sequence that is at least 90% identical to any one of SEQ NOs: 15-23 or 52. Preferably, the recombinant vector construct comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 52, and the AAV capsid is an AAV5 type capsid.

在一些實施例中，rAAV顆粒包含重組載體建構體，其中該重組載體建構體包含SEQ ID NO: 52之一個、兩個、三個或全部元件。在某些實施例中，rAAV顆粒包含重組載體建構體，其中該重組載體建構體包含與SEQ ID NO: 52至少90%一致的核苷酸序列。在一些實施例中，rAAV顆粒包含重組載體建構體，其中該重組載體建構體包含與SEQ ID NO: 52至少95%一致的核苷酸序列。在某些實施例中，rAAV顆粒包含重組載體建構體，其中該重組載體建構體包含SEQ ID NO: 52之核苷酸序列。在具體實施例中，rAAV顆粒包含AAV衣殼，其為AAV5型衣殼。In some embodiments, the rAAV particle comprises a recombinant vector construct, wherein the recombinant vector construct comprises one, two, three, or all of the elements of SEQ ID NO:52. In certain embodiments, the rAAV particle comprises a recombinant vector construct, wherein the recombinant vector construct comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:52. In some embodiments, the rAAV particle comprises a recombinant vector construct, wherein the recombinant vector construct comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:52. In certain embodiments, the rAAV particle comprises a recombinant vector construct, wherein the recombinant vector construct comprises the nucleotide sequence of SEQ ID NO:52. In specific embodiments, the rAAV particles comprise an AAV capsid, which is an AAV5-type capsid.

本文所提供之呈單股形式之AAV載體建構體的長度小於約7.0 kb、或長度小於6.5 kb、或長度小於6.4 kb、或長度小於6.3 kb、或長度小於6.2 kb、或長度小於6.0 kb、或長度小於5.8 kb、或長度小於5.6 kb、或長度小於5.5 kb、或長度小於5.4 kb、或長度小於5.3 kb、或長度小於5.2 kb、或長度小於5.0 kb、或長度小於4.8 kb、或長度小於4.6 kb、或長度小於4.5 kb、或長度小於4.4 kb、或長度小於4.3 kb、或長度小於4.2 kb、或長度小於4.1 kb、或長度小於4.0 kb、或長度小於3.9 kb、或長度小於3.8 kb、或長度小於3.7 kb、或長度小於3.6 kb、或長度小於3.5 kb、或長度小於3.4 kb、或長度小於3.3 kb、或長度小於3.2 kb、或長度小於3.1 kb、或長度小於3.0 kb。本文所提供之呈單股形式之AAV載體建構體的長度在約5.0 kb至約6.5 kb之範圍內、或長度在約4.8 kb至約5.2 kb之範圍內、或長度為4.8 kb至5.3 kb、或長度在約4.9 kb至約5.5 kb之範圍內、或長度為約4.8 kb至約6.0 kb、或長度為約5.0 kb至約6.2 kb、或長度為約5.1 kb至約6.3 kb、或長度為約5.2 kb至約6.4 kb、或長度為約5.5 kb至約6.5 kb、或長度在約4.0 kb至約5.0 kb之範圍內、或長度在約3.8 kb至約4.8 kb之範圍內、或長度為約3.6 kb至約4.6 kb、或長度在約3.4 kb至約4.4 kb之範圍內、或長度在約3.2 kb至約4.2 kb之範圍內、或長度在約3.0 kb至4.0 kb之範圍內、或長度在約3.5 kb至約4.0 kb之範圍內、或長度在約3.0 kb至約3.5 kb之範圍內、或長度在約4 kb至約4.5 kb之範圍內。The AAV vector constructs provided herein in single-stranded form are less than about 7.0 kb in length, or less than 6.5 kb in length, or less than 6.4 kb in length, or less than 6.3 kb in length, or less than 6.2 kb in length, or less than 6.0 kb in length, or less than 5.8 kb in length, or less than 5.6 kb in length, or less than 5.5 kb in length, or less than 5.4 kb in length, or less than 5.3 kb in length, or less than 5.2 kb in length, or less than 5.0 kb in length, or less than 4.8 kb in length, or Less than 4.6 kb, or less than 4.5 kb in length, or less than 4.4 kb in length, or less than 4.3 kb in length, or less than 4.2 kb in length, or less than 4.1 kb in length, or less than 4.0 kb in length, or less than 3.9 kb in length, or less than 3.8 in length kb, or less than 3.7 kb in length, or less than 3.6 kb in length, or less than 3.5 kb in length, or less than 3.4 kb in length, or less than 3.3 kb in length, or less than 3.2 kb in length, or less than 3.1 kb in length, or less than 3.0 kb in length. The AAV vector constructs provided herein in single-stranded form range from about 5.0 kb to about 6.5 kb in length, or from about 4.8 kb to about 5.2 kb in length, or from 4.8 kb to 5.3 kb in length, or in the range of about 4.9 kb to about 5.5 kb in length, or about 4.8 kb to about 6.0 kb in length, or about 5.0 kb to about 6.2 kb in length, or about 5.1 kb to about 6.3 kb in length, or about 5.2 kb to about 6.4 kb, or about 5.5 kb to about 6.5 kb in length, or about 4.0 kb to about 5.0 kb in length, or about 3.8 kb to about 4.8 kb in length, or about 3.6 kb to about 4.6 kb, or in the range of about 3.4 kb to about 4.4 kb in length, or in the range of about 3.2 kb to about 4.2 kb in length, or in the range of about 3.0 kb to 4.0 kb in length, or The length is in the range of about 3.5 kb to about 4.0 kb, or the length is in the range of about 3.0 kb to about 3.5 kb, or the length is in the range of about 4 kb to about 4.5 kb.

當AAV載體由過大的重組載體建構體產生時，其可能缺乏重組載體建構體之5'或3'端之一部分。因為AAV為單股DNA病毒，且封裝有義或反義股，所以過大AAV載體中之有義股缺少5' AAV ITR及編碼目標蛋白之基因之5'端的可能部分，且過大AAV載體中之反義股缺少3' ITR及編碼目標蛋白之基因之3'端的可能部分。功能性轉基因藉由目標細胞內之有義及反義截短基因體之黏接產生於過大AAV載體感染細胞中。因此，在某些實施例中，本發明之rAAV顆粒可包含重組載體建構體，其包含至少一個ITR及編碼功能性hPAH之核苷酸序列的實質部分，諸如SEQ ID NO: 7、8、9、10、11、12或13中之任一者的片段，其大於核苷酸序列長度之50%、60%、70%、80%或90%。本發明之rAAV顆粒亦可包含SEQ ID NO: 15至23或52中之任一者之實質部分，例如大於SEQ ID NO: 15至23或52中之任一者中所闡述之核苷酸序列長度之50%、60%、70%、80%或90%的片段。When an AAV vector is produced from an oversized recombinant vector construct, it may lack a portion of the 5' or 3' end of the recombinant vector construct. Because AAV is a single-stranded DNA virus and encapsulates sense or antisense strands, the sense strand in the oversized AAV vector lacks the 5' AAV ITR and possibly a portion of the 5' end of the gene encoding the target protein, and the sense strand in the oversized AAV vector lacks the 5' end of the gene encoding the target protein. The antisense strand lacks the 3' ITR and a possible portion of the 3' end of the gene encoding the protein of interest. Functional transgenes are produced in oversized AAV vector-infected cells by cohesion of sense and antisense truncated gene bodies within the target cells. Thus, in certain embodiments, the rAAV particles of the invention may comprise a recombinant vector construct comprising at least one ITR and a substantial portion of a nucleotide sequence encoding a functional hPAH, such as SEQ ID NOs: 7, 8, 9 A fragment of any of , 10, 11, 12, or 13 that is greater than 50%, 60%, 70%, 80%, or 90% of the length of the nucleotide sequence. The rAAV particles of the invention may also comprise a substantial portion of any one of SEQ ID NOs: 15 to 23 or 52, eg, greater than the nucleotide sequence set forth in any one of SEQ ID NOs: 15 to 23 or 52 Fragments of 50%, 60%, 70%, 80% or 90% of their length.

包括經修飾之形式之聚核苷酸及多肽可使用各種標準選殖、重組DNA技術經由熟習此項技術者已知之細胞表現或活體外轉譯及化學合成技術(Sambrook等人,Molecular Cloning : A Laboratory Manual ,第2版)製得。可使用此項技術中熟知的任何適合之基因工程化技術完成載體建構體之產生，該等技術包括但不限於限制性核酸內切酶消化、接合、轉型、質體純化及DNA測序之標準技術，例如，如Sambrook等人(Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989))中所描述。Polynucleotides and polypeptides, including modified forms, can be expressed by cells or in vitro translation and chemical synthesis techniques known to those skilled in the art using a variety of standard selection, recombinant DNA techniques (Sambrook et al., Molecular Cloning : A Laboratory Manual , 2nd edition). Generation of vector constructs can be accomplished using any suitable genetic engineering technique well known in the art, including but not limited to standard techniques of restriction endonuclease digestion, ligation, transformation, plastid purification and DNA sequencing , eg, as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)).

AAV載體建構體可以複製及包裝成感染性AAV顆粒，較佳地複製缺乏型AAV顆粒，當存在於經編碼及表現rep及cap基因產物之聚核苷酸轉染的宿主細胞中時。 5.2 重組AAV顆粒及AAV衣殼AAV vector constructs can replicate and package into infectious AAV particles, preferably replication-deficient AAV particles, when present in host cells transfected with polynucleotides encoding and expressing the rep and cap gene products. 5.2 Recombinant AAV particles and AAV capsids

AAV顆粒之生產需要AAV「rep」及「cap」基因，其分別為編碼複製及衣殼化蛋白之基因。AAV rep及cap基因已發現於到目前為止檢查之所有AAV血清型中，且描述於本文及所引用之參考文獻中。在野生型AAV中，一般發現rep及cap基因在病毒基因體中彼此鄰接(亦即，其以鄰接或重疊轉錄單元「偶聯」在一起)，且其一般在AAV血清型中為保守的。AAV rep及cap基因亦個別地且統稱為「AAV封裝基因」。本文中所用之AAV cap基因編碼Cap蛋白，其能夠在rep及腺輔助功能存在下封裝AAV載體且能夠結合目標細胞受體。在一些實施例中，AAV cap基因編碼具有衍生自特定AAV血清型之胺基酸序列的衣殼蛋白。The production of AAV particles requires the AAV "rep" and "cap" genes, which are genes encoding replication and encapsidation proteins, respectively. AAV rep and cap genes have been found in all AAV serotypes examined so far and are described herein and in the references cited. In wild-type AAV, the rep and cap genes are generally found adjacent to each other in the viral genome (ie, they are "coupled" together in contiguous or overlapping transcriptional units), and they are generally conserved among AAV serotypes. The AAV rep and cap genes are also individually and collectively referred to as "AAV packaging genes". The AAV cap gene used herein encodes a Cap protein that is capable of encapsulating AAV vectors in the presence of rep and glandular helper functions and capable of binding to target cell receptors. In some embodiments, the AAV cap gene encodes a capsid protein having an amino acid sequence derived from a particular AAV serotype.

用於產生AAV的AAV序列可以源自任何AAV血清型的基因體。通常，AAV血清型的基因體序列在胺基酸及核酸層面上具有顯著同源性，提供一組類似基因功能，產生基本上在實體上及功能上等效的病毒顆粒，並藉由幾乎相同的機制複製及組裝。關於AAV血清型之基因體序列及基因體類似性之討論。(參見例如GenBank寄存編號U89790；GenBank寄存編號J01901；GenBank寄存編號AF043303；GenBank寄存編號AF085716；Chiorini等人,J . Vir . (1997)第71卷, 第6823頁至第6833頁；Srivastava等人, J . Vir . (1983)第45卷, 第555頁至第564頁；Chiorini等人,J . Vir . (1999)第73卷, 第1309頁至第1319頁；Rutledge等人,J . Vir . (1998)第72卷, 第309頁至第319頁；及Wu等人,J . Vir . (2000)第74卷, 第8635頁至第8647頁)。The AAV sequence used to generate the AAV can be derived from the gene body of any AAV serotype. In general, the gene body sequences of AAV serotypes share significant homology at the amino acid and nucleic acid levels, provide a set of similar gene functions, produce substantially physically and functionally equivalent viral particles, and are mechanism of replication and assembly. Discussion of gene body sequences and gene body similarity of AAV serotypes. (See, e.g., GenBank Accession No. U89790; GenBank Accession No. J01901; GenBank Accession No. AF043303; GenBank Accession No. AF085716;. Chiorini et al., J Vir (1997) Vol. 71, pages 6823 to page 6833;. Srivastava et al., .. J Vir (1983) Vol. 45, page 555 to page 564;. Chiorini et al., J Vir (1999) Vol. 73, page 1309 to page 1319;.. Rutledge et al., J Vir. (1998) vol. 72, page 309 to page 319;. and Wu et al., J Vir (2000) Vol. 74, pages 8635 to page 8647).

所有已知AAV血清型之基因體組織極為類似。AAV之基因體為長度小於約5,000個核苷酸(nt)之線性、單股DNA分子。反向末端重複序列(ITR)側接用於非結構複製(Rep)蛋白及結構(VP)蛋白之獨特編碼核苷酸序列。VP蛋白形成衣殼。組裝活化蛋白(AAP)會快速伴隨蛋白衣殼組裝且防止游離衣殼蛋白降解(Grosse等人, J. Virol. 91(20):e01198-17, 2017)。末端145 nt為自身互補型且經組織以使得可形成形成T形髮夾之能量穩定的分子內雙螺旋體。此等髮夾結構充當病毒DNA複製來源，充當細胞DNA聚合酶複合物之引子。Rep基因編碼Rep蛋白Rep78、Rep68、Rep52及Rep40。Rep78及Rep68由p5啟動子轉錄，且Rep 52及Rep40由p19啟動子轉錄。cap基因編碼VP蛋白VP1、VP2及VP3。cap基因由p40啟動子轉錄。本發明實施例之載體中採用的ITR可對應於與相關cap基因相同之血清型，或可不同。在一個實施例中，本文中採用的ITR對應於AAV2血清型，且cap基因對應於AAV5血清型。The genome organization of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) are flanked by unique coding nucleotide sequences for non-structural replication (Rep) proteins and structural (VP) proteins. VP proteins form the capsid. Assembly-activating proteins (AAPs) rapidly accompany protein capsid assembly and prevent degradation of free capsid proteins (Grosse et al., J. Virol. 91(20):e01198-17, 2017). The terminal 145 nt are self-complementary and organized so that an energetically stable intramolecular duplex that forms a T-shaped hairpin can be formed. These hairpin structures serve as a source of viral DNA replication and serve as primers for cellular DNA polymerase complexes. The Rep gene encodes the Rep proteins Rep78, Rep68, Rep52 and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep 52 and Rep40 are transcribed from the p19 promoter. The cap gene encodes the VP proteins VP1, VP2 and VP3. The cap gene is transcribed from the p40 promoter. The ITR employed in the vectors of the embodiments of the present invention may correspond to the same serotype as the relevant cap gene, or may be different. In one embodiment, the ITR employed herein corresponds to the AAV2 serotype and the cap gene corresponds to the AAV5 serotype.

已知AAV VP蛋白用於測定AAV病毒顆粒之細胞毒性。比起不同AAV血清型中之Rep蛋白及基因，VP蛋白編碼序列之保守性顯著較低。Rep及ITR序列交叉互補其他血清型之對應序列之能力允許產生包含血清型(例如，AAV1、5或8)之衣殼蛋白及另一AAV血清型(例如，AAV2)之Rep及/或ITR序列的假模式化AAV顆粒。該等假模式化rAAV顆粒為本發明之一部分。The AAV VP protein is known to be used to measure the cytotoxicity of AAV virus particles. The VP protein coding sequence is significantly less conserved than the Rep proteins and genes in different AAV serotypes. The ability of Rep and ITR sequences to cross-complement corresponding sequences from other serotypes allows the generation of Rep and/or ITR sequences comprising capsid proteins of a serotype (eg, AAV1, 5, or 8) and another AAV serotype (eg, AAV2) of pseudo-patterned AAV particles. These pseudo-patterned rAAV particles are part of the present invention.

本文所描述之AAV顆粒(及編碼AAV載體基因體)可包含WO 2018/022608或PCT/US19/32097中所描述之衣殼蛋白中之任一者，該等文獻以全文引用之方式併入本文中用於其揭示人類及猿猴AAV衣殼及其特性，諸如轉導效率、組織向性、糖結合及IVIG中和之抗性，包括(但不限於)序列清單中之衣殼中之任一者及其變異體，例如具有嵌合調換可變區及/或聚醣結合序列及/或GH迴路。The AAV particles (and encoding AAV vector gene bodies) described herein may comprise any of the capsid proteins described in WO 2018/022608 or PCT/US19/32097, which are incorporated herein by reference in their entirety for its disclosure of human and simian AAV capsids and their properties, such as transduction efficiency, tissue tropism, sugar binding, and resistance to IVIG neutralization, including but not limited to any of the capsids in the Sequence Listing and variants thereof, eg, having chimeric swapped variable regions and/or glycan binding sequences and/or GH loops.

在一個實施例中，用於本發明之上下文中之AAV ITR序列來源於AAV1、AAV2、AAV4及/或AAV6。同樣，在一個實施例中，Rep (例如Rep78及Rep52)編碼序列來源於AAV1、AAV2、AAV4及/或AAV6。然而，編碼用於本發明之上下文中之VP1、VP2及VP3衣殼蛋白的序列可獲自任何血清型，諸如AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11或AAV12，或獲自猿猴AAV，包括WO 2018/022608或PCT/US19/AAV32097中所描述之衣殼蛋白中之任一者，或藉由衣殼調換技術及AAV衣殼文庫獲得之新研發之AAV類顆粒，或與SEQ ID NO: 35至51中之任一者至少90%一致的任何衣殼。In one embodiment, the AAV ITR sequences used in the context of the present invention are derived from AAV1, AAV2, AAV4 and/or AAV6. Likewise, in one embodiment, the Rep (eg, Rep78 and Rep52) coding sequences are derived from AAV1, AAV2, AAV4, and/or AAV6. However, the sequences encoding the VP1, VP2 and VP3 capsid proteins for use in the context of the present invention can be obtained from any serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12, or obtained from simian AAV, including any of the capsid proteins described in WO 2018/022608 or PCT/US19/AAV32097, or a newly developed AAV obtained by capsid swapping techniques and AAV capsid libraries particle-like, or any capsid that is at least 90% identical to any one of SEQ ID NOs: 35-51.

舉例而言，公開各種衣殼之胺基酸序列。參見例如，For example, the amino acid sequences of various capsids are disclosed. See for example,

AAVRh.1 / hu.14 / AAV9 AAS99264.1 (SEQ ID NO: 35)AAVRh.1/hu.14/AAV9 AAS99264.1 (SEQ ID NO: 35)

美國專利公開案2013/0045186之AAVRh.8 SEQ97 (SEQ ID NO: 36)AAVRh.8 SEQ97 (SEQ ID NO: 36) of U.S. Patent Publication 2013/0045186

美國專利公開案2013/0045186之AAVRh.10 SEQ81 (SEQ ID NO: 37)AAVRh.10 SEQ 81 (SEQ ID NO: 37) of US Patent Publication 2013/0045186

國際專利公開案WO 2013/123503之AAVRh.74 SEQ 1 (SEQ ID NO: 38)AAVRh.74 SEQ 1 (SEQ ID NO: 38) of International Patent Publication WO 2013/123503

AAV1 AAB_95452.1 (SEQ ID NO: 39)AAV1 AAB_95452.1 (SEQ ID NO: 39)

AAV2 YP_680426.1 (SEQ ID NO: 40)AAV2 YP_680426.1 (SEQ ID NO: 40)

AAV3 NP_043941.1 (SEQ ID NO: 41)AAV3 NP_043941.1 (SEQ ID NO: 41)

AAV3B AAB95452.1 (SEQ ID NO: 42)AAV3B AAB95452.1 (SEQ ID NO: 42)

AAV4 NP_044927.1 (SEQ ID NO: 43)AAV4 NP_044927.1 (SEQ ID NO: 43)

AAV5 YP_068409.1 (SEQ ID NO: 44)AAV5 YP_068409.1 (SEQ ID NO: 44)

AAV6 AAB95450.1 (SEQ ID NO: 45)AAV6 AAB95450.1 (SEQ ID NO: 45)

AAV7 YP_077178.1 (SEQ ID NO: 46)AAV7 YP_077178.1 (SEQ ID NO: 46)

AAV8 YP_077180.1 (SEQ ID NO: 47)AAV8 YP_077180.1 (SEQ ID NO: 47)

AAV10 AAT46337.1 (SEQ ID NO: 48)AAV10 AAT46337.1 (SEQ ID NO: 48)

AAV11 AAT46339.1 (SEQ ID NO: 49)AAV11 AAT46339.1 (SEQ ID NO: 49)

AAV12 ABI16639.1 (SEQ ID NO: 50)AAV12 ABI16639.1 (SEQ ID NO: 50)

AAV13 ABZ10812.1 (SEQ ID NO: 51)AAV13 ABZ10812.1 (SEQ ID NO: 51)

其中AAV衣殼包含與SEQ ID NO: 35至51中之任一者至少85%一致之胺基酸序列。wherein the AAV capsid comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 35-51.

較佳地，AAV衣殼為具有肝趨向性之AAV衣殼。在一些情況下，具有肝趨向性之AAV衣殼不包括AAV8及/或AAVHSC15。較佳地，具有肝趨向性之AAV衣殼為AAV5型衣殼，視情況與SEQ ID NO: 44至少85%、90%或95%一致。在具體實施例中，AAV衣殼包含SEQ ID NO: 44之序列。Preferably, the AAV capsid is an AAV capsid with liver tropism. In some cases, the AAV capsid with liver tropism does not include AAV8 and/or AAVHSC15. Preferably, the AAV capsid with liver tropism is an AAV5 type capsid that is at least 85%, 90% or 95% identical to SEQ ID NO: 44, as appropriate. In a specific embodiment, the AAV capsid comprises the sequence of SEQ ID NO:44.

經修飾之「AAV」序列亦可以用於本發明之上下文中，例如，用於產生AAV基因療法載體。此類經修飾之序列，例如與AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8或AAV9 ITR、Rep或VP具有至少約70%、至少約75%、至少約80%、至少約85%、至少約90%、至少約95%或更多的核苷酸及/或胺基酸序列一致性之序列(例如，具有約75至99%的核苷酸序列一致性之序列)，其可以用於取代野生型AAV ITR、Rep或VP序列。Modified "AAV" sequences can also be used in the context of the present invention, eg, in the production of AAV gene therapy vectors. Such modified sequences, for example, have at least about 70%, at least about 75%, at least about 80%, at least about 85% with AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep or VP %, at least about 90%, at least about 95% or more nucleotide and/or amino acid sequence identity sequences (e.g., sequences having about 75 to 99% nucleotide sequence identity), which Can be used to replace wild-type AAV ITR, Rep or VP sequences.

在一些實施例中，編碼AAV衣殼蛋白之核酸序列可操作地連接至表現控制序列，用於在特定細胞類型中表現，諸如Sf9或HEK細胞。可以使用本領域中熟習此項技術者已知用於在昆蟲宿主細胞或哺乳動物宿主細胞中表現外源基因之技術來實踐實施例。用於分子工程及昆蟲細胞中表現多肽的方法描述於例如Summers及Smith (1986) A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. 第7555號, College Station, Tex.; Luckow (1991) In Prokop等人，Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152；King, L. A.及R. D. Possee (1992) The baculovirus expression system, Chapman and Hall, United Kingdom；O'Reilly, D. R., L. K. Miller, V. A. Luckow (1992) Baculovirus Expression Vectors: A Laboratory Manual, New York；W.H. Freeman及Richardson, C. D. (1995) Baculovirus Expression Protocols, Methods in Molecular Biology, 第39卷；美國專利公開案第4,745,051號；US2003148506；及WO 03/074714，其內容皆以全文引用之方式併入本文中。用於編碼AAV衣殼蛋白之核苷酸序列之轉錄的尤其適合之啟動子為例如多面體啟動子。然而，本領域中已知在昆蟲細胞中具有活性之其他啟動子，例如p10、p35或IE-1啟動子，且亦涵蓋以上參考文獻中所描述之其他啟動子。In some embodiments, the nucleic acid sequence encoding an AAV capsid protein is operably linked to an expression control sequence for expression in a particular cell type, such as Sf9 or HEK cells. The embodiments can be practiced using techniques known to those skilled in the art for expressing foreign genes in insect host cells or mammalian host cells. Methods for molecular engineering and expression of polypeptides in insect cells are described, for example, in Summers and Smith (1986) A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex.; Luckow (1991) In Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152; King, LA and RD Possee (1992) The baculovirus expression system, Chapman and Hall, United Kingdom; O'Reilly, DR, LK Miller, VA Luckow (1992) Baculovirus Expression Vectors: A Laboratory Manual, New York; WH Freeman and Richardson, CD (1995) Baculovirus Expression Protocols, Methods in Molecular Biology, Vol. 39; USA Patent Publication No. 4,745,051; US2003148506; and WO 03/074714, the contents of which are all incorporated herein by reference in their entirety. A particularly suitable promoter for transcription of the nucleotide sequence encoding the AAV capsid protein is, for example, the polyhedral promoter. However, other promoters that are active in insect cells are known in the art, such as the p10, p35 or IE-1 promoters, and other promoters described in the above references are also encompassed.

使用昆蟲細胞表現異源蛋白質為有據可查的，因為其作為將核酸，如載體，例如昆蟲-細胞相容載體引入至此類細胞中之方法及將此類細胞維持於培養物中之方法。(參見例如METHODS IN MOLECULAR BIOLOGY, Richard編, Humana Press, N J (1995)；O'Reilly等人, BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994)；Samulski等人,J . Vir . (1989)第63卷, 第3822頁至第3828頁；Kajigaya等人,Proc . Nat ' l . Acad . Sci . USA (1991)第88卷, 第4646頁至第4650頁；Ruffing等人,J . Vir . (1992)第66卷, 第6922頁至第6930頁；Kirnbauer等人,Vir . (1996)第219卷, 第37頁至第44頁；Zhao等人,Vir . (2000)第272卷, 第382頁至第393頁；及美國專利第6,204,059號)。在一些實施例中，昆蟲細胞中編碼AAV之核酸建構體為昆蟲細胞相容載體。如本文所用之「昆蟲細胞相容載體」或「載體」係指能夠有效轉型或轉染昆蟲或昆蟲細胞的核酸分子。例示性生物載體包括質體、線性核酸分子及重組病毒。可以使用任何載體，只要其為昆蟲細胞相容的。載體可整合至昆蟲細胞基因體中，但載體無需永久存在於昆蟲細胞中，並且亦包括短暫游離型載體。載體可藉由任何已知方法引入，例如藉由化學處理細胞、電穿孔或感染。在一些實施例中，載體為桿狀病毒、病毒載體或質體。在一個實施例中，載體為桿狀病毒，亦即建構體為桿狀病毒載體。桿狀病毒載體及其使用方法描述於上文所引用之關於昆蟲細胞之分子工程改造的參考文獻中。 5.3 用於產生重組AAV顆粒之方法The use of insect cells to express heterologous proteins is well documented as a method for introducing nucleic acids, such as vectors, eg insect-cell compatible vectors, into such cells and for maintaining such cells in culture. (See, e.g., METHODS IN MOLECULAR BIOLOGY, Richard ed, Humana Press, NJ (1995) ;. O'Reilly et al., BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL , Oxford Univ Press (1994);. Samulski et al., J Vir (. 1989) Vol. 63, page 3822 to page 3828; Kajigaya et al., Proc Nat 'l Acad Sci USA (1991) volume 88, pages 4646 to page 4650;.... Ruffing et al., J. Vir . (1992) vol. 66, pp. 6922-6930; Kirnbauer et al., Vir . (1996) vol. 219, pp. 37-44; Zhao et al., Vir . (2000) vol. 272 , pp. 382-393; and US Patent No. 6,204,059). In some embodiments, the nucleic acid construct encoding the AAV in the insect cell is an insect cell-compatible vector. An "insect cell-compatible vector" or "vector" as used herein refers to a nucleic acid molecule capable of efficiently transforming or transfecting insects or insect cells. Exemplary biological vectors include plastids, linear nucleic acid molecules, and recombinant viruses. Any carrier can be used as long as it is compatible with insect cells. The vector can be integrated into the insect cell genome, but the vector need not be permanently present in the insect cell and also includes transient episomal vectors. The vector can be introduced by any known method, such as by chemical treatment of cells, electroporation or infection. In some embodiments, the vector is a baculovirus, a viral vector, or a plastid. In one embodiment, the vector is a baculovirus, ie, the construct is a baculovirus vector. Baculovirus vectors and methods of use are described in the references cited above for molecular engineering of insect cells. 5.3 Methods for producing recombinant AAV particles

在另一實施例中，提供產生包含本文所提供之AAV載體建構體中之任一者之重組型腺相關病毒(AAV)顆粒之方法。該方法包含以下步驟：培養已經本文所提供之AAV載體建構體中之任一者(與各種AAV cap及rep基因相關)轉染之細胞且自經轉染的細胞或經轉染的細胞上清液中回收重組治療性AAV顆粒。In another embodiment, methods of producing recombinant adeno-associated virus (AAV) particles comprising any of the AAV vector constructs provided herein are provided. The method comprises the steps of culturing cells that have been transfected with any of the AAV vector constructs provided herein (associated with the various AAV cap and rep genes) and supernatant from the transfected cells or transfected cells Recombinant therapeutic AAV particles are recovered from the liquid.

適用於本文所提供之重組AAV產生之細胞為易受桿狀病毒感染之任何細胞類型，包括諸如High Five、Sf9、Se301、SeIZD2109、SeUCR1、Sf9、Sf900+、Sf21、BTI-TN-5B1-4、MG-1、Tn368、HzAm1、BM-N、Ha2302、Hz2E5及Ao38之昆蟲細胞。在另一實施例中，可以使用哺乳動物細胞，諸如HEK293、HeLa、CHO、NSO、SP2/0、PER.C6、Vero、RD、BHK、HT 1080、A549、Cos-7、ARPE-19及MRC-5。Cells suitable for the production of recombinant AAV provided herein are any cell type susceptible to baculovirus infection, including, for example, High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, Insect cells of MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5 and Ao38. In another embodiment, mammalian cells such as HEK293, HeLa, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19 and MRC can be used -5.

本發明提供用於在昆蟲或哺乳動物細胞中產生重組型AAV顆粒之材料及方法，該等昆蟲或哺乳動物細胞包含本文所描述之載體建構體中之任一者。在一些實施例中，載體建構體進一步包含啟動子及啟動子下游之限制位點以允許***編碼一或多種所關注蛋白質之聚核苷酸，其中該啟動子及該限制位點位於5' AAV ITR之下游及3' AAV ITR之上游。在一些實施例中，載體建構體進一步包含在限制位點下游及3' AAV ITR上游之轉錄後調節元件。在一些實施例中，載體建構體進一步包含***限制位點且與啟動子可操作地連接之聚核苷酸，其中該聚核苷酸包含所關注蛋白質之編碼區。如熟習此項技術者將瞭解，本申請案中所揭示之AAV載體建構體中的任一者可以用於產生重組AAV顆粒之方法中。The present invention provides materials and methods for producing recombinant AAV particles in insect or mammalian cells comprising any of the vector constructs described herein. In some embodiments, the vector construct further comprises a promoter and a restriction site downstream of the promoter to allow insertion of polynucleotides encoding one or more proteins of interest, wherein the promoter and the restriction site are located at the 5' AAV Downstream of the ITR and upstream of the 3' AAV ITR. In some embodiments, the vector construct further comprises post-transcriptional regulatory elements downstream of the restriction site and upstream of the 3' AAV ITR. In some embodiments, the vector construct further comprises a polynucleotide inserted into the restriction site and operably linked to the promoter, wherein the polynucleotide comprises the coding region for the protein of interest. As will be appreciated by those skilled in the art, any of the AAV vector constructs disclosed in this application can be used in methods of producing recombinant AAV particles.

在一些實施例中，輔助功能由包含腺病毒或桿狀病毒輔助基因之一或多個輔助質體或輔助病毒提供。腺病毒或桿狀病毒輔助基因之非限制性實例包括但不限於E1A、E1B、E2A、E4及VA，其可以向AAV封裝提供輔助功能。In some embodiments, the helper function is provided by a helper plastid or helper virus comprising one or more adenovirus or baculovirus helper genes. Non-limiting examples of adenoviral or baculoviral helper genes include, but are not limited to, E1A, E1B, E2A, E4, and VA, which can provide helper functions to AAV encapsulation.

AAV之輔助病毒為本領域中已知的，且包括例如來自腺病毒科(Adenoviridae)及疱疹病毒科(Herpes viridae)之病毒。AAV之輔助病毒之實例包括但不限於美國公開案第20110201088號(其揭示內容以引用之方式併入本文中)中所描述之SAdV-13輔助病毒及SAdV-13樣輔助病毒及輔助載體pHELP (Applied Viromics)。本領域熟習此項技術者將瞭解，本文中可以使用任何可為AAV提供足夠輔助功能的AAV之輔助病毒或輔助質體。Helper viruses for AAV are known in the art and include, for example, viruses from the families Adenoviridae and Herpes viridae. Examples of helper viruses for AAV include, but are not limited to, the SAdV-13 helper virus and SAdV-13-like helper virus and the helper vector pHELP ( Applied Viromics). Those skilled in the art will appreciate that any helper virus or helper plastid for AAV that provides sufficient helper function for the AAV can be used herein.

在一些實施例中，AAV cap基因存在於質體中。質體可以進一步包含AAV rep基因，其可以對應於或不對應於與cap基因相同之血清型。本文中可以使用來自任何AAV血清型(包括但不限於AAV1、AAV2、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13及其任何變異體)之cap基因及/或rep基因以產生重組AAV。在一些實施例中，AAV cap基因編碼來自血清型1、血清型2、血清型4、血清型5、血清型6、血清型7、血清型8、血清型9、血清型10、血清型11、血清型12、血清型13或其變異體的衣殼。In some embodiments, the AAV cap gene is present in the plastid. The plastid may further comprise an AAV rep gene, which may or may not correspond to the same serotype as the cap gene. Cap genes and/or reps from any AAV serotype (including but not limited to AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 and any variants thereof) can be used herein gene to produce recombinant AAV. In some embodiments, the AAV cap gene encodes a gene from serotype 1, serotype 2, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 , serotype 12, serotype 13, or the capsid of a variant thereof.

在一些實施例中，昆蟲或哺乳動物細胞可以經輔助質體或輔助病毒、載體建構體及編碼AAV cap基因之質體轉染；且可以在共轉染之後的各個時間點收集重組AAV病毒。舉例而言，重組AAV病毒可以在共轉染之後約12小時、約24小時、約36小時、約48小時、約72小時、約96小時、約120小時或其中任何兩個時間點之間的時間收集。In some embodiments, insect or mammalian cells can be transfected with a helper plastid or helper virus, a vector construct, and a plastid encoding the AAV cap gene; and recombinant AAV virus can be collected at various time points after co-transfection. For example, the recombinant AAV virus can be co-transfected at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, or between any two time points therein. time collection.

重組AAV顆粒亦可以使用任何此項技術中已知適合於產生感染性重組AAV之習知方法產生。在一些情況下，可以藉由使用穩定表現AAV顆粒產生所需之一些組件的昆蟲或哺乳動物細胞來產生重組AAV。舉例而言，可以將包含AAV rep及cap基因之質體(或多個質體)及選擇標記(諸如新黴素抗性基因)整合至細胞的基因體中。昆蟲或哺乳動物細胞隨後可以經輔助病毒(例如，提供輔助功能之腺病毒或桿狀病毒)及包含5'及3' AAV ITR(以及必要時，編碼異源蛋白質之核苷酸序列)之病毒載體建構體共感染。此方法之優點為細胞為可選的且適合於大規模產生重組AAV顆粒。作為另一非限制性實例，腺病毒或桿狀病毒而非質體可以用於將rep及cap基因引入至封裝細胞中。作為又另一非限制性實例，含有5'及3' AAV LTR之病毒載體建構體及rep-cap基因均可以穩定整合至生產細胞之DNA中，且輔助功能可以由野生型腺病毒提供以產生重組AAV。Recombinant AAV particles can also be produced using any conventional method known in the art to be suitable for producing infectious recombinant AAV. In some cases, recombinant AAV can be produced by using insect or mammalian cells that stably express some of the components required for AAV particle production. For example, a plastid (or plastids) comprising the AAV rep and cap genes and a selectable marker (such as a neomycin resistance gene) can be integrated into the genome of the cell. Insect or mammalian cells can then be infected with helper viruses (eg, adenovirus or baculovirus that provide helper functions) and viruses comprising 5' and 3' AAV ITRs (and, if necessary, nucleotide sequences encoding heterologous proteins) Vector construct co-infection. The advantage of this method is that cells are optional and suitable for large-scale production of recombinant AAV particles. As another non-limiting example, adenovirus or baculovirus, rather than plastids, can be used to introduce rep and cap genes into encapsulating cells. As yet another non-limiting example, both viral vector constructs containing 5' and 3' AAV LTRs and rep-cap genes can be stably integrated into the DNA of producer cells, and helper functions can be provided by wild-type adenovirus for production Recombinant AAV.

本文提供用於產生適用作基因遞送載體之AAV顆粒之方法，該方法包含以下步驟： (a)為允許AAV複製之細胞(例如昆蟲細胞或哺乳動物細胞)提供一或多種核酸建構體，該一或多種核酸建構體包含： (i)本文所提供之核酸分子(重組載體建構體)，其具有至少一個側接AAV反向末端重複序列核苷酸序列； (ii)編碼一或多種AAV Rep蛋白之核苷酸序列，其可操作地連接至能夠驅動該細胞中之該一或多種Rep蛋白之表現的啟動子； (iii)編碼一或多種AAV衣殼蛋白之核苷酸序列，其可操作地連接至能夠驅動該細胞中之該一或多種衣殼蛋白之表現的啟動子； (iv)及視情況選用之含於VP2/3 mRNA中之AAP及MAAP； (b)在允許表現Rep及衣殼蛋白之條件下培養(a)中定義之細胞；及，視情況選用之(c)回收AAV顆粒，及視情況選用之(d)純化該AAV顆粒。舉例而言，(i)之重組載體建構體包含(1)至少一種AAV ITR；(2)異源肝特異性轉錄調節區；及(3)編碼功能性人類***酸羥化酶(hPAH)之核酸。Provided herein are methods for producing AAV particles suitable for use as gene delivery vectors comprising the steps of: (a) providing cells (eg, insect cells or mammalian cells) that permit replication of the AAV with one or more nucleic acid constructs comprising: (i) a nucleic acid molecule (recombinant vector construct) provided herein having at least one nucleotide sequence flanking an AAV inverted terminal repeat; (ii) a nucleotide sequence encoding one or more AAV Rep proteins operably linked to a promoter capable of driving expression of the one or more Rep proteins in the cell; (iii) a nucleotide sequence encoding one or more AAV capsid proteins operably linked to a promoter capable of driving expression of the one or more capsid proteins in the cell; (iv) and optionally AAP and MAAP contained in VP2/3 mRNA; (b) culturing the cells defined in (a) under conditions permitting expression of the Rep and capsid proteins; and, Optionally (c) recover AAV particles, and Optionally (d) purify the AAV particles. For example, (i) a recombinant vector construct comprising (1) at least one AAV ITR; (2) a heterologous liver-specific transcriptional regulatory region; and (3) a functional human phenylalanine hydroxylase (hPAH) encoding nucleic acid.

隨後，用於產生AAV基因遞送載體之本文所提供之方法通常包含：在足以將載體基因體複製及封裝至AAV衣殼中之條件下向允許AAV複製的細胞提供：(a)編碼用於產生載體基因體之模板的核苷酸序列(例如本發明之載體建構體) (如在本文中詳細描述)；(b)足以複製模板以產生載體基因體(上文所定義之第一表現卡匣)的核苷酸序列；(c)足以將載體基因體封裝至AAV衣殼(上文所定義之第二表現卡匣)中之核苷酸序列，由此包含在AAV衣殼內衣殼化之載體基因體之AAV顆粒在細胞中產生。Subsequently, the methods provided herein for producing an AAV gene delivery vector generally comprise: providing to a cell permissive for AAV replication under conditions sufficient to replicate and encapsulate the vector gene body into the AAV capsid: (a) encoding for production of The nucleotide sequence of the template of the vector genome (eg, the vector construct of the invention) (as described in detail herein); (b) sufficient to replicate the template to generate the vector genome (first expression cassette as defined above) ); (c) a nucleotide sequence sufficient to encapsulate the vector genome into an AAV capsid (second expression cassette as defined above), thereby encapsulating within the AAV capsid AAV particles of the vector genome are produced in cells.

使用桿狀病毒表現載體系統(BEVS) (Mietzsch等人, Hum. Gene Ther. 25: 212-22 (2014))之黏附HEK293細胞之短暫轉染(Chahal等人, J. Virol. Meth. 196: 163-73 (2014))及Sf9細胞之轉染為兩種最常用的產生AAV載體之方法。Transient transfection of adherent HEK293 cells (Chahal et al., J. Virol. Meth. 196: 163-73 (2014)) and transfection of Sf9 cells are the two most commonly used methods to generate AAV vectors.

本文所提供之方法可包含使用抗AAV抗體(在一個實施例中為固定抗體)對(包含)重組小病毒(rAAV)載體建構體(之病毒粒子)進行親和力純化的步驟。在另一實施例中，抗AAV抗體為單株抗體。本文中所用之一種抗體為單鏈駱駝抗體或其片段，如例如可獲自駱駝或駱馬(參見例如，Muyldermans, 2001, Biotechnol. 74: 277-302)。用於使rAAV親和力純化之抗體為特異性結合AAV衣殼蛋白上之抗原決定基之抗體，由此在一個實施例中，抗原決定基為存在於超過一種AAV血清型之衣殼蛋白上之抗原決定基。舉例而言，該抗體可基於特異性結合至AAV5衣殼而升高或選擇，但同時其亦可特異性結合至AAV1、AAV2、AAV3、AAV6、AAV8或AAV9衣殼。The methods provided herein can comprise the step of affinity purification of (including) a recombinant parvoviral (rAAV) vector construct (virion) using an anti-AAV antibody (in one embodiment an immobilized antibody). In another embodiment, the anti-AAV antibody is a monoclonal antibody. As used herein, an antibody is a single-chain camelid antibody or fragment thereof, as can be obtained, for example, from camels or llamas (see, eg, Muyldermans, 2001, Biotechnol. 74: 277-302). The antibody used for affinity purification of rAAV is an antibody that specifically binds an epitope on the AAV capsid protein, whereby in one embodiment the epitope is an antigen present on the capsid protein of more than one AAV serotype decision base. For example, the antibody can be raised or selected based on specific binding to the AAV5 capsid, but at the same time it can also specifically bind to the AAV1, AAV2, AAV3, AAV6, AAV8 or AAV9 capsid.

可使用任何允許產生AAV或生物產物且可維持於培養物中之無脊椎動物細胞類型產生包含本文所描述之載體建構體的病毒顆粒。舉例而言，所用昆蟲細胞系可以來自草地夜蛾(Spodoptera frugiperda)(諸如SF9、SF21、SF900+)、果蠅細胞系、蚊細胞系(例如白紋伊蚊(Aedes albopictus)衍生的細胞系)、家蠶細胞系(例如桑蠶(Bombyx mori)細胞系)、粉紋夜蛾(Trichoplusia)細胞系(諸如High Five細胞)，或鱗翅目細胞系(諸如黑巫婆飛蛾(Ascalapha odorata)細胞系)。在一個實施例中，昆蟲細胞為來自易受桿狀病毒感染之昆蟲物種之細胞，包括High Five、Sf9、Se301、SeIZD2109、SeUCR1、Sf9、Sf900+、Sf21、BTI-TN-5B1-4、MG-1、Tn368、HzAm1、BM-N、Ha2302、Hz2E5及Ao38。Viral particles comprising the vector constructs described herein can be produced using any invertebrate cell type that allows the production of AAV or biological products and can be maintained in culture. For example, the insect cell line used may be from Spodoptera frugiperda (such as SF9, SF21, SF900+), Drosophila cell line, mosquito cell line (eg Aedes albopictus derived cell line), Bombyx mori cell line (eg Bombyx mori cell line), Trichoplusia cell line (such as High Five cells), or Lepidopteran cell line (such as Ascalapha odorata cell line). In one embodiment, the insect cell is a cell from an insect species susceptible to baculovirus infection, including High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG- 1. Tn368, HzAm1, BM-N, Ha2302, Hz2E5 and Ao38.

桿狀病毒為節肢動物之包膜DNA病毒，其兩個成員為用於在細胞培養物中產生重組蛋白之熟知表現載體。桿狀病毒具有環狀雙鏈基因體(80至200 kbp)，其可經工程改造以允許將大基因體內含物傳遞至特定細胞。用作載體之病毒一般為加洲苜蓿夜蛾(Autographa californica)多衣殼核多角體病毒(AcMNPV)或家蠶核多角體病毒(BmNPV) (Kato等人, (2010), Applied Microbiology and Biotechnology,第85卷, 第3期, 第459頁至第470頁)。Baculoviruses are arthropod enveloped DNA viruses, two members of which are well known expression vectors for the production of recombinant proteins in cell culture. Baculoviruses have circular duplex genomes (80 to 200 kbp) that can be engineered to allow delivery of large genome contents to specific cells. The viruses used as vectors are typically Autographa californica polycapsid nuclear polyhedrosis virus (AcMNPV) or silkworm nuclear polyhedrosis virus (BmNPV) (Kato et al., (2010), Applied Microbiology and Biotechnology, p. 85, No. 3, pp. 459-470).

桿狀病毒通常用於感染昆蟲細胞以表現重組蛋白。特定言之，在昆蟲中表現異源基因可如例如以下中所描述完成：美國專利第4,745,051號；EP 127,839；EP 155,476；Vlak等人, (1988), Journal of General Virology,第68卷, 第765頁至第776頁；Miller等人, (1988), Annual Review of Microbiology,第42卷, 第177頁至第179頁；Carbonell等人, (1998), Gene,第73卷, 第2期, 第409頁至第418頁；Maeda等人, (1985), Nature,第315卷, 第592頁至第594頁；Lebacq-Veheyden等人, (1988), Molecular and Cellular Biology, 第8卷, 第8號, 第3129頁至第3135頁；Smith等人, (1985), PNAS, 第82卷, 第8404頁至第8408頁；及Miyajima等人, (1987), Gene,第58卷, 第273頁至第281頁。可以用於蛋白質產生之許多桿狀病毒菌株及變異體及對應容許昆蟲宿主細胞描述於Luckow等人, (1988), Nature Biotechnology, 第6卷, 第47頁至第55頁；Maeda等人, (1985), Nature, 第315卷, 第592頁至第594頁；及McKenna等人, (1998), Journal of Invertebrate Pathology,第71卷, 第1期, 第82頁至第90頁中。Baculoviruses are commonly used to infect insect cells to express recombinant proteins. In particular, expression of heterologous genes in insects can be accomplished as described, for example, in US Pat. No. 4,745,051; EP 127,839; EP 155,476; Vlak et al., (1988), Journal of General Virology, Vol. 68, p. 765-776; Miller et al., (1988), Annual Review of Microbiology, Vol. 42, pp. 177-179; Carbonell et al., (1998), Gene, Vol. 73, No. 2, 409-418; Maeda et al., (1985), Nature, vol. 315, pp. 592-594; Lebacq-Veheyden et al., (1988), Molecular and Cellular Biology, vol. 8, p. No. 8, pp. 3129-3135; Smith et al., (1985), PNAS, vol. 82, pp. 8404-8408; and Miyajima et al., (1987), Gene, vol. 58, pp. 273 page to page 281. A number of baculovirus strains and variants that can be used for protein production and corresponding permissive insect host cells are described in Luckow et al., (1988), Nature Biotechnology, Vol. 6, pp. 47-55; Maeda et al., ( 1985), Nature, Vol. 315, pp. 592-594; and in McKenna et al., (1998), Journal of Invertebrate Pathology, Vol. 71, No. 1, pp. 82-90.

在另一實施例中，用允許複製AAV或產生生物產物且可維持在培養物中之任何哺乳動物細胞類型進行本文所提供之方法。在一個實施例中，可以使用哺乳動物細胞，HEK293、HeLa、CHO、NSO、SP2/0、PER.C6、Vero、RD、BHK、HT 1080、A549、Cos-7、ARPE-19及MRC-5細胞。In another embodiment, the methods provided herein are performed with any mammalian cell type that allows replication of AAV or production of biological products and can be maintained in culture. In one embodiment, mammalian cells can be used, HEK293, HeLa, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19 and MRC-5 cell.

本文所提供之用於產生rAAV顆粒之方法產生rAAV顆粒群。在一些實施例中，藉由減少空衣殼之數目的步驟，使包含全長或幾乎全長載體基因體之顆粒富集為群。The methods provided herein for producing rAAV particles produce populations of rAAV particles. In some embodiments, particles comprising full-length or nearly full-length vector gene bodies are enriched into a population by the step of reducing the number of empty capsids.

藉由本文所提供之方法產生之rAAV顆粒群用於例如以減少本文所描述之人類個體中之血漿Phe含量的任何方法及治療本文所描述之PKU的任何方法投與。在某些實施例中，rAAV顆粒提供於實例2之液體調配物中。在某些實施例中，調配物適用於靜脈內投與以用於根據如實例3或實例4中所描述之方案治療PKU之方法。 5.4 醫藥調配物Populations of rAAV particles produced by the methods provided herein are used for administration, eg, in any method of reducing plasma Phe levels in a human subject described herein and in any method of treating PKU described herein. In certain embodiments, rAAV particles are provided in the liquid formulation of Example 2. In certain embodiments, the formulations are suitable for intravenous administration for a method of treating PKU according to a regimen as described in Example 3 or Example 4. 5.4 Pharmaceutical formulations

在一個實施例中，提供一種醫藥組合物，其包含本文所提供之核酸或載體及醫藥學上可接受之稀釋劑、賦形劑、載劑及/或其他藥劑、藥學製劑或佐劑等。In one embodiment, there is provided a pharmaceutical composition comprising the nucleic acid or vector provided herein and a pharmaceutically acceptable diluent, excipient, carrier and/or other agent, pharmaceutical formulation or adjuvant, and the like.

「醫藥學上可接受」意謂不為生物學上或以其他方式非所需的材料，亦即可向個體投與該材料而不會引起任何非所需生物效應。因此，醫藥組合物可例如用於活體外細胞之轉染或用於直接向個體投與病毒顆粒或細胞。"Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, ie, the material can be administered to an individual without causing any undesirable biological effect. Thus, the pharmaceutical composition can be used, for example, for transfection of cells in vitro or for direct administration of viral particles or cells to an individual.

載劑可適用於非經腸投與，其包括靜脈內、腹膜內或肌內投與。可替代地，載劑可適用於舌下或經口投與。醫藥學上可接受之載劑包括無菌水溶液或分散液及用於臨時製備無菌可注射溶液或分散液之無菌粉劑。此類介質及試劑用於醫藥活性物質之用途為此項技術中熟知的。除非任何習知介質或試劑與活性化合物不相容，否則考慮將其用於本文所提供之醫藥組合物中。The carrier may be suitable for parenteral administration, including intravenous, intraperitoneal or intramuscular administration. Alternatively, the carrier may be suitable for sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, it is contemplated for use in the pharmaceutical compositions provided herein.

在其他實施例中，本文提供AAV顆粒之醫藥組合物(亦即調配物)，其適用於向罹患遺傳病症之個體投與以遞送編碼所關注蛋白質之基因。在某些實施例中，本文所提供之醫藥調配物為包含包括含本文所揭示之載體建構體中之任一者的重組AAV顆粒的液體調配物。調配物中重組AAV病毒粒子之濃度可變化。在某些實施例中，調配物中之重組AAV顆粒的濃度可在1×10¹³ 至約1×10¹⁴ vg/ml，例如6×10¹³ vg/ml的範圍內。在某些實施例中，調配物中重組AAV顆粒之濃度如實例2中所描述。In other embodiments, provided herein are pharmaceutical compositions (ie, formulations) of AAV particles suitable for administration to individuals suffering from a genetic disorder to deliver a gene encoding a protein of interest. In certain embodiments, the pharmaceutical formulations provided herein are liquid formulations comprising recombinant AAV particles comprising any of the vector constructs disclosed herein. The concentration of recombinant AAV virions in the formulation can vary. In certain embodiments, the concentration of recombinant AAV particles in the formulation may be about 1 × 10 ¹³ to 1 × 10 ¹⁴ vg / ml, for example in the range of 6 × 10 ¹³ vg / ml of. In certain embodiments, the concentration of recombinant AAV particles in the formulation is as described in Example 2.

在其他實施例中，本文所提供之AAV顆粒醫藥調配物包含一或多種醫藥學上可接受之賦形劑，以提供具有用於儲存及/或向個體投與以治療遺傳病症之有利特性的調配物。在某些實施例中，本文所提供之醫藥調配物能夠在低於約-60℃ (攝氏零下60度)下儲存至少6個月、1.5年或2年之時間，而穩定性無明顯變化。另外，本文所提供之醫藥調配物在適合加速儲存條件下為穩定的。實例加速條件包括在約40℃及約75%濕度下儲存持續例如6、9、12、18及/或24個月之時段，或在約25℃及約60%濕度下持續例如6、9、12、18及/或24個月之時段，或(對於意欲用於儲存於冷凍器中之藥物物質)在約-20℃下持續例如12個月之時段。參見例如FDA Guidance for Industry: Stability Testing of New Drug Substances and Products, 2003年11月。In other embodiments, the AAV particle pharmaceutical formulations provided herein include one or more pharmaceutically acceptable excipients to provide an AAV with favorable properties for storage and/or administration to an individual for the treatment of genetic disorders formulation. In certain embodiments, the pharmaceutical formulations provided herein can be stored below about -60°C (minus 60°C) for a period of at least 6 months, 1.5 years, or 2 years without significant change in stability. Additionally, the pharmaceutical formulations provided herein are stable under conditions suitable for accelerated storage. Example accelerated conditions include storage at about 40°C and about 75% humidity for periods of time such as 6, 9, 12, 18 and/or 24 months, or at about 25°C and about 60% humidity for periods such as 6, 9, Periods of 12, 18 and/or 24 months, or (for drug substances intended for storage in a freezer) at about -20°C for a period of, for example, 12 months. See eg FDA Guidance for Industry: Stability Testing of New Drug Substances and Products, November 2003.

就此而言，術語「穩定」意謂存在於調配物中之重組型AAV顆粒在儲存期間基本上保持其物理穩定性、化學穩定性及/或生物活性。在某些實施例中，醫藥調配物中存在之重組AAV顆粒在儲存期間，在-65℃下在人類個體中保持至少約80%其vg/ml(或至少約80%其感染性rAAV顆粒)持續測定時段，在其他實施例中在人類個體中之至少約85%、90%、95%、98%或99%其vg/ml，或可替代地感染性rAAV顆粒。In this regard, the term "stable" means that the recombinant AAV particles present in the formulation substantially retain their physical stability, chemical stability and/or biological activity during storage. In certain embodiments, the recombinant AAV particles present in the pharmaceutical formulation retain at least about 80% of their vg/ml (or at least about 80% of their infectious rAAV particles) in human subjects during storage at -65°C For the assay period, in other embodiments at least about 85%, 90%, 95%, 98% or 99% of their vg/ml, or alternatively infectious rAAV particles, in a human subject.

在某些實施例中，如實例2中所描述評估本文所描述之調配物之穩定性。在某些實施例中，如藉由實例2中所描述之分析所評估，當在2至8℃ (例如4℃)下儲存至少6個月(例如6至9個月、9至12個月或6至12個月)時，本文所描述之調配物係穩定的。在一些實施例中，如藉由實例2中所描述之分析所評估，當在≤-60℃ (例如-60℃、-65℃、-70℃、-75℃或-80℃)下儲存至少12個月(例如，12至18個月、12至24個月、18至36個月、24至48個月、36至48個月或12至48個月)時，本文所描述之調配物係穩定的。在某些實施例中，如藉由實例2中所描述之分析所評估，當在≤-60℃(例如-60℃、-65℃、-70℃、-75℃或-80℃)下儲存至少24個月(例如24至36個月、24至48個月或36至48個月)時，本文所描述之調配物係穩定的。In certain embodiments, the stability of the formulations described herein is assessed as described in Example 2. In certain embodiments, as assessed by the assay described in Example 2, when stored at 2 to 8°C (eg, 4°C) for at least 6 months (eg, 6 to 9 months, 9 to 12 months) or 6 to 12 months), the formulations described herein are stable. In some embodiments, as assessed by the assay described in Example 2, when stored at ≤ -60°C (eg, -60°C, -65°C, -70°C, -75°C, or -80°C) for at least At 12 months (eg, 12 to 18 months, 12 to 24 months, 18 to 36 months, 24 to 48 months, 36 to 48 months, or 12 to 48 months), the formulations described herein is stable. In certain embodiments, as assessed by the assay described in Example 2, when stored at ≤ -60°C (eg, -60°C, -65°C, -70°C, -75°C, or -80°C) The formulations described herein are stable for at least 24 months (eg, 24 to 36 months, 24 to 48 months, or 36 to 48 months).

在一些實施例中，如藉由量測重組AAV顆粒之脫醯胺含量(脫醯胺百分比)所評估，當在2至8℃ (例如4℃)下儲存至少6個月(例如6至9個月、9至12個月或6至12個月)時，包含本文所描述之重組AAV顆粒(例如重組AAV5顆粒)的本文所描述之調配物係穩定的。在某些實施例中，如藉由量測重組AAV顆粒之脫醯胺含量(脫醯胺百分比)所評估，當在≤-60℃ (例如-60℃、-65℃、-70℃、-75℃或-80℃)下儲存至少12個月(例如12至18個月、12至24個月、18至36個月、24至48個月、36至48個月或12至48個月)時，包含本文所描述之重組AAV顆粒(例如重組AAV5顆粒)的本文所描述之調配物係穩定的。在某些實施例中，如藉由量測重組AAV顆粒之脫醯胺含量(脫醯胺百分比)所評估，當在≤-60℃ (例如-60℃、-65℃、-70℃、-75℃或-80℃)下儲存至少24個月(例如24至36個月、24至48個月、36至48個月)時，包含本文所描述之重組AAV顆粒(例如重組AAV5顆粒)的本文所描述之調配物係穩定的。熟習此項技術者已知之標準技術可用於量測脫醯胺。In some embodiments, when stored at 2-8°C (eg, 4°C) for at least 6 months (eg, 6-9 Formulations described herein comprising recombinant AAV particles described herein (eg, recombinant AAV5 particles) are stable at 6 to 12 months, 9 to 12 months, or 6 to 12 months. In certain embodiments, as assessed by measuring the deamidation content (percent deamidation) of the recombinant AAV particles, when at ≤ -60°C (eg -60°C, -65°C, -70°C, - Store at 75°C or -80°C for at least 12 months (e.g. 12 to 18 months, 12 to 24 months, 18 to 36 months, 24 to 48 months, 36 to 48 months or 12 to 48 months ), formulations described herein comprising recombinant AAV particles described herein (eg, recombinant AAV5 particles) are stable. In certain embodiments, as assessed by measuring the deamidation content (percent deamidation) of the recombinant AAV particles, when at ≤ -60°C (eg -60°C, -65°C, -70°C, - When stored at 75°C or -80°C for at least 24 months (eg 24 to 36 months, 24 to 48 months, 36 to 48 months), a recombinant AAV particle (eg recombinant AAV5 particle) described herein The formulations described herein are stable. Standard techniques known to those skilled in the art can be used to measure the deamidation.

在某些實施例中，VP1蛋白在其N端處之脫醯胺含量藉由液相層析-質譜分析(LC-MS)定量。該分析精確量測AAV5病毒蛋白1(VP1)之N端區處之脫醯胺百分比，特定言之N50及N56處之脫醯胺百分比。調配之主體藥物物質或藥品中之衣殼顆粒經變性以解離病毒蛋白且在LC-MS分析之前消化成肽。藉由相對於未經修飾及脫醯胺肽峰面積之總和量測脫醯胺肽峰面積之強度來計算脫醯胺百分比。In certain embodiments, the deamidation content of the VP1 protein at its N-terminus is quantified by liquid chromatography-mass spectrometry (LC-MS). This assay precisely measures the percent deamidation at the N-terminal region of AAV5 viral protein 1 (VP1), specifically at N50 and N56. Capsid particles in the formulated host drug substance or drug product are denatured to dissociate viral proteins and digested into peptides prior to LC-MS analysis. The percent deamidation was calculated by measuring the intensity of the peak areas of the deamidated peptides relative to the sum of the unmodified and deamidated peptide peak areas.

在一些實施例中，如藉由量測聚集所評估，本文所描述之包含本文所描述之重組AAV顆粒(例如重組AAV5顆粒)的調配物在2至8℃ (例如4℃)下穩定至少6個月(例如6至9個月、9至12個月或6至12個月)。在某些實施例中，如藉由量測聚集所評估，當在≤-60℃ (例如-60℃、-65℃、-70℃、-75℃或-80℃)下儲存至少12個月(例如12至18個月、12至24個月、18至36個月、24至48個月、36至48個月或12至48個月)時，包含本文所描述之重組AAV顆粒(例如重組AAV5顆粒)的本文所描述之調配物係穩定的。在某些實施例中，如藉由量測聚集所評估，當在≤-60℃ (例如-60℃、-65℃、-70℃、-75℃或-80℃)下儲存至少24個月(例如24至36個月、24至48個月或36至48個月)時，包含本文所描述之重組AAV顆粒(例如重組AAV5顆粒)的本文所描述之調配物係穩定的。熟習此項技術者已知之標準技術可用於量測聚集，諸如衣殼蛋白聚集。In some embodiments, formulations described herein comprising recombinant AAV particles described herein (eg, recombinant AAV5 particles) are stable at 2 to 8°C (eg, 4°C) for at least 6, as assessed by measuring aggregation months (eg, 6 to 9 months, 9 to 12 months, or 6 to 12 months). In certain embodiments, when stored at ≤ -60°C (eg, -60°C, -65°C, -70°C, -75°C, or -80°C) for at least 12 months, as assessed by measuring aggregation (eg, 12 to 18 months, 12 to 24 months, 18 to 36 months, 24 to 48 months, 36 to 48 months, or 12 to 48 months), recombinant AAV particles described herein (eg, The formulations described herein of recombinant AAV5 particles) are stable. In certain embodiments, when stored at ≤ -60°C (eg, -60°C, -65°C, -70°C, -75°C, or -80°C) for at least 24 months, as assessed by measuring aggregation Formulations described herein comprising recombinant AAV particles described herein (eg, recombinant AAV5 particles) are stable at (eg, 24 to 36 months, 24 to 48 months, or 36 to 48 months). Standard techniques known to those skilled in the art can be used to measure aggregation, such as capsid protein aggregation.

在某些實施例中，衣殼蛋白聚集藉由尺寸排阻層析高效液相層析法(SEC-HPLC)監測。衣殼蛋白顆粒藉由UV在280 nm下監測且根據尺寸以三聚體、二聚體及單體之次序溶離。樣品緩衝液中之賦形劑及鹽，諸如泊洛沙姆，在單體峰之後溶離。聚集含量以多聚體%報告，其中多聚體%為二聚體及三聚體峰面積相對於總峰面積(單體、二聚體及三聚體)之總和。參考在每個分析下運行以確認分析效能。In certain embodiments, capsid protein aggregation is monitored by size exclusion chromatography high performance liquid chromatography (SEC-HPLC). Capsid protein particles were monitored by UV at 280 nm and eluted in the order of trimers, dimers and monomers according to size. Excipients and salts in the sample buffer, such as poloxamers, elute after the monomer peak. Aggregate content is reported as % multimer, where % multimer is the sum of the dimer and trimer peak areas relative to the total peak area (monomer, dimer, and trimer). Reference runs under each assay to confirm assay performance.

在一些實施例中，如藉由量測重組AAV顆粒之脫醯胺含量(脫醯胺百分比)及聚集所評估，當在2至8℃ (例如4℃)下儲存至少6個月(例如6至9個月、9至12個月或6至12個月)時，包含本文所描述之重組AAV顆粒(例如重組AAV5顆粒)的本文所描述之調配物係穩定的。在某些實施例中，如藉由量測重組AAV顆粒之脫醯胺含量(脫醯胺百分比)及聚集所評估，當在≤-60℃ (例如-60℃、-65℃、-70℃、-75℃或-80℃)下儲存至少12個月(例如12至18個月、12至24個月、18至36個月、24至48個月、36至48個月或12至48個月)時，包含本文所描述之重組AAV顆粒(例如重組AAV5顆粒)的本文所描述之調配物係穩定的。熟習此項技術者已知之標準技術可用於量測脫醯胺及聚集。在某些實施例中，衣殼蛋白聚集藉由尺寸排阻層析高效液相層析法(SEC-HPLC)監測且脫醯胺藉由LC-MS量測，諸如本文所描述。In some embodiments, when stored at 2 to 8° C. (eg, 4° C.) for at least 6 months (eg, 6 Formulations described herein comprising recombinant AAV particles described herein (eg, recombinant AAV5 particles) are stable by 9 months, 9 to 12 months, or 6 to 12 months). In certain embodiments, as assessed by measuring the deamidation content (percent deamidation) and aggregation of the recombinant AAV particles, when ≤ -60°C (eg -60°C, -65°C, -70°C) , -75°C or -80°C) for at least 12 months (e.g. 12 to 18 months, 12 to 24 months, 18 to 36 months, 24 to 48 months, 36 to 48 months or 12 to 48 months Months), formulations described herein comprising recombinant AAV particles described herein (eg, recombinant AAV5 particles) are stable. Standard techniques known to those skilled in the art can be used to measure deamidation and aggregation. In certain embodiments, capsid protein aggregation is monitored by size exclusion chromatography high performance liquid chromatography (SEC-HPLC) and deamidation is measured by LC-MS, such as described herein.

在某些態樣中，包含重組AAV顆粒之調配物進一步包含一或多種緩衝劑。In certain aspects, formulations comprising recombinant AAV particles further comprise one or more buffers.

在另一實施例中，本文所提供之重組AAV顆粒調配物可包含一或多種等張劑，諸如氯化鈉。此項技術中已知之其他緩衝劑及等張劑為適合的且可慣例地採用於本文所提供之調配物中。In another embodiment, the recombinant AAV particle formulations provided herein can include one or more isotonic agents, such as sodium chloride. Other buffers and isotonic agents known in the art are suitable and can be routinely employed in the formulations provided herein.

在另一實施例中，本文所提供之重組AAV顆粒調配物可包含一或多種增積劑，包括低溫保護劑。例示性增積劑包括(但不限於)甘露醇、蔗糖、聚葡萄糖、乳糖、海藻糖及聚維酮(PVP K24)。In another embodiment, the recombinant AAV particle formulations provided herein can include one or more bulking agents, including cryoprotectants. Exemplary bulking agents include, but are not limited to, mannitol, sucrose, polydextrose, lactose, trehalose, and povidone (PVP K24).

在又另一實施例中，本文所提供之重組AAV顆粒調配物可包含一或多種界面活性劑，其可為非離子界面活性劑。例示性界面活性劑包括離子界面活性劑、非離子界面活性劑及其組合。舉例而言，界面活性劑可為(但不限於)TWEEN 80(亦稱為聚山梨醇酯80，或其化學名稱聚氧乙烯脫水山梨糖醇單油酸酯)、十二烷基硫酸鈉、硬脂酸鈉、十二烷基硫酸銨、TRITON AG 98(Rhone-Poulenc)、泊洛沙姆407、泊洛沙姆188及其類似物，及其組合。In yet another embodiment, the recombinant AAV particle formulations provided herein can include one or more surfactants, which can be nonionic surfactants. Exemplary surfactants include ionic surfactants, nonionic surfactants, and combinations thereof. For example, the surfactant can be, but is not limited to, TWEEN 80 (also known as polysorbate 80, or its chemical name polyoxyethylene sorbitan monooleate), sodium lauryl sulfate, Sodium stearate, ammonium lauryl sulfate, TRITON AG 98 (Rhone-Poulenc), Poloxamer 407, Poloxamer 188, and analogs thereof, and combinations thereof.

本文所提供之重組AAV顆粒調配物係穩定的且可在無不可接受的品質、效能或純度變化的情況下儲存延長時段。在一個態樣中，調配物在約5℃ (例如2℃至8℃)之溫度下穩定至少1個月，例如至少1個月、至少3個月、至少6個月、至少12個月、至少18個月、至少24個月或更久。在另一實施例中，調配物在低於或等於約-20℃之溫度下穩定至少6個月，例如至少6個月、至少12個月、至少18個月、至少24個月、至少36個月或更久。在另一實施例中，調配物在低於或等於約-40℃之溫度下穩定至少6個月，例如至少6個月、至少12個月、至少18個月、至少24個月、至少36個月或更久。在另一實施例中，調配物在低於或等於約-60℃之溫度下穩定至少6個月，例如至少6個月、至少12個月、至少18個月、至少24個月、至少36個月或更久。在另一實施例中，調配物在-60℃、-65℃、-70℃、-75℃或-80℃、或-60℃至-80℃之溫度下穩定至少6個月，例如至少6個月、至少12個月、至少18個月、至少24個月、至少36個月或更久。在另一實施例中，調配物在-60℃、-65℃、-70℃、-75℃或-80℃、或-60℃至-80℃之溫度下穩定6至12個月、9至12個月、12至18個月、12至24個月、18至24個月、24至36個月、36至48個月或24至48個月。The recombinant AAV particle formulations provided herein are stable and can be stored for extended periods of time without unacceptable changes in quality, potency, or purity. In one aspect, the formulation is stable at a temperature of about 5°C (eg, 2°C to 8°C) for at least 1 month, eg, at least 1 month, at least 3 months, at least 6 months, at least 12 months, At least 18 months, at least 24 months or longer. In another embodiment, the formulation is stable at temperatures below or equal to about -20°C for at least 6 months, eg, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months months or more. In another embodiment, the formulation is stable at temperatures below or equal to about -40°C for at least 6 months, eg, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months months or more. In another embodiment, the formulation is stable at temperatures below or equal to about -60°C for at least 6 months, eg, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months months or more. In another embodiment, the formulation is stable at a temperature of -60°C, -65°C, -70°C, -75°C, or -80°C, or -60°C to -80°C for at least 6 months, eg, at least 6 months month, at least 12 months, at least 18 months, at least 24 months, at least 36 months or more. In another embodiment, the formulation is stable for 6 to 12 months, 9 to 12 months, 12 to 18 months, 12 to 24 months, 18 to 24 months, 24 to 36 months, 36 to 48 months, or 24 to 48 months.

醫藥組合物在製造及儲存條件下通常無菌且穩定。醫藥組合物可調配為溶液、微乳液、脂質體或適於容納高藥物濃度之其他有序結構。載劑可為含有例如水、乙醇、多元醇(例如，丙三醇、丙二醇及液體聚乙二醇及其類似物)及其適合混合物之溶劑或分散介質。適當流動性可以例如藉由使用諸如卵磷脂之包衣、藉由在分散液之情況下維持所需粒度及藉由使用界面活性劑來維持。在一些實施例中，等張劑，例如糖、多元醇(諸如甘露醇、山梨糖醇)或氯化鈉包括於組合物中。藉由使組合物中包括延遲吸收之藥劑(例如單硬脂酸鹽及明膠)，可得到可注射組合物之延長吸收。在某些實施例中，本文所提供之核酸或載體建構體可在時間或受控釋放調配物中，例如在包括防止化合物快速釋放之緩慢釋放聚合物或其他載劑之組合物(包括植入物及微囊封遞送系統)中投與。可例如使用生物可降解、生物相容性聚合物，諸如乙烯乙酸乙烯酯、聚酸酐、聚乙醇酸、膠原蛋白、聚原酸酯、聚乳酸及聚乳酸、聚乙醇酸共聚物(PLG)。Pharmaceutical compositions are generally sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for accommodating high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the desired particle size in the case of dispersions, and by the use of surfactants. In some embodiments, isotonic agents such as sugars, polyols (such as mannitol, sorbitol), or sodium chloride are included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, such as monostearate salts and gelatin. In certain embodiments, the nucleic acid or carrier constructs provided herein can be in time or controlled release formulations, such as in compositions including slow release polymers or other carriers that prevent rapid release of the compound (including implants) drug and microencapsulated delivery system). Biodegradable, biocompatible polymers can be used, for example, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic acid, polyglycolic acid copolymers (PLG).

在又另一態樣中，本發明提供醫藥組合物，其包含濃度為至少1E13 vg/ml，例如約1E13 vg/ml至約5E14 vg/ml、約2E13 vg/ml至約2E14 vg/ml、約1E13 vg/ml、約2E13 vg/ml、約6E13 vg/ml或約2E14 vg/ml之rAAV顆粒；緩衝劑；等張劑；冷凍保存劑；及界面活性劑，該醫藥組合物在約-60℃(攝氏零下六十度)或更低溫度下儲存期間穩定持續至少約1年、1.5年或2年。在一些實施例中，界面活性劑為濃度小於0.2% w/v，或小於0.15% w/v，例如約0.1% w/v之泊洛沙姆，或可替代地聚山梨醇酯。在一些實施例中，冷凍保存劑為糖，例如海藻糖。In yet another aspect, the present invention provides a pharmaceutical composition comprising a concentration of at least 1E13 vg/ml, such as about 1E13 vg/ml to about 5E14 vg/ml, about 2E13 vg/ml to about 2E14 vg/ml, rAAV particles of about 1E13 vg/ml, about 2E13 vg/ml, about 6E13 vg/ml, or about 2E14 vg/ml; buffers; isotonic agents; cryopreservatives; and surfactants, the pharmaceutical composition is at about- Stable for at least about 1 year, 1.5 years or 2 years during storage at 60°C (minus sixty degrees Celsius) or lower. In some embodiments, the surfactant is a poloxamer at a concentration of less than 0.2% w/v, or less than 0.15% w/v, eg, about 0.1% w/v, or alternatively a polysorbate. In some embodiments, the cryopreservative is a sugar, such as trehalose.

在一些實施例中，醫藥組合物為水性的且包含濃度為至少1E13 vg/ml，例如約1E13 vg/ml至約5E14 vg/ml、約2E13 vg/ml至約2E14 vg/ml、約1E13 vg/ml、約2E13 vg/ml、約6E13 vg/ml或約2E14 vg/ml之rAAV顆粒；濃度為約5至約15 mM之磷酸鈉；濃度為約100 mM至約165 mM之氯化鈉；為糖，視情況為海藻糖之冷凍保存劑；及濃度為小於0.2% w/v之泊洛沙姆。磷酸鈉可包含磷酸氫二鈉及/或磷酸二氫鈉。視情況，糖為濃度為約60 mM至約90 mM、或約60 mM至約80 mM、或約70 mM至約90 mM、或約70 mM至約80 mM之海藻糖。視情況，泊洛沙姆為濃度為約0.05% w/v至0.15% w/v之泊洛沙姆188。在某些實施例中，醫藥組合物包含實例2中所描述之調配物。In some embodiments, the pharmaceutical composition is aqueous and comprises a concentration of at least 1E13 vg/ml, eg, about 1E13 vg/ml to about 5E14 vg/ml, about 2E13 vg/ml to about 2E14 vg/ml, about 1E13 vg rAAV particles per ml, about 2E13 vg/ml, about 6E13 vg/ml, or about 2E14 vg/ml; sodium phosphate at a concentration of about 5 to about 15 mM; sodium chloride at a concentration of about 100 mM to about 165 mM; is a sugar, and optionally a cryopreservative for trehalose; and a poloxamer at a concentration of less than 0.2% w/v. The sodium phosphate may comprise disodium hydrogen phosphate and/or sodium dihydrogen phosphate. Optionally, the sugar is trehalose at a concentration of about 60 mM to about 90 mM, or about 60 mM to about 80 mM, or about 70 mM to about 90 mM, or about 70 mM to about 80 mM. A Poloxamer is Poloxamer 188 at a concentration of about 0.05% w/v to 0.15% w/v, as appropriate. In certain embodiments, the pharmaceutical composition comprises the formulation described in Example 2.

在一些實施例中，磷酸鈉之濃度為約5至約15 mM。在一些實施例中，氯化鈉之濃度為約100至約140 mM。在一些實施例中，糖(例如海藻糖)之濃度為約60至約90 mM。在一些實施例中，泊洛沙姆(例如泊洛沙姆188)之濃度為約0.05% w/v至約0.15% w/v。In some embodiments, the concentration of sodium phosphate is from about 5 to about 15 mM. In some embodiments, the concentration of sodium chloride is from about 100 to about 140 mM. In some embodiments, the concentration of the sugar (eg, trehalose) is from about 60 to about 90 mM. In some embodiments, the concentration of the poloxamer (eg, poloxamer 188) is from about 0.05% w/v to about 0.15% w/v.

在一些實施例中，磷酸鈉之濃度為約8至約12 mM。在一些實施例中，氯化鈉之濃度為約110至約130 mM。在一些實施例中，糖(例如海藻糖)之濃度為約70至約80 mM。在一些實施例中，泊洛沙姆(例如泊洛沙姆188)之濃度為約0.08% w/v至約0.12% w/v，或約0.1% w/v。In some embodiments, the concentration of sodium phosphate is from about 8 to about 12 mM. In some embodiments, the concentration of sodium chloride is from about 110 to about 130 mM. In some embodiments, the concentration of the sugar (eg, trehalose) is from about 70 to about 80 mM. In some embodiments, the concentration of the poloxamer (eg, poloxamer 188) is from about 0.08% w/v to about 0.12% w/v, or about 0.1% w/v.

在一些實施例中，磷酸二氫鈉(二水合物)之濃度為大於0.1 mg/mL且小於1 mg/mL，且該磷酸氫二鈉(十二水合物)之濃度為大於0.5 mg/ml且小於5 mg/ml。在一些實施例中，氯化鈉之濃度為大於5 mg/ml且小於10 mg/ml。在一些實施例中，糖為濃度大於20 mg/ml至小於40 mg/ml之海藻糖(二水合物)。在一些實施例中，泊洛沙姆188之濃度為約1.5 mg/ml或更小，或約1 mg/ml。In some embodiments, the concentration of sodium dihydrogen phosphate (dihydrate) is greater than 0.1 mg/mL and less than 1 mg/mL, and the concentration of disodium hydrogen phosphate (dodecahydrate) is greater than 0.5 mg/ml and less than 5 mg/ml. In some embodiments, the concentration of sodium chloride is greater than 5 mg/ml and less than 10 mg/ml. In some embodiments, the sugar is trehalose (dihydrate) at a concentration of greater than 20 mg/ml to less than 40 mg/ml. In some embodiments, the concentration of poloxamer 188 is about 1.5 mg/ml or less, or about 1 mg/ml.

在一些實施例中，磷酸二氫鈉(二水合物)之濃度為大於0.1 mg/mL且小於0.5 mg/mL，視情況約0.3至約0.4 mg/mL，且磷酸氫二鈉(十二水合物)之濃度為大於2.5 mg/ml且小於3 mg/ml，視情況約2.7 mg/ml。在一些實施例中，氯化鈉之濃度為大於5 mg/ml且小於8 mg/ml，視情況約7 mg/ml。在一些實施例中，糖為濃度大於20 mg/ml至小於40 mg/ml，或約25 mg/ml至約35 mg/ml，或約28 mg/ml之海藻糖(二水合物)。在一些實施例中，泊洛沙姆188之濃度為小於1.5 mg/ml或約1 mg/ml。In some embodiments, the concentration of sodium dihydrogen phosphate (dihydrate) is greater than 0.1 mg/mL and less than 0.5 mg/mL, optionally from about 0.3 to about 0.4 mg/mL, and disodium hydrogen phosphate (dodecahydrate) The concentration of the substance) is greater than 2.5 mg/ml and less than 3 mg/ml, depending on the situation, about 2.7 mg/ml. In some embodiments, the concentration of sodium chloride is greater than 5 mg/ml and less than 8 mg/ml, optionally about 7 mg/ml. In some embodiments, the sugar is trehalose (dihydrate) at a concentration of greater than 20 mg/ml to less than 40 mg/ml, or about 25 mg/ml to about 35 mg/ml, or about 28 mg/ml. In some embodiments, the concentration of poloxamer 188 is less than 1.5 mg/ml or about 1 mg/ml.

在一些實施例中，醫藥組合物包含濃度為約1E13 vg/ml至約5E14 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM海藻糖(二水合物)及0.1% w/v泊洛沙姆188。在一些實施例中，醫藥組合物包含濃度為約2E13 vg/ml至約2E14 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM海藻糖(二水合物)及0.1% w/v泊洛沙姆188。在一些實施例中，醫藥組合物包含濃度為約1E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM海藻糖(二水合物)及0.1% w/v泊洛沙姆188。在一些實施例中，醫藥組合物包含濃度為約2E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM海藻糖(二水合物)及0.1% w/v泊洛沙姆188。在一些實施例中，醫藥組合物包含濃度為約6E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM海藻糖(二水合物)及0.1% w/v泊洛沙姆188。在一些實施例中，醫藥組合物包含濃度為約2E14 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM海藻糖(二水合物)及0.1% w/v泊洛沙姆188。舉例而言，小瓶可包含8 mL水溶液，該水溶液包括4.8 E14 vg之rAAV顆粒、3.1 mg磷酸二氫鈉一元二水合物、21.6 mg磷酸氫二鈉十二水合物、56.1 mg氯化鈉、224 mg二水合海藻糖及8 mg泊洛沙姆188。In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 1E13 vg/ml to about 5E14 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v Poloxamer 188. In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 2E13 vg/ml to about 2E14 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v Poloxamer 188. In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 1E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxa Mu 188. In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 2E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxa Mu 188. In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 6E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxa Mu 188. In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration of about 2E14 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxa Mu 188. For example, a vial may contain 8 mL of an aqueous solution comprising 4.8 E14 vg of rAAV particles, 3.1 mg sodium dihydrogen phosphate monobasic dihydrate, 21.6 mg disodium hydrogen phosphate dodecahydrate, 56.1 mg sodium chloride, 224 mg mg trehalose dihydrate and 8 mg poloxamer 188.

在某些實施例中，本發明提供一種醫藥組合物，其包含單位劑量為480×10¹³ vg之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM海藻糖(二水合物)及0.1% w/v泊洛沙姆188。在一些實施例中，本發明提供一種醫藥組合物，其包含單位劑量為316×10¹³ vg之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM海藻糖(二水合物)及0.1% w/v泊洛沙姆188。在某些實施例中，本發明提供一種醫藥組合物，其包含單位劑量為約1.6×10¹⁶ vg之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM海藻糖(二水合物)及0.1% w/v泊洛沙姆188。在一些實施例中，本發明提供一種醫藥組合物，其包含單位劑量為約316×10¹³ vg至約1.6×10¹⁶ vg之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM海藻糖(二水合物)及0.1% w/v泊洛沙姆188。在某些實施例中，本發明提供一種醫藥組合物，其包含單位劑量為約250×10¹³ vg至約2×10¹⁶ vg之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM海藻糖(二水合物)及0.1% w/v泊洛沙姆188。In certain embodiments, the present invention provides a pharmaceutical composition comprising a unit dose of 480×10 ¹³ vg of rAAV particles, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v Poloxamer 188. In some embodiments, the present invention provides a pharmaceutical composition comprising a unit dose of 316 x 10 ¹³ vg of rAAV particles, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1 % w/v Poloxamer 188. In certain embodiments, the present invention provides a pharmaceutical composition comprising a unit dose of about 1.6 x 10 ¹⁶ vg of rAAV particles, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate) and 0.1% w/v Poloxamer 188. In some embodiments, the present invention provides a pharmaceutical composition comprising rAAV particles, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM seaweed at a ^{unit dose of about 316 x 10 13} vg to about 1.6 x 10 ^{16 vg} Sugar (dihydrate) and Poloxamer 188 at 0.1% w/v. In certain embodiments, the present invention provides a pharmaceutical composition comprising ^{rAAV particles at a unit dose of about 250 x 10 13} vg to about 2 x 10 ¹⁶ vg, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM Trehalose (dihydrate) and Poloxamer 188 at 0.1% w/v.

在具體實施例中，本文所描述之醫藥組合物包含重組AAV顆粒，其中該重組AAV顆粒包含AAV衣殼及重組載體建構體，其中該重組AAV顆粒包含編碼功能性***酸羥化酶(PAH)之核酸及視情況選用之異源肝特異性轉錄調節區。在另一具體實施例中，重組AAV載體，且其中重組AAV載體包含：(a)以下中之一或兩者：(i) AAV 5'反向末端重複序列(ITR)及(ii)AAV 3' ITR；(b)異源肝特異性轉錄調節區；及(c)編碼功能性人類***酸羥化酶(hPAH)之核酸，視情況其中該等AAV ITR為AAV2 ITR。在一個實施例中，編碼功能性hPAH之核酸編碼與SEQ ID NO: 2至少95%一致之胺基酸序列。在另一實施例中，編碼功能性hPAH之核酸包含與SEQ ID NO: 1或7至13至少90%一致之核苷酸序列。在某些實施例中，編碼PAH之核酸可操作地連接至包含hAAT啟動子之片段的啟動子及HCR增強子/ApoE增強子之片段。在一些實施例中，肝特異性轉錄調節區包含與SEQ ID NO: 3、4或24中之任一者至少90%一致或可替代地與SEQ ID NO: 25或26中之任一者至少90%一致的核苷酸序列。在具體實施例中，重組載體建構體包含與SEQ ID NO: 6至少90%一致之核苷酸序列。在某些實施例中，重組載體建構體進一步包含內含子。在一些實施例中，內含子包含與SEQ ID NO: 14或27或29或34至少90%一致之核苷酸序列。在某些實施例中，重組載體建構體進一步包含多腺苷酸化信號。在一些實施例中，重組載體建構體包含牛生長激素(bGH)多腺苷酸化信號。在具體實施例中，重組AAV顆粒包含與SEQ NO: 15至23或52中之任一者至少90%一致的重組載體建構體。在另一具體實施例中，重組AAV顆粒包含與SEQ NO: 15至23或52中之任一者至少95%一致的重組載體建構體。在另一具體實施例中，重組AAV顆粒包含包括SEQ NO: 15至23或52中之任一者之核苷酸序列的重組載體建構體。在另一具體實施例中，AAV衣殼包含與SEQ ID NO: 35至51中之任一者至少85%一致之胺基酸序列。在另一具體實施例中，AAV衣殼包含與SEQ ID NO: 35至51中之任一者至少90%一致之胺基酸序列。在另一具體實施例中，AAV衣殼包含與SEQ ID NO: 35至51中之任一者至少95%一致之胺基酸序列。在另一具體實施例中，AAV衣殼為具有肝趨向性之AAV衣殼。在某些實施例中，具有肝趨向性之AAV衣殼不包括AAV8及/或AAVHSC15。在一些實施例中，具有肝趨向性之AAV衣殼為AAV5型衣殼，視情況與SEQ ID NO: 44至少85%、90%或95%一致。在具體實施例中，具有肝趨向性之AAV衣殼為AAV5型衣殼，其中該AAV5型衣殼包含SEQ ID NO: 44之序列。In specific embodiments, the pharmaceutical compositions described herein comprise a recombinant AAV particle, wherein the recombinant AAV particle comprises an AAV capsid and a recombinant vector construct, wherein the recombinant AAV particle comprises encoding a functional phenylalanine hydroxylase (PAH) nucleic acid and optionally a heterologous liver-specific transcriptional regulatory region. In another specific embodiment, the recombinant AAV vector, and wherein the recombinant AAV vector comprises: (a) one or both of the following: (i) AAV 5' inverted terminal repeat (ITR) and (ii) AAV 3 ' ITR; (b) a heterologous liver-specific transcriptional regulatory region; and (c) a nucleic acid encoding a functional human phenylalanine hydroxylase (hPAH), optionally wherein the AAV ITRs are AAV2 ITRs. In one embodiment, the nucleic acid encoding a functional hPAH encodes an amino acid sequence that is at least 95% identical to SEQ ID NO:2. In another embodiment, the nucleic acid encoding a functional hPAH comprises a nucleotide sequence that is at least 90% identical to SEQ ID NOs: 1 or 7-13. In certain embodiments, the nucleic acid encoding the PAH is operably linked to a promoter comprising a fragment of the hAAT promoter and a fragment of the HCR enhancer/ApoE enhancer. In some embodiments, the liver-specific transcriptional regulatory region comprises at least 90% identical to any of SEQ ID NO: 3, 4 or 24 or alternatively at least any of SEQ ID NO: 25 or 26 90% identical nucleotide sequences. In specific embodiments, the recombinant vector construct comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:6. In certain embodiments, the recombinant vector construct further comprises an intron. In some embodiments, the intron comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14 or 27 or 29 or 34. In certain embodiments, the recombinant vector construct further comprises a polyadenylation signal. In some embodiments, the recombinant vector construct comprises a bovine growth hormone (bGH) polyadenylation signal. In specific embodiments, the recombinant AAV particle comprises a recombinant vector construct that is at least 90% identical to any one of SEQ NOs: 15-23 or 52. In another specific embodiment, the recombinant AAV particle comprises a recombinant vector construct that is at least 95% identical to any one of SEQ NOs: 15-23 or 52. In another specific embodiment, the recombinant AAV particle comprises a recombinant vector construct comprising the nucleotide sequence of any one of SEQ NOs: 15 to 23 or 52. In another specific embodiment, the AAV capsid comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 35-51. In another specific embodiment, the AAV capsid comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 35-51. In another specific embodiment, the AAV capsid comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 35-51. In another specific embodiment, the AAV capsid is an AAV capsid with liver tropism. In certain embodiments, the AAV capsid with liver tropism does not include AAV8 and/or AAVHSC15. In some embodiments, the AAV capsid with liver tropism is an AAV5-type capsid that is at least 85%, 90%, or 95% identical to SEQ ID NO: 44, as appropriate. In a specific embodiment, the AAV capsid having liver tropism is an AAV5-type capsid, wherein the AAV5-type capsid comprises the sequence of SEQ ID NO:44.

在具體實施例中，本文所描述之醫藥組合物包含重組AAV顆粒，其中該重組AAV顆粒包含AAV5衣殼及重組載體建構體，其中該重組載體建構體包含與SEQ ID NO: 18至少90%一致之核苷酸序列。在另一具體實施例中，本文所描述之醫藥組合物包含重組AAV顆粒，其中該等重組AAV顆粒包含AAV5衣殼及重組載體建構體，其中該重組載體建構體包含與SEQ ID NO: 18至少95%一致之核苷酸序列。在另一具體實施例中，本文所描述之醫藥組合物包含重組AAV顆粒，其中該重組AAV顆粒包含AAV5衣殼及重組載體建構體，其中該重組載體建構體包含包括SEQ ID NO: 18之核苷酸序列的重組載體建構體。在一些實施例中，AAV5衣殼與SEQ ID NO: 44至少85%、90%或95%一致。在具體實施例中，AAV5衣殼包含SEQ ID NO: 44之序列。In specific embodiments, the pharmaceutical compositions described herein comprise recombinant AAV particles, wherein the recombinant AAV particles comprise an AAV5 capsid and a recombinant vector construct, wherein the recombinant vector construct comprises at least 90% identity to SEQ ID NO: 18 the nucleotide sequence. In another specific embodiment, the pharmaceutical compositions described herein comprise recombinant AAV particles, wherein the recombinant AAV particles comprise an AAV5 capsid and a recombinant vector construct, wherein the recombinant vector construct comprises at least SEQ ID NO: 18 95% identical nucleotide sequences. In another specific embodiment, the pharmaceutical composition described herein comprises a recombinant AAV particle, wherein the recombinant AAV particle comprises an AAV5 capsid and a recombinant vector construct, wherein the recombinant vector construct comprises a core comprising SEQ ID NO: 18 Recombinant vector constructs of nucleotide sequences. In some embodiments, the AAV5 capsid is at least 85%, 90%, or 95% identical to SEQ ID NO:44. In a specific embodiment, the AAV5 capsid comprises the sequence of SEQ ID NO:44.

在具體實施例中，本文所描述之醫藥組合物包含重組AAV顆粒，其中該重組AAV顆粒包含AAV5衣殼及重組載體建構體，其中該重組載體建構體包含與SEQ ID NO: 52至少90%一致之核苷酸序列。在另一具體實施例中，本文所描述之醫藥組合物包含重組AAV顆粒，其中該等重組AAV顆粒包含AAV5衣殼及重組載體建構體，其中該重組載體建構體包含與SEQ ID NO: 52至少95%一致之核苷酸序列。在另一具體實施例中，本文所描述之醫藥組合物包含重組AAV顆粒，其中該重組AAV顆粒包含AAV5衣殼及重組載體建構體，其中該重組載體建構體包含SEQ ID NO: 52之核苷酸序列。在一些實施例中，AAV5衣殼與SEQ ID NO: 44至少85%、90%或95%一致。在具體實施例中，AAV5衣殼包含SEQ ID NO: 44之序列。In specific embodiments, the pharmaceutical compositions described herein comprise recombinant AAV particles, wherein the recombinant AAV particles comprise an AAV5 capsid and a recombinant vector construct, wherein the recombinant vector construct comprises at least 90% identity to SEQ ID NO: 52 the nucleotide sequence. In another specific embodiment, the pharmaceutical compositions described herein comprise recombinant AAV particles, wherein the recombinant AAV particles comprise an AAV5 capsid and a recombinant vector construct, wherein the recombinant vector construct comprises at least the SEQ ID NO: 52 95% identical nucleotide sequences. In another specific embodiment, the pharmaceutical composition described herein comprises a recombinant AAV particle, wherein the recombinant AAV particle comprises an AAV5 capsid and a recombinant vector construct, wherein the recombinant vector construct comprises the nucleosides of SEQ ID NO: 52 acid sequence. In some embodiments, the AAV5 capsid is at least 85%, 90%, or 95% identical to SEQ ID NO:44. In a specific embodiment, the AAV5 capsid comprises the sequence of SEQ ID NO:44.

在具體實施例中，本文所描述之醫藥組合物包含本文所描述之重組AAV顆粒。在具體實施例中，本文所描述之醫藥組合物係用於向個體靜脈內投與。在具體實施例中，本文所描述之醫藥組合物係用於輸注至個體中。In specific embodiments, the pharmaceutical compositions described herein comprise recombinant AAV particles described herein. In specific embodiments, the pharmaceutical compositions described herein are for intravenous administration to a subject. In specific embodiments, the pharmaceutical compositions described herein are used for infusion into an individual.

在具體實施例中，本文所描述之醫藥組合物係用於本文所描述之方法(例如部分5、實例3或實例4)。在某些實施例中，本文所描述之醫藥組合物係用於減少有需要之人類個體中之血漿***酸(Phe)含量的方法。在一些實施例中，本文所描述之醫藥組合物係用於治療患有苯酮尿症之人類個體的方法。關於可投與本文所描述之醫藥組合物之個體，參見例如部分5.5及實例3及4。In specific embodiments, the pharmaceutical compositions described herein are used in the methods described herein (eg, Section 5, Example 3, or Example 4). In certain embodiments, the pharmaceutical compositions described herein are methods for reducing plasma phenylalanine (Phe) levels in a human subject in need thereof. In some embodiments, the pharmaceutical compositions described herein are used in a method of treating a human subject suffering from phenylketonuria. See, eg, Section 5.5 and Examples 3 and 4 for individuals to which the pharmaceutical compositions described herein can be administered.

在具體實施例中，醫藥組合物包含實例2中之調配物。 5.5 治療方法In a specific embodiment, the pharmaceutical composition comprises the formulation of Example 2. 5.5 Treatment methods

在實施例中之任一者中，個體患有苯酮尿症(PKU)，視情況為典型PKU或重度PKU。在一些實施例中，在該投與之前該個體之血漿Phe含量為600 μmol/L或更高，或700、800、900、1000或1000 μmol/L或更高。在一些實施例中，在該投與之前該個體之血漿Phe含量為1200 μmol/L或更高。在某些實施例中，待治療之個體將根據實例3或實例4中所描述之方案治療。In any of the embodiments, the individual suffers from phenylketonuria (PKU), classic PKU or severe PKU, as the case may be. In some embodiments, the subject's plasma Phe level prior to the administration is 600 μmol/L or higher, or 700, 800, 900, 1000 or 1000 μmol/L or higher. In some embodiments, the subject has plasma Phe levels of 1200 μmol/L or greater prior to the administration. In certain embodiments, the individual to be treated will be treated according to the regimen described in Example 3 or Example 4.

在一些實施例中，個體為小於2歲之嬰兒。在一些實施例中，個體為以下年齡之人類，舉例而言In some embodiments, the individual is an infant less than 2 years old. In some embodiments, the individual is a human of the following ages, for example

(1) 15歲至18歲、12歲至＜15歲、5-歲＜12歲或0(出生)歲至＜5歲(1) 15 to 18 years old, 12 years old to <15 years old, 5-year olds <12 years old or 0 (birth) years old to <5 years old

(2) 15歲至18歲、12歲至＜15歲、9歲至＜12歲、5歲至9歲、2歲至＜5歲或0歲至＜2歲(2) 15 years old to 18 years old, 12 years old to <15 years old, 9 years old to <12 years old, 5 years old to 9 years old, 2 years old to <5 years old or 0 years old to <2 years old

(3) 12歲至＜18歲、6歲至＜12歲、0歲至＜6歲(3) 12 years old to <18 years old, 6 years old to <12 years old, 0 years old to <6 years old

(4) 15歲至＜18歲、12歲至＜15歲、9歲至＜12歲、6歲至＜9歲、3歲至＜6歲、0歲至＜3歲。(4) 15 years old to <18 years old, 12 years old to <15 years old, 9 years old to <12 years old, 6 years old to <9 years old, 3 years old to <6 years old, 0 years old to <3 years old.

在一些實施例中，個體為人類，其年齡為：In some embodiments, the individual is a human whose age is:

(a) 12歲至＜15歲。(a) 12 years old to <15 years old.

(b)5歲至＜12歲；或9歲至＜12歲；或5歲至＜9歲(b) 5 years old to <12 years old; or 9 years old to <12 years old; or 5 years old to <9 years old

(c) 0歲至＜5歲；或2歲至＜5歲或0歲至＜2歲。(c) 0 years old to <5 years old; or 2 years old to <5 years old or 0 years old to <2 years old.

在一些實施例中，個體為15歲或更大或18歲或更大。在一些實施例中，個體為成人。在一些實施例中，個體為男性。在一些實施例中，個體為女性，例如非妊娠女性。In some embodiments, the individual is 15 years old or older or 18 years old or older. In some embodiments, the individual is an adult. In some embodiments, the individual is male. In some embodiments, the individual is female, eg, a non-pregnant female.

在實施例中之任一者中，該個體在編碼PAH之內在基因中具有突變，視情況為突變F39L、L48S、I65T、R68S、A104D、S110C、D129G、E178G、V190A、P211T、R241C、R261Q、A300S、L308F、A313T、K320N、A373T、V388M E390G、A395P、P407S及Y414C。In any of the embodiments, the individual has a mutation in a gene encoding a PAH, optionally a mutation F39L, L48S, I65T, R68S, A104D, S110C, D129G, E178G, V190A, P211T, R241C, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G, A395P, P407S and Y414C.

在一些實施例中，當投與rAAV顆粒時個體未接受治療PKU之藥物療法。舉例而言，個體在該投與前至少30天未接受培格瓦力，及/或個體在該投與前至少30天未接受大型中性胺基酸(LNAA)，及/或個體在該投與前至少7天未接受沙丙蝶呤。在一些實施例中，個體在該投與之前至少30天未接受類固醇。In some embodiments, the individual is not receiving drug therapy to treat PKU when the rAAV particles are administered. For example, the subject has not received pegvar for at least 30 days prior to the administration, and/or the subject has not received a large neutral amino acid (LNAA) for at least 30 days prior to the administration, and/or the subject has not received a large neutral amino acid (LNAA) for at least 30 days prior to the administration No sapropterin was received for at least 7 days prior to administration. In some embodiments, the individual has not received steroids for at least 30 days prior to the administration.

在一些實施例中，個體在投與rAAV顆粒時在血液中不具有可檢測之抗AAV衣殼抗體(例如並非AAV5血清反應呈陽性)。抗AAV中和抗體為不合需要的，因為其可阻斷細胞轉導或以其他方式降低治療之整體效率。In some embodiments, the individual does not have detectable anti-AAV capsid antibodies in blood (eg, is not seropositive for AAV5) when the rAAV particles are administered. Anti-AAV neutralizing antibodies are undesirable because they can block cell transduction or otherwise reduce the overall efficiency of the treatment.

在某些實施例中，在向個體投與本文所描述之rAAV顆粒之前，個體在來自個體之血液樣品中不具有任何可檢測之抗AAV衣殼抗體(例如抗AAV-5衣殼抗體)。在一些實施例中，在向個體投與本文所描述之rAAV顆粒之後，個體在來自個體之血液樣品中不具有任何可檢測之抗AAV衣殼抗體(例如抗AAV-5衣殼抗體)。在某些實施例中，在向個體投與本文所描述之rAAV顆粒之前及之後，個體在來自個體之血液樣品中不具有任何可檢測之抗AAV衣殼抗體(例如抗AAV-5衣殼抗體)。In certain embodiments, the individual does not have any detectable anti-AAV capsid antibodies (eg, anti-AAV-5 capsid antibodies) in a blood sample from the individual prior to administration of the rAAV particles described herein to the individual. In some embodiments, after administration of the rAAV particles described herein to the individual, the individual does not have any detectable anti-AAV capsid antibodies (eg, anti-AAV-5 capsid antibodies) in a blood sample from the individual. In certain embodiments, the individual does not have any detectable anti-AAV capsid antibodies (eg, anti-AAV-5 capsid antibodies) in a blood sample from the individual before and after administration of the rAAV particles described herein to the individual ).

在某些實施例中，在向個體投與rAAV顆粒之前，個體在來自個體之血液樣品中不具有任何可檢測之抗PAH抗體。在一些實施例中，在向個體投與rAAV顆粒之後，個體在來自個體之血液樣品中不具有任何可檢測之抗PAH抗體。在某些實施例中，在向個體投與rAAV顆粒之前及之後，個體在來自個體之血液樣品中不具有任何可檢測之抗PAH抗體。In certain embodiments, the individual does not have any detectable anti-PAH antibodies in a blood sample from the individual prior to administration of the rAAV particles to the individual. In some embodiments, after administration of rAAV particles to the individual, the individual does not have any detectable anti-PAH antibodies in a blood sample from the individual. In certain embodiments, the individual does not have any detectable anti-PAH antibodies in a blood sample from the individual before and after administration of the rAAV particles to the individual.

在某些實施例中，在向個體投與本文所描述之rAAV顆粒之前，個體在來自個體之血液樣品中不具有任何可檢測之抗AAV衣殼抗體(例如抗AAV-5衣殼抗體)，且在向個體投與rAAV顆粒之前，個體在來自個體之血液樣品中不具有任何可檢測之抗PAH抗體。在一些實施例中，在向個體投與本文所描述之rAAV顆粒之後，個體在來自個體之血液樣品中不具有任何可檢測之抗AAV衣殼抗體(例如抗AAV-5衣殼抗體)，且在向個體投與rAAV顆粒之後，個體在來自個體之血液樣品中不具有任何可檢測之抗PAH抗體。在某些實施例中，在向個體投與本文所描述之rAAV顆粒之前及之後，個體在來自個體之血液樣品中不具有任何可檢測之抗AAV衣殼抗體(例如抗AAV-5衣殼抗體)，且在向個體投與rAAV顆粒之前及之後，個體在來自個體之血液樣品中不具有任何可檢測之抗PAH抗體。In certain embodiments, the individual does not have any detectable anti-AAV capsid antibodies (eg, anti-AAV-5 capsid antibodies) in a blood sample from the individual prior to administration of the rAAV particles described herein to the individual, And prior to administration of the rAAV particles to the individual, the individual did not have any detectable anti-PAH antibodies in the blood sample from the individual. In some embodiments, the individual does not have any detectable anti-AAV capsid antibodies (eg, anti-AAV-5 capsid antibodies) in a blood sample from the individual following administration of the rAAV particles described herein to the individual, and Following administration of rAAV particles to the subject, the subject does not have any detectable anti-PAH antibodies in the blood sample from the subject. In certain embodiments, the individual does not have any detectable anti-AAV capsid antibodies (eg, anti-AAV-5 capsid antibodies) in a blood sample from the individual before and after administration of the rAAV particles described herein to the individual ), and the individual did not have any detectable anti-PAH antibodies in the blood sample from the individual before and after administration of the rAAV particles to the individual.

在一些實施例中，個體不具有(1)活性感染或免疫抑制病症之證據；(2)癌症病史；(3)物質使用病症、重度抑鬱症、精神病或躁鬱症；或(4)對皮質類固醇之禁忌。在一些實施例中，個體先前未感染B型或C型肝炎或肺結核。在一些實施例中，個體不具有大於或等於1.5 mg/dL之血清肌酐。In some embodiments, the individual has no evidence of (1) an active infection or an immunosuppressive disorder; (2) a history of cancer; (3) a substance use disorder, major depressive disorder, psychosis, or bipolar disorder; or (4) resistance to corticosteroids taboo. In some embodiments, the individual is not previously infected with hepatitis B or C or tuberculosis. In some embodiments, the individual does not have a serum creatinine greater than or equal to 1.5 mg/dL.

在一些實施例中，個體在該投與之前不具有臨床上顯著之肝病。在一些實施例中，個體不具有展示如按0至4之等級評級之3或4之顯著纖維化的先前肝切片。在一些實施例中，個體之丙胺酸轉胺酶(ALT)、天冬胺酸轉胺酶(AST)、γ-麩胺醯轉化酶(GGT)或膽紅素中之任一者的升高不超過正常值上限(ULN) 1.25倍，或其國際標準化比率等於或大於1.2。In some embodiments, the subject does not have clinically significant liver disease prior to the administration. In some embodiments, the individual does not have a previous liver section that exhibits significant fibrosis as 3 or 4 on a scale of 0 to 4. In some embodiments, the individual has an increase in any one of alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamine converting enzyme (GGT), or bilirubin Not more than 1.25 times the upper limit of normal (ULN), or its international normalized ratio equal to or greater than 1.2.

在某些實施例中，個體實現實例3或實例4中之納入標準中之一者、兩者、三者或更多者。在一些實施例中，個體不滿足實例3或實例4中根據排除標準列舉之準則中之一者、兩者、三者或更多者。在某些實施例中，個體實現實例3或實例4中之納入標準中之一者、兩者、三者或更多者，且個體不滿足實例3或實例4中之根據排除標準列舉之準則中之一者、兩者、三者或更多者。In certain embodiments, the individual implements one, both, three or more of the inclusion criteria in Example 3 or Example 4. In some embodiments, the individual does not meet one, both, three or more of the criteria recited in Example 3 or Example 4 according to the exclusion criteria. In certain embodiments, the individual fulfills one, both, three or more of the inclusion criteria in Example 3 or Example 4, and the individual does not meet the criteria listed in Example 3 or Example 4 according to the exclusion criteria one, two, three or more of them.

在一些實施例中，個體滿足實例3或實例4中根據排除標準列舉之標準中之一者、兩者、三者或更多者。在某些實施例中，個體實現實例3或實例4中之納入標準中之一者、兩者、三者或更多者，且個體滿足實例3或實例4中之根據排除標準列舉之準則中之一者、兩者、三者或更多者。In some embodiments, the individual meets one, two, three, or more of the criteria listed in Example 3 or Example 4 according to the exclusion criteria. In certain embodiments, the individual fulfills one, both, three or more of the inclusion criteria in Example 3 or Example 4, and the individual meets the criteria listed in Example 3 or Example 4 according to the exclusion criteria one, two, three or more.

在輸注rAAV顆粒之前，對個體評估以下各者：(1)基線物理檢驗；(2)基線臨床實驗室測試，其包括(a)血漿Phe含量、(b)血漿酪胺酸(Tyr)含量及(c)肝酶測試，其包括ALT、AST、GGT及膽紅素；(d)及基線AAV5抗體檢測；(3)自完整食物及自醫療食品攝入之基線蛋白；(4)量測注意力不集中及/或執行功能，例如，注意力不足過動症等級量表(ADHD-RS IV)，其為調查員分級之注意力不集中得分，劍橋神經心理學測試自動電池(CANTAB)得分(包括快速視覺處理、停止信號及空間工作記憶)；(5)量測健康相關生活品質(HRQoL)，例如，苯酮尿症影響及治療生活品質調查表(PKU-QOL)得分或生活品質享受及滿意調查表(Q-LES-Q-SF)得分；(6)在研究期間監測之其他參數的基線含量；及(7)PAH基因分型，若允許。在某些實施例中，在輸注rAAV顆粒之前，如實例3或實例4中所描述評估個體。Prior to infusion of rAAV particles, subjects were assessed for (1) baseline physical tests; (2) baseline clinical laboratory tests including (a) plasma Phe levels, (b) plasma tyrosine (Tyr) levels and (c) Liver enzyme tests, including ALT, AST, GGT, and bilirubin; (d) and baseline AAV5 antibody tests; (3) baseline protein intake from whole foods and from medical foods; (4) measurement cautions Inattention and/or executive functioning, e.g., Attention Deficit Hyperactivity Disorder Rating Scale (ADHD-RS IV), which is an investigator-rated inattention score, Cambridge Neuropsychological Test Automated Battery (CANTAB) score (including rapid visual processing, stop signals, and spatial working memory); (5) measure health-related quality of life (HRQoL), e.g., phenylketonuria impact and treatment quality of life questionnaire (PKU-QOL) score or quality of life enjoyment and Satisfaction Questionnaire (Q-LES-Q-SF) scores; (6) baseline levels of other parameters monitored during the study; and (7) PAH genotyping, if allowed. In certain embodiments, the subject is assessed as described in Example 3 or Example 4 prior to infusion of rAAV particles.

在本發明之方法中，rAAV顆粒以單次給藥經靜脈內投與。在一些實施例中，載體建構體或重組AAV顆粒藉由靜脈內注射以單次推注形式或經延長時段投與，該延長時段可為至少約1、5、10、15、30、45、60、75、90、120、150、180、210或240分鐘或更久。In the methods of the present invention, the rAAV particles are administered intravenously in a single dose. In some embodiments, the vector construct or recombinant AAV particle is administered by intravenous injection as a single bolus injection or over an extended period of time, which may be at least about 1, 5, 10, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210 or 240 minutes or more.

在一些實施例中，rAAV顆粒以在每公斤個體體重約1E13至約5E14載體基因體(vg/kg)，2E13至約2E14 (vg/kg)範圍內之劑量，例如約1E13 vg/kg之劑量、或約2E13 vg/kg之劑量、或約6E13 vg/kg之劑量、或約2E14 vg/kg之劑量投與。在某些實施例中，rAAV顆粒以實例3及4中指定之劑量投與。在一些實施例中，投與rAAV顆粒之個體為約80 kg。在其他實施例中，投與rAAV顆粒之個體為約60至85 kg(例如60、65、70、75、80或85 kg)。In some embodiments, the rAAV particles are in a dose ranging from about 1E13 to about 5E14 vector gene body (vg/kg), 2E13 to about 2E14 (vg/kg) per kilogram of body weight of the subject, eg, a dose of about 1E13 vg/kg , or a dose of about 2E13 vg/kg, or a dose of about 6E13 vg/kg, or a dose of about 2E14 vg/kg was administered. In certain embodiments, the rAAV particles are administered at the doses specified in Examples 3 and 4. In some embodiments, the subject to which the rAAV particles are administered is about 80 kg. In other embodiments, the subject to which the rAAV particles are administered is about 60 to 85 kg (eg, 60, 65, 70, 75, 80, or 85 kg).

在一些實施例中，rAAV顆粒以在約200E13至約2E16載體基因體或316E13至約1.6E16載體基因體範圍內之單位劑量，例如約316E13 vg之單位劑量，或約480E13 vg之單位劑量，或約1.6E16 vg之單位劑量投與。In some embodiments, the rAAV particles are in a unit dose in the range of about 200E13 to about 2E16 vector genomes or 316E13 to about 1.6E16 vector genomes, such as a unit dose of about 316E13 vg, or a unit dose of about 480E13 vg, or A unit dose of about 1.6E16 vg is administered.

在輸注rAAV顆粒之後，方法可進一步包含監測不同參數之步驟，例如每週量測參數。量測可替代地每1、2、3、4、5或6天或每週或每兩週或每三週或每月進行。參數可經由第24週、第48週、第96週或更久而監測。方法可包括量測個體之血漿Phe含量。舉例而言，量測血漿Phe含量，且在第8週、第12週及第24週觀測到平均血漿Phe含量自基線之減少。視情況，方法包括對個體進行Phe挑戰測試或Phe呼吸測試。觀測到Phe活性之提高，如藉由Phe呼吸測試上Phe氧化之提高速率所量測。Following infusion of the rAAV particles, the method may further comprise the step of monitoring various parameters, eg, measuring the parameters weekly. Measurements may alternatively be taken every 1, 2, 3, 4, 5 or 6 days or every week or every two weeks or every three weeks or monthly. Parameters can be monitored via Week 24, Week 48, Week 96 or more. The method may comprise measuring the plasma Phe level of the subject. For example, plasma Phe levels were measured and reductions in mean plasma Phe levels from baseline were observed at weeks 8, 12, and 24. Methods include subjecting the individual to a Phe challenge test or a Phe breath test, as appropriate. An increase in Phe activity was observed, as measured by the increased rate of Phe oxidation on the Phe breath test.

方法亦可進一步包含量測個體之一或多種神經傳遞質或神經傳遞質代謝物之血漿含量的步驟。可量測酪胺酸(Tyr)含量，且可計算Phe/Tyr比率。舉例而言，該一或多種神經傳遞質或神經傳遞質代謝物為苯乙胺、苯乙醇胺、酪胺、多巴胺、正腎上腺素、腎上腺素、色胺、羥基色胺、苯乙酸、苯乙醯基麩醯胺酸、杏仁酸、羥基苯乙酸、DOPAC、高香草酸、DOMA、MOPEG、香草基杏仁酸、吲哚乙酸及5-羥基吲哚乙酸。在一個實例中，監測苯基乙醯基麩醯胺酸[PAG]、高香草酸[HVA]、3-甲氧基-4-羥苯基二醇[MOPEG]及5-羥基吲哚乙酸[5HIAA]。The method may also further comprise the step of measuring the plasma level of one or more neurotransmitters or neurotransmitter metabolites in the subject. Tyrosine (Tyr) content can be measured and the Phe/Tyr ratio can be calculated. For example, the one or more neurotransmitters or neurotransmitter metabolites are phenylethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid, phenylacetylene glutamic acid, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillyl mandelic acid, indoleacetic acid and 5-hydroxyindoleacetic acid. In one example, phenylacetoxyglutamic acid [PAG], homovanillic acid [HVA], 3-methoxy-4-hydroxyphenyldiol [MOPEG], and 5-hydroxyindoleacetic acid [ 5HIAA].

方法可進一步包含監測耐受膳食蛋白質攝入增加及/或醫療食物攝入減少(Phe減少或無Phe食物)之能力的步驟。舉例而言，在根據本文所描述之方法投與rAAV顆粒之後，在第48週輸注後個體可以消耗來自完整食物之至少0.8 g/kg蛋白質攝入，同時維持血漿Phe小於或等於360 μmol/L，及/或個體可以不消耗醫療食品。Phe攝入之此類改善可例如在第24週、第48週或第96週發生。The method may further comprise the step of monitoring the ability to tolerate increased dietary protein intake and/or decreased medical food intake (Phe reduced or Phe free food). For example, following administration of rAAV particles according to the methods described herein, an individual can consume at least 0.8 g/kg protein intake from intact food at week 48 post infusion, while maintaining plasma Phe less than or equal to 360 μmol/L , and/or the individual may not consume the medical food. Such improvement in Phe intake can occur, for example, at week 24, week 48 or week 96.

方法可進一步包含監測注意力不集中之症狀及量測執行功能之步驟，例如如藉由ADHD-RS IV(調查員分級之注意力不集中得分)、CANTAB得分(快速視覺處理、停止信號及空間工作記憶)所量測，或健康相關生活品質(HRQoL)，例如如藉由PKU-QOL得分或Q-LES-Q-SF得分所量測。例如藉由第24週、第48週或第96週觀測到此等參數中之任一者之臨床上顯著的改善。The method may further comprise the steps of monitoring symptoms of inattention and measuring executive function, such as by ADHD-RS IV (Investigator-rated inattention score), CANTAB score (rapid visual processing, stop signals, and spatial working memory), or health-related quality of life (HRQoL), eg, as measured by the PKU-QOL score or the Q-LES-Q-SF score. A clinically significant improvement in any of these parameters is observed, eg, by Week 24, Week 48, or Week 96.

額外參數包括營養標記物及/或空腹血脂。PKU患者已展示缺乏數種營養標記物，且包括：25-羥基(OH)維生素D、甲基丙二酸(B-12缺乏之指示符)、血清鐵蛋白(鐵缺乏之指示符)、硒及鋅。例如藉由第24週、第48週或第96週觀測到此等參數中之任一者之改善。Additional parameters include nutritional markers and/or fasting lipids. PKU patients have been shown to be deficient in several nutritional markers and include: 25-hydroxy(OH) vitamin D, methylmalonic acid (an indicator of B-12 deficiency), serum ferritin (an indicator of iron deficiency), selenium and zinc. Improvements in any of these parameters were observed, eg, by Week 24, Week 48, or Week 96.

該等方法亦可進一步包含監測游離基因體形成之步驟，該等步驟包含視情況藉由PCR或南方墨點法自個體之肝細胞提取DNA及檢測環形載體基因體。The methods may also further comprise the steps of monitoring the formation of episomal bodies comprising, as appropriate, extracting DNA from individual hepatocytes by PCR or Southern blotting and detecting circular vector gene bodies.

在輸注後第24週之後，使在相隔至少一週的兩次連續評估中達成血漿Phe小於或等於360 μmol/L之個體如下增加完整蛋白質攝入：After Week 24 post-infusion, subjects who achieved a plasma Phe less than or equal to 360 μmol/L in two consecutive assessments at least one week apart were allowed to increase their intact protein intake as follows:

(a)若個體已自完整來源之蛋白質攝入大於2倍膳食參考攝入DRI(大於1.6 g/kg)，則個體可以維持來自完整來源之蛋白質攝入且中斷來自醫療食品之蛋白質攝入。(a) If the subject has consumed more than 2 times the dietary reference intake DRI (greater than 1.6 g/kg) of protein from intact sources, then the subject can maintain protein intake from intact sources and discontinue protein intake from medical foods.

(b)若個體之蛋白質攝入為0.5倍至2倍DRI(0.4至1.6 g/kg)，則個體可將來自完整來源之蛋白質攝入增加10 g/天，且減少來自醫療食品之蛋白質攝入10 g/天。(b) If the subject's protein intake is 0.5 to 2 times the DRI (0.4 to 1.6 g/kg), the subject may increase protein intake from intact sources by 10 g/day and decrease protein intake from medical foods 10 g/day.

(c)若個體之蛋白質攝入小於0.5倍DRI(小於0.4 g/kg)，則個體可將來自完整來源之蛋白質攝入增加20 g/天且減少來自醫療食物之蛋白質攝入20 g/天。(c) If the subject's protein intake is less than 0.5 times the DRI (less than 0.4 g/kg), the subject may increase protein intake from intact sources by 20 g/day and decrease protein intake from medical foods by 20 g/day .

在膳食調節之後，若個體維持血漿Phe小於或等於360 μmol/L持續兩週，則可以根據以上a、b及c進行進一步調節。若個體在膳食調節之後尚未能夠維持血漿Phe小於或等於360 μmol/L，則個體應藉由與最後一次增加類似的調節來減少蛋白質攝入。After dietary adjustment, if the individual maintains plasma Phe less than or equal to 360 μmol/L for two weeks, further adjustments can be made according to a, b and c above. If the individual has not been able to maintain plasma Phe less than or equal to 360 μmol/L after dietary adjustment, the individual should reduce protein intake by a similar adjustment to the last increase.

若血漿Phe含量小於30 μmol/L且在重複血漿Phe量測(在大約2週內進行)之後得到證實，則營養學家可以考慮以下調整：(a)若個體消耗小於2倍DRI(1.6 g/kg/天)，則營養學家可指導個體將其完整蛋白質增加20 g/天且使其醫療食品蛋白質減少20 g/天，(b)若個體消耗2倍或更多DRI，則營養學家可指導個體將其完整蛋白質增加10 g/天且使其醫療食品蛋白質減少10 g/天。If the plasma Phe level is less than 30 μmol/L and confirmed after repeated plasma Phe measurements (performed within approximately 2 weeks), the nutritionist may consider the following adjustments: (a) if the individual consumes less than 2 times the DRI (1.6 g /kg/day), the nutritionist may instruct the individual to increase their complete protein by 20 g/day and decrease their medical food protein by 20 g/day, (b) if the individual consumes 2 times or more DRI, the nutritionist Homes can instruct individuals to increase their complete protein by 10 g/day and decrease their medical food protein by 10 g/day.

在不存在同時藥物療法之情況下，本發明之方法可導致血漿Phe含量(例如平均血漿Phe含量，或兩個連續血漿Phe含量之平均值)臨床上顯著降低。舉例而言，在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與之後8週降低至360 μmol/L或更低，或在該投與之後24、48或96週或2、3或4年降低至360 μmol/L或更低。舉例而言，在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與之後8週在120與360 μmol/L之間。在一些實施例中，在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與之後8週降低至120 μmol/L或更低，或在未同時藥物療法之情況下，在該投與之後24、48或96週或2、3或4年降低至120 μmol/L或更低。In the absence of concurrent drug therapy, the methods of the present invention can result in a clinically significant reduction in plasma Phe levels (eg, mean plasma Phe levels, or the average of two consecutive plasma Phe levels). For example, in the absence of concomitant drug therapy, the subject's plasma Phe levels decrease to 360 μmol/L or less 8 weeks after the administration, or 24, 48 or 96 weeks or 2 weeks after the administration , 3 or 4 years to 360 μmol/L or less. For example, without concomitant drug therapy, the subject's plasma Phe levels were between 120 and 360 μmol/L 8 weeks after the administration. In some embodiments, the subject's plasma Phe levels decrease to 120 μmol/L or less 8 weeks after the administration without concomitant drug therapy, or, without concomitant drug therapy, at the Reduce to 120 μmol/L or less 24, 48 or 96 weeks or 2, 3 or 4 years after administration.

本發明之方法可允許個體經受來自完整食物來源之Phe攝入增加。舉例而言，在該投與之後該個體之血漿Phe含量在120與360 μmol/L之間，且與基線處之Phe受限膳食相比，個體耐受Phe攝入增加。在一些實施例中，個體可經允許增加所消耗之完整蛋白質且減少醫療食品蛋白質，諸如實例3或實例4中所描述。The methods of the present invention may allow individuals to experience increased intake of Phe from whole food sources. For example, the subject's plasma Phe levels were between 120 and 360 μmol/L following the administration, and the subject tolerated increased Phe intake compared to a Phe-restricted diet at baseline. In some embodiments, the individual may be allowed to increase the intact protein consumed and decrease the medical food protein, such as described in Example 3 or Example 4.

在該投與之後，本發明之方法可降低個體之神經傳遞質或神經傳遞質代謝物之血漿含量。舉例而言，該一或多種神經傳遞質或神經傳遞質代謝物為苯乙胺、苯乙醇胺、酪胺、多巴胺、正腎上腺素、腎上腺素、色胺、羥基色胺、苯乙酸、苯乙醯基麩醯胺酸、杏仁酸、羥基苯乙酸、DOPAC、高香草酸、DOMA、MOPEG、香草基杏仁酸、吲哚乙酸及5-羥基吲哚乙酸。Following such administration, the methods of the present invention can reduce plasma levels of neurotransmitters or neurotransmitter metabolites in the subject. For example, the one or more neurotransmitters or neurotransmitter metabolites are phenylethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid, phenylacetylene glutamic acid, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillyl mandelic acid, indoleacetic acid and 5-hydroxyindoleacetic acid.

本發明之方法可使得該個體之生活品質在該投與之後改善，視情況如藉由PKU-QOL或Q-Q-SF調查表所量測。本發明之方法可使得該個體之神經認知症狀或量測在該投與之後改善。較佳地，該個體在該投與之後未罹患低***酸血症。The methods of the present invention may result in an improvement in the subject's quality of life following the administration, as measured by the PKU-QOL or Q-Q-SF questionnaire, as appropriate. The methods of the present invention may result in an improvement in the individual's neurocognitive symptoms or measures following the administration. Preferably, the individual does not suffer from hypophenylalaninemia following the administration.

本發明之方法以安全方式，例如無臨床上顯著治療引發之嚴重不良事件，不繼續發生低***酸血症(血漿Phe發生率在2次連續量測時小於30 μmol/L)，及在標準臨床實驗室值或肝毒性標記物(諸如AST及/或ALT)中無臨床顯著變化(或若發生變化，則大部分在用全身性免疫抑制劑治療之後為短暫的或分解)之方式投與rAAV顆粒。方法亦可提供降低的針對AAV衣殼及/或PAH轉基因之免疫反應。方法亦可提供改善的血液生物分佈，或降低的以尿液、大便、***或唾液排出之載體。The method of the present invention is in a safe manner, for example, without serious adverse events caused by clinically significant treatment, without continued hypophenylalanineemia (the incidence of plasma Phe is less than 30 μmol/L in 2 consecutive measurements), and in standard Administered in a manner without clinically significant changes in clinical laboratory values or markers of hepatotoxicity (such as AST and/or ALT) (or, if changes, were mostly transient or resolved following treatment with systemic immunosuppressants) rAAV particles. The methods may also provide reduced immune responses to AAV capsids and/or PAH transgenes. The methods may also provide improved blood biodistribution, or reduced carrier excretion in urine, stool, semen or saliva.

在本發明之一態樣中，例如經由短暫肝轉胺酶升高所檢測之肝毒性可藉由預防性免疫抑制治療或治療性免疫抑制治療減少或避免。根據此等態樣，除了投與治療有效量之AAV病毒之外，個體可在防治上、治療上或其兩者上用皮質類固醇治療以預防及/或治療與投與AAV病毒相關之任何肝毒性。預防性免疫抑制治療 In one aspect of the invention, hepatotoxicity detected, for example, by transient elevation of hepatic transaminases can be reduced or avoided by prophylactic immunosuppressive therapy or therapeutic immunosuppressive therapy. According to these aspects, in addition to administering a therapeutically effective amount of AAV virus, the subject may be prophylactically, therapeutically, or both treated with corticosteroids to prevent and/or treat any liver disease associated with administration of AAV virus toxicity. preventive immunosuppressive therapy

本發明之方法可進一步包含向個體投與預防有效量之皮質類固醇以預防肝毒性，隨後檢測肝毒性(例如，如藉由高於正常值上限(ULN)或至少2倍基線ALT之ALT升高所檢測)。在一些實施例中，與投與本發明之rAAV顆粒同時投與預防有效量之免疫抑制劑(例如，皮質類固醇)。如本文所用之「並行」意謂同一天，例如或在投與rAAV顆粒之一天或一週內(在其之前或在其之後)。在其他實施例中，在投與rAAV顆粒之後開始投與預防有效量之免疫抑制劑(例如皮質類固醇)，例如在投與rAAV顆粒之後3、4、5、6、7、8、9或10週開始，但在檢測肝毒性之前。The methods of the invention may further comprise administering to the subject a prophylactically effective amount of a corticosteroid to prevent liver toxicity, followed by detection of liver toxicity (eg, as by an ALT elevation above the upper limit of normal (ULN) or at least 2 times baseline ALT) detected). In some embodiments, a prophylactically effective amount of an immunosuppressant (eg, a corticosteroid) is administered concurrently with the administration of the rAAV particles of the invention. "Concurrent" as used herein means the same day, eg, or within a day or a week (before or after) the day or week in which the rAAV particles are administered. In other embodiments, administration of a prophylactically effective amount of an immunosuppressant (eg, a corticosteroid) begins after administration of the rAAV particles, eg, 3, 4, 5, 6, 7, 8, 9, or 10 after administration of the rAAV particles week, but before testing for hepatotoxicity.

皮質類固醇或其他免疫抑制劑可投與預防性治療時段，例如持續至少約3至13週之時段(3、4、5、6、7、8、9、10、11、12、13週)，且較佳地接著為遞減時段，在該時段期間，投與遞減量之皮質類固醇或其他免疫抑制劑，例如持續約2至4週，或約2、3或4週之時段。舉例而言，皮質類固醇之預防有效量為10毫克/天至40毫克/天之普賴松等效物劑量持續至少約3至13週(3、4、5、6、7、8、9、10、11、12、13)之時段，接著遞減量之該皮質類固醇持續約2、3或4週之時段。在一些實施例中，投與預防有效量之皮質類固醇持續約13週之時段，接著遞減量之該皮質類固醇持續約3週之時段。舉例而言，與該持續約13週之時段的投與同時投與40毫克/天之普賴松等效物劑量，接著遞減量之該普賴松等效物持續約3週之時段(例如30毫克/天之普賴松等效物劑量持續一週、20毫克/天持續一週及10毫克/天持續一週)。Corticosteroids or other immunosuppressive agents can be administered for periods of prophylactic treatment, for example, for periods of at least about 3 to 13 weeks (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 weeks), And preferably followed by a tapering period during which tapering amounts of corticosteroids or other immunosuppressants are administered, eg, for a period of about 2 to 4 weeks, or about 2, 3 or 4 weeks. For example, a prophylactically effective amount of a corticosteroid is a prisone equivalent dose of 10 mg/day to 40 mg/day for at least about 3 to 13 weeks (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13), followed by decreasing amounts of the corticosteroid for a period of about 2, 3, or 4 weeks. In some embodiments, a prophylactically effective amount of a corticosteroid is administered for a period of about 13 weeks, followed by a decreasing amount of the corticosteroid for a period of about 3 weeks. For example, a dose of 40 mg/day of the prisone equivalent is administered concurrently with the administration for a period of about 13 weeks, followed by a decreasing amount of the prisone equivalent for a period of about 3 weeks (e.g., Prisone equivalent doses of 30 mg/day for one week, 20 mg/day for one week and 10 mg/day for one week).

在一些實施例中，以40毫克/天之起始普賴松等效物劑量向個體投與16週預防性皮質類固醇療程，在第1天以40毫克/天開始給藥數小時之預輸注rAAV顆粒持續13週之時段，接著在第14週開始遞減之3週劑量(至30毫克/天之普賴松等效物劑量持續一週，20毫克/天持續一週，及10毫克/天持續一週)。在輸注當天，應在rAAV顆粒輸注之前最少3小時投與預防性皮質類固醇。每週監測ALT及AST含量。若ALT升高至大於正常值上限(ULN)或大於2倍基線ALT值，則在前12週期間，對皮質類固醇給藥之調節係基於臨床判斷，且可更頻繁地監測肝臟酶。In some embodiments, subjects are administered a 16-week course of prophylactic corticosteroids at an initial prisone equivalent dose of 40 mg/day, with a pre-infusion of 40 mg/day dosed over several hours on Day 1 rAAV particles for a period of 13 weeks, followed by 3-week doses that are tapered beginning at week 14 (to 30 mg/day of prisone equivalent doses for one week, 20 mg/day for one week, and 10 mg/day for one week ). On the day of infusion, prophylactic corticosteroids should be administered a minimum of 3 hours prior to rAAV particle infusion. ALT and AST levels were monitored weekly. If ALT rises to greater than the upper limit of normal (ULN) or greater than 2 times the baseline ALT value, during the first 12 weeks, adjustments to corticosteroid dosing are based on clinical judgment and liver enzymes may be monitored more frequently.

治療性免疫抑制治療therapeutic immunosuppressive therapy

在一些情況下，本發明之AAV顆粒之投與可引起可觀測程度之肝毒性。肝毒性可藉由各種熟知的常規使用技術量測，例如在AAV投與之前(亦即，基線)及AAV投與之後，量測個體血流中某些肝相關酶(例如丙胺酸轉胺酶，ALT)之濃度。AAV投與之後(與投與之前相比)ALT濃度之可觀測的增加指示藥物誘導之肝毒性。本發明之方法可包含在檢測肝毒性之後向個體投與治療有效量之皮質類固醇或其他全身性免疫抑制劑以治療肝毒性。In some instances, administration of the AAV particles of the invention can cause observable levels of liver toxicity. Hepatotoxicity can be measured by a variety of well-known and routinely used techniques, such as measuring certain liver-related enzymes (eg, alanine transaminase) in the bloodstream of an individual before AAV administration (ie, baseline) and after AAV administration. , ALT) concentration. An observable increase in ALT concentrations following AAV administration (compared to before administration) is indicative of drug-induced hepatotoxicity. The methods of the present invention may comprise administering to the subject a therapeutically effective amount of a corticosteroid or other systemic immunosuppressive agent to treat hepatotoxicity following detection of hepatotoxicity.

反應性免疫抑制劑(例如皮質類固醇)療法可在預防性方案完成之後開始，或回應於符合預先指定標準之輕微ALT升高，或基於臨床判斷。在一些實施例中，若ALT在72小時內之兩次連續評估中大於ULN或大於2倍基線，或在48小時內之兩次連續評估中為3倍ULN，則開始。在一些實施例中，反應性免疫抑制(例如皮質類固醇)方案之總持續時間為8週，其中5週40毫克/天普賴松等效物給藥，接著3週劑量遞減，其中ALT均小於或等於ULN且小於或等於2倍基線值。在中斷反應性免疫抑制療法之後的期間，歷經4週每週監測肝臟酶，或若ALT值高於ULN，則更頻繁地監測肝臟酶。Reactive immunosuppressive (eg, corticosteroid) therapy can be initiated after completion of a prophylactic regimen, or in response to mild ALT elevations meeting prespecified criteria, or based on clinical judgment. In some embodiments, start if ALT is greater than ULN or greater than 2 times baseline in two consecutive assessments within 72 hours, or 3 times ULN in two consecutive assessments within 48 hours. In some embodiments, the total duration of the reactive immunosuppressive (eg, corticosteroid) regimen is 8 weeks, with 5 weeks of 40 mg/day prisone equivalent administration followed by 3 weeks of dose escalation, wherein ALT is less than or equal to the ULN and less than or equal to 2 times the baseline value. During the period following discontinuation of reactive immunosuppressive therapy, liver enzymes were monitored weekly for 4 weeks, or more frequently if ALT values were above ULN.

本發明之方法可進一步包含以下步驟：(a)在該投與之前，視情況在該投與之前約一個月測定該個體之血液中的肝毒性標記之基線含量，及(b)在該投與之後，視情況每週或每1、2、3、4、5或6天測定該個體之血液中的投與後該肝毒性標記之含量。The methods of the invention may further comprise the steps of: (a) prior to the administration, optionally about one month prior to the administration, determining a baseline level of a marker of hepatotoxicity in the blood of the individual, and (b) at the time of the administration and thereafter, the post-administration level of the hepatotoxic marker in the subject's blood is determined weekly or every 1, 2, 3, 4, 5 or 6 days as appropriate.

此類方法可進一步包含以下步驟：(c)在生物化學或臨床症狀檢測肝毒性之後，向個體投與治療有效量之免疫抑制劑(例如皮質類固醇)持續治療處理時段，例如持續至少約5至約8週或更長(例如5、6、7或8週)之時段，且較佳地接著為遞減時段，在該遞減時段期間投與遞減量之免疫抑制劑(例如皮質類固醇)持續約2至4週之時段(例如3週)。舉例而言，步驟(c)包含在藉由(i)大於正常值上限(ULN)之投與後該肝毒性標記之含量，或(ii)大於或等於該肝毒性標記的基線含量兩倍之投與後該肝毒性標記之含量來檢測肝毒性之後，向該個體投與治療有效量之皮質類固醇持續至少約5至約8週(例如，5、6、7或8週或更久)或更久之時段，接著遞減量之皮質類固醇持續約2、3或4週之時段。在此類實施例中之任一者中，肝毒性標記為ALT及/或AST，較佳地ALT。在一些實施例中，在該檢測之後，普賴松等效物以約40毫克/天之普賴松等效物劑量投與持續約5週之時段，接著以遞減量之普賴松等效物持續約3週之時段。Such methods may further comprise the step of: (c) administering to the individual a therapeutically effective amount of an immunosuppressant (eg, a corticosteroid) for a therapeutic treatment period, eg, for at least about 5 to A period of about 8 weeks or longer (eg, 5, 6, 7, or 8 weeks), and preferably followed by a taper period during which a taper amount of an immunosuppressant (eg, corticosteroid) is administered for about 2 Periods up to 4 weeks (eg 3 weeks). For example, step (c) comprises following administration by (i) greater than the upper limit of normal (ULN) level of the hepatotoxic marker, or (ii) greater than or equal to twice the baseline level of the hepatotoxic marker After administration of the level of the hepatotoxic marker to detect hepatotoxicity, the subject is administered a therapeutically effective amount of a corticosteroid for at least about 5 to about 8 weeks (eg, 5, 6, 7, or 8 weeks or more) or Longer periods, followed by tapering doses of corticosteroids for periods of about 2, 3 or 4 weeks. In any of such embodiments, the hepatotoxicity marker is ALT and/or AST, preferably ALT. In some embodiments, following the testing, the prisone equivalent is administered at a dose of about 40 mg/day of the prisone equivalent for a period of about 5 weeks, followed by decreasing amounts of the prisone equivalent Things last for a period of about 3 weeks.

「預防性」皮質類固醇或全身性免疫抑制劑治療係指投與皮質類固醇或免疫抑制劑以預防肝毒性及/或預防個體中之量測ALT含量增加。「治療性」皮質類固醇治療係指投與皮質類固醇或免疫抑制劑以降低由投AVV病毒引起之肝毒性及/或降低由投與AAV病毒引起之個體血流中之較高ALT濃度。在某些實施例中，預防性或治療性皮質類固醇治療可包含向個體投與至少5、10、15、20、25、30、35、40、45、50、55、60或更多毫克/天之普賴松等效物劑量，例如在約10毫克/天與約60毫克/天的皮質類固醇之間的普賴松等效物劑量。在某些實施例中，個體之預防性或治療性皮質類固醇治療可經至少約3、4、5、6、7、8、9、10、11、12、13週或更長之連續時段進行，接著持續投與遞減量之時段。可用於本文所描述之方法中的皮質類固醇包括任何已知或常規所用皮質類固醇，其包括例如以等效物劑量之***、普賴松、普賴蘇穠、氟可體松、氫化可體松、布***及其類似物持續相同時段。"Prophylactic" corticosteroid or systemic immunosuppressive therapy refers to the administration of corticosteroids or immunosuppressive agents to prevent liver toxicity and/or prevent an increase in measured ALT levels in an individual. "Therapeutic" corticosteroid therapy refers to the administration of corticosteroids or immunosuppressive agents to reduce hepatotoxicity caused by administration of AVV virus and/or to reduce the higher ALT concentrations in the bloodstream of an individual caused by administration of AAV virus. In certain embodiments, prophylactic or therapeutic corticosteroid treatment can comprise administering to the individual at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more mg/m A daily dose of prisone equivalent, eg, a dose of prisone equivalent between about 10 mg/day and about 60 mg/day of corticosteroids. In certain embodiments, prophylactic or therapeutic corticosteroid treatment of an individual may be administered over a continuous period of at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 weeks or longer , followed by a period of continuous delivery of decreasing amounts. Corticosteroids useful in the methods described herein include any known or conventionally used corticosteroids including, for example, dexamethasone, prisone, prisulol, flucortisone, hydrocortisone in equivalent doses Body pine, budesonide, and their analogs continued for the same period.

可以預防有效或治療有效劑量投與以預防或減少肝毒性之其他全身性免疫抑制劑包括(1)鈣調神經磷酸酶抑制劑，例如他克莫司或環孢素；(2)抗增殖劑或IMDH抑制劑，例如黴酚酸酯、來氟米特或硫唑嘌呤；(3) mTOR抑制劑，例如西羅莫司或依維莫司；(4)詹納斯激酶抑制劑，例如托法替尼；或(5)免疫抑制劑抗體。 5.6 檢測抗AAV抗體Other systemic immunosuppressants that can be administered in prophylactically effective or therapeutically effective doses to prevent or reduce liver toxicity include (1) calcineurin inhibitors such as tacrolimus or cyclosporine; (2) antiproliferative agents or IMDH inhibitors such as mycophenolate mofetil, leflunomide or azathioprine; (3) mTOR inhibitors such as sirolimus or everolimus; (4) Janus kinase inhibitors such as TOR Fatinib; or (5) immunosuppressive antibodies. 5.6 Detection of anti-AAV antibodies

為了使藉由全身性AAV介導之治療性基因轉移之成功肝轉導的可能性最大化，在針對如上文所描述之人類患者的治療方案中投與AAV顆粒之前，可針對能夠阻斷細胞轉導或以其他方式降低治療方案之總效率之抗AAV衣殼抗體的存在評估預期患者。此類抗體可存在於預期患者之血清中且可係針對任何血清型之AAV衣殼。在一個實施例中，預先存在的抗體所針對的血清型為AAV5。To maximize the likelihood of successful hepatic transduction by systemic AAV-mediated therapeutic gene transfer, prior to administration of AAV particles in a therapeutic regimen for human patients as described above, cells capable of blocking Prospective patients are assessed for the presence of anti-AAV capsid antibodies that transduce or otherwise reduce the overall efficacy of the treatment regimen. Such antibodies may be present in the sera of the intended patient and may be directed against the AAV capsid of any serotype. In one embodiment, the serotype to which the pre-existing antibody is directed is AAV5.

檢測預先存在之AAV免疫性之方法已為所熟知且慣例地採用於此項技術中且包括基於細胞之活體外轉導抑制(TI)檢定、活體內(例如小鼠內)TI檢定及總抗衣殼抗體(TAb)之基於ELISA之檢測(參見例如Masat等人,Discov . Med . , 第15卷, 第379頁至第389頁及Boutin等人, (2010)Hum . Gene Ther . , 第21卷, 第704頁至第712頁)。TI分析可採用已預先引入AAV誘導性報告載體之宿主細胞。報告載體可包含在藉由AAV病毒轉導宿主細胞時誘導表現之誘導性報告基因，諸如GFP等。能夠防止/減少宿主細胞轉導存在於人類血清中之抗AAV衣殼抗體將進而減少報告基因於系統中之總體表現。因此，此類分析可用於檢測能夠藉由治療性AAV顆粒來預防/減少細胞轉導之抗AAV衣殼抗體在人類血清中之存在。Methods to detect pre-existing AAV immunity are well known and routinely employed in the art and include cell-based in vitro transduction inhibition (TI) assays, in vivo (eg, in mice) TI assays, and total antibody ELISA-based detection of capsid antibodies (TAbs) (see eg Masat et al., Discov . Med . , Vol. 15, pp. 379-389 and Boutin et al., (2010) Hum . Gene Ther . , pp. 21 Volume, pp. 704 to 712). TI assays can employ host cells that have been pre-introduced with an AAV-inducible reporter vector. The reporter vector may contain an inducible reporter gene, such as GFP and the like, that induces expression upon transduction of host cells by the AAV virus. The ability to prevent/reduce host cell transduction of anti-AAV capsid antibodies present in human serum will in turn reduce the overall performance of the reporter gene in the system. Thus, such assays can be used to detect the presence in human serum of anti-AAV capsid antibodies capable of preventing/reducing cell transduction by therapeutic AAV particles.

檢測抗AAV衣殼抗體之分析可採用固相結合AAV衣殼作為人類血清越過之「捕獲劑」，進而允許存在於血清中之抗衣殼抗體結合至固相結合衣殼「捕獲劑」。一旦洗滌去除非特異性結合，則可以使用「檢測劑」來檢測與捕捉劑結合之抗衣殼抗體的存在。檢測劑可為抗體、AAV衣殼或其類似物，且可經可檢測地標記以幫助檢測及定量經結合抗衣殼抗體。在一個實施例中，檢測劑藉由可使用電化學發光技術及設備檢測之釕或釕複合物標記。Assays for the detection of anti-AAV capsid antibodies may employ solid phase bound AAV capsids as a "capture agent" that human serum crosses, thereby allowing the anti-capsid antibodies present in the serum to bind to the solid phase bound capsid "capture agent". Once non-specific binding is removed by washing, a "detector" can be used to detect the presence of anti-capsid antibodies bound to the capture agent. The detection agent can be an antibody, AAV capsid, or analog thereof, and can be detectably labeled to aid in the detection and quantification of bound anti-capsid antibodies. In one embodiment, the detection agent is labeled with ruthenium or a ruthenium complex detectable using electrochemiluminescence techniques and devices.

相同上述方法可用於評估及檢測預先用所關注的治療性AAV病毒治療之患者中之抗AAV衣殼免疫反應之產生。因此，在用治療性AAV病毒治療之前，不僅可以利用此等技術來評估抗AAV衣殼抗體的存在，而且可以在投藥之後利用其評估及量測針對所投與之治療性AAV病毒之免疫反應的誘導。因此，本文涵蓋將檢測人類血清中之抗AAV衣殼抗體之技術與治療PKU之治療性AAV病毒之投與組合的方法，其中檢測人類血清中之抗AAV衣殼抗體之技術可在投與治療性AAV病毒之前或之後進行。 5.7 試劑盒The same methods described above can be used to assess and detect the generation of anti-AAV capsid immune responses in patients previously treated with a therapeutic AAV virus of interest. Thus, these techniques can be utilized not only to assess the presence of anti-AAV capsid antibodies prior to treatment with a therapeutic AAV virus, but also after administration to assess and measure the immune response to the administered therapeutic AAV virus induction. Thus, covered herein are methods of combining techniques for detecting anti-AAV capsid antibodies in human serum with the administration of a therapeutic AAV virus for the treatment of PKU, wherein the techniques for detecting anti-AAV capsid antibodies in human serum may be useful in administering therapeutics Sexual AAV virus before or after. 5.7 Kits

在某些實施例中，本文提供包含本文所描述之醫藥調配物(例如實例4中所描述之調配物)的容器。容器可為通常用於儲存重組AAV顆粒調配物之任何類型，諸如小瓶。在某些實施例中，醫藥組合物為凍乾調配物。在其他實施例中，醫藥組合物為液體調配物。在一些實施例中，本文提供包含5至25 ml(例如5 ml、10 ml、15 ml、20 ml或25 ml)本文所描述之醫藥調配物的容器(例如輸液袋或小瓶)。在某些實施例中，本文提供包含10至50 ml(例如10 ml、20 ml、30 ml、40 ml或50 ml)本文所描述之醫藥調配物的容器(例如輸液袋或小瓶)。在一些實施例中，本文提供包含50至100 ml本文所描述之醫藥調配物的容器(例如輸液袋或小瓶)。在某些實施例中，本文提供包含100至500 ml本文所描述之醫藥調配物的容器(例如輸液袋或小瓶)。在一些實施例中，本文提供包含500至1000 ml本文所描述之醫藥調配物的容器(例如輸液袋或小瓶)。在某些實施例中，本文提供包含250至500 ml本文所描述之醫藥調配物的容器(例如輸液袋或小瓶)。在某些實施例中，重組AAV顆粒之濃度為約1×10¹³ vg/ml至約4×10¹⁴ vg/ml(例如2×10¹³ vg/ml，6×10¹³ vg/ml或2×10¹⁴ vg/ml)。在一些實施例中，重組AAV顆粒之濃度足以向個體(例如60至85 kg之個體，諸如60 kg、65 kg、70 kg、75 kg、80 kg或85 kg)投與2×10¹³ vg/ml、6×10¹³ vg/ml或2×10¹⁴ vg/ml之劑量。In certain embodiments, provided herein are containers comprising a pharmaceutical formulation described herein (eg, a formulation described in Example 4). The container can be of any type commonly used to store recombinant AAV particle formulations, such as vials. In certain embodiments, the pharmaceutical composition is a lyophilized formulation. In other embodiments, the pharmaceutical composition is a liquid formulation. In some embodiments, provided herein are containers (eg, infusion bags or vials) comprising 5 to 25 ml (eg, 5 ml, 10 ml, 15 ml, 20 ml, or 25 ml) of a pharmaceutical formulation described herein. In certain embodiments, provided herein are containers (eg, infusion bags or vials) comprising 10 to 50 ml (eg, 10 ml, 20 ml, 30 ml, 40 ml, or 50 ml) of a pharmaceutical formulation described herein. In some embodiments, provided herein are containers (eg, infusion bags or vials) comprising 50 to 100 ml of a pharmaceutical formulation described herein. In certain embodiments, provided herein are containers (eg, infusion bags or vials) comprising 100 to 500 ml of a pharmaceutical formulation described herein. In some embodiments, provided herein are containers (eg, infusion bags or vials) comprising 500 to 1000 ml of a pharmaceutical formulation described herein. In certain embodiments, provided herein are containers (eg, infusion bags or vials) comprising 250 to 500 ml of a pharmaceutical formulation described herein. In certain embodiments, the concentration of recombinant AAV particles is from about 1×10 ¹³ vg/ml to about 4×10 ¹⁴ vg/ml (eg, 2×10 ¹³ vg/ml, 6×10 ¹³ vg/ml or 2× 10 ¹⁴ vg/ml). ^{In some embodiments, the concentration of recombinant AAV particles is sufficient to administer 2 x 10 13} vg/g to an individual (eg, an individual of 60 to 85 kg, such as 60 kg, 65 kg, 70 kg, 75 kg, 80 kg, or 85 kg) ml, 6 x 10 ¹³ vg/ml or 2 x 10 ¹⁴ vg/ml.

在一些實施例中，包含本文所描述之醫藥調配物的容器附有說明書或產品插頁，其描述例如醫藥調配物之投與方法、劑量及用途。舉例而言，說明書或產品插頁可描述醫藥調配物之輸注速率、向個體投與醫藥調配物之標準及/或評估輸注後之一種、兩種或更多種事物，諸如本文所描述(例如實例3或實例4中)。In some embodiments, containers containing the pharmaceutical formulations described herein are accompanied by instructions or product inserts that describe, for example, methods of administration, dosages, and uses of the pharmaceutical formulations. For example, the instructions or product inserts can describe the infusion rate of the pharmaceutical formulation, the criteria for administering the pharmaceutical formulation to an individual, and/or assess one, two or more things after infusion, such as described herein (eg, Example 3 or Example 4).

在某些實施例中，本文提供一種試劑盒，其在容器(例如輸液袋或小瓶)中包含本文所描述之醫藥調配物。在一些實施例中，試劑盒進一步包含說明書或產品插頁，其描述例如醫藥調配物之投與方法、劑量及用途。試劑盒可進一步包含以下中之一者、兩者或更多者：注射器、IV極、IV管、敷料、膠帶、防腐劑溶液。In certain embodiments, provided herein is a kit comprising a pharmaceutical formulation described herein in a container (eg, an infusion bag or vial). In some embodiments, the kit further comprises instructions or product inserts describing, eg, methods of administration, dosages, and uses of the pharmaceutical formulations. The kit may further comprise one, two or more of the following: syringe, IV pole, IV tube, dressing, tape, antiseptic solution.

本發明之其他態樣及優點將在考慮以下說明性實例後理解。 6. 實例 6.1 實例1：向非人類靈長類動物投與AAV顆粒Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples. 6. Examples 6.1 Example 1: Administration of AAV particles to non-human primates

食蟹獼猴以每公斤體重多達4E14載體基因體(vg/kg)之劑量投與包含本文所描述之重組載體建構體及AAV型衣殼的媒劑或rAAV顆粒。監測安全性量測，包括每週身體特徵，體重量測，針對抗AAV5抗體反應、抗PAH抗體反應及諸如ALT及AST之肝臟酶含量進行監測。監測靈長類動物之不良臨床症狀，且評估所有主要器官之病理。 6.2 實例2：醫藥調配物Cynomolgus monkeys were administered vehicle or rAAV particles comprising the recombinant vector constructs described herein and AAV-type capsids at doses of up to 4E14 vector genomes per kilogram of body weight (vg/kg). Monitoring of safety measures, including weekly physical characteristics, weight measurement, monitoring for anti-AAV5 antibody response, anti-PAH antibody response, and levels of liver enzymes such as ALT and AST. Primates were monitored for adverse clinical symptoms, and all major organs were assessed for pathology. 6.2 Example 2: Pharmaceutical Formulations

包含AAV5型衣殼及本文所描述之重組載體建構體(SEQ ID NO: 15至23或52中之一者)之rAAV顆粒提供於適合作為靜脈內投與之生理上相容IV溶液的液體調配物中，其在≤60℃下冷凍(在約-60℃或更低)時穩定長時段，例如1或2年。液體調配物在適當加速儲存條件下亦穩定持續例如至少6或12個月之時段。rAAV particles comprising AAV5-type capsids and the recombinant vector constructs described herein (one of SEQ ID NOs: 15 to 23 or 52) are provided in liquid formulations suitable for intravenous administration as physiologically compatible IV solutions therewith Among others, it is stable for extended periods of time, such as 1 or 2 years, when frozen at < 60°C (at about -60°C or lower). Liquid formulations are also stable under appropriate accelerated storage conditions for periods of, eg, at least 6 or 12 months.

VP1蛋白在其N端處之脫醯胺含量藉由液相層析-質譜分析(LC-MS)定量。該分析精確量測AAV5病毒蛋白1(VP1)之N端區處之脫醯胺百分比，特定言之N50及N56處之脫醯胺百分比。經調配之主體藥物物質或藥品中之衣殼顆粒係經變性以便解離病毒蛋白且在LC-MS分析之前消化成肽。藉由相對於未經修飾及脫醯胺肽峰面積之總和，量測脫醯胺肽峰面積之強度來計算脫醯胺百分比。The deamidation content of VP1 protein at its N-terminus was quantified by liquid chromatography-mass spectrometry (LC-MS). This assay precisely measures the percent deamidation at the N-terminal region of AAV5 viral protein 1 (VP1), specifically at N50 and N56. The formulated host drug substance or capsid particles in the drug product are denatured to dissociate viral proteins and digest into peptides prior to LC-MS analysis. The percent deamidation was calculated by measuring the intensity of the peak areas of the deamidated peptides relative to the sum of the unmodified and deamidated peptide peak areas.

以加速及加壓穩定性研究來評估在pH範圍為7至8(在pH 7.1、7.5及7.9下)下緩衝液pH對rAAV5顆粒之脫醯胺含量的影響。將樣品在4℃下儲存30天或在室溫(RT)下儲存15天。圖1A及圖1B中所示之結果指示，在增加pH值及較高儲存溫度下脫醯胺含量升高。儘管與鹼性pH條件相比在酸性pH值下存在更大熱穩定性，但產生接近生理pH(pH 7.4)之最終調配物的調配物為較佳的。對於在無稀釋劑的情況下投與之醫藥調配物，醫藥調配物之較佳pH經選擇為pH 7.2。Accelerated and pressurized stability studies were performed to evaluate the effect of buffer pH on the deamidation content of rAAV5 particles over a pH range of 7 to 8 (at pH 7.1, 7.5 and 7.9). Samples were stored at 4°C for 30 days or at room temperature (RT) for 15 days. The results shown in Figures 1A and 1B indicate that the deamidation levels increased at increasing pH and higher storage temperatures. Formulations that yield final formulations near physiological pH (pH 7.4) are preferred despite greater thermal stability at acidic pH compared to alkaline pH conditions. For pharmaceutical formulations to be administered without diluents, the preferred pH of the pharmaceutical formulation is chosen to be pH 7.2.

選擇磷酸鈉緩衝液以維持溶液之目標pH(7.2)。10 mM磷酸鈉濃度展現足以將pH維持在長期及加速穩定性測試條件下。在三種不同儲存條件下評估調配物之pH穩定性：長期(≤-60℃)、加速(2至8℃)及加壓(25℃/60% RH)。對於所有測試條件，pH隨時間推移無顯著變化。Sodium phosphate buffer was chosen to maintain the target pH of the solution (7.2). The 10 mM sodium phosphate concentration appeared to be sufficient to maintain pH under long-term and accelerated stability test conditions. The pH stability of the formulations was evaluated under three different storage conditions: long term (≤-60°C), accelerated (2 to 8°C) and pressurized (25°C/60% RH). For all conditions tested, pH did not change significantly over time.

在某些濃度範圍內之氯化鈉保持衣殼膠態穩定性及溶液透明度。在無NaCl存在下，rAAV顆粒可能會自溶液中沈澱出來。含有至少50 mM NaCl之水溶液為降低rAAV顆粒溶液之總體濁度且維持rAAV顆粒之可溶性所必需的。將NaCl濃度從50增加至100 mM改良了穩定性，而將NaCl濃度從100 mM增加至165 mM展示類似結果。選擇在彼範圍內之120 mM NaCl之濃度以維持穩定效應同時維持等張溶液。研究熱穩定性與NaCl濃度之函數關係，並將分析結果提供於表1中。與NaCl濃度增加相對應的起始溫度升高進一步證實氯化鈉對AAV穩定性具有顯著影響。Sodium chloride within a certain concentration range maintains the colloidal stability of the capsid and the clarity of the solution. In the absence of NaCl, rAAV particles may precipitate out of solution. An aqueous solution containing at least 50 mM NaCl is necessary to reduce the overall turbidity of the rAAV particle solution and maintain the solubility of the rAAV particles. Increasing the NaCl concentration from 50 to 100 mM improved stability, while increasing the NaCl concentration from 100 mM to 165 mM showed similar results. The concentration of 120 mM NaCl within that range was chosen to maintain a stabilizing effect while maintaining an isotonic solution. Thermal stability as a function of NaCl concentration was investigated and the analytical results are provided in Table 1. The increase in onset temperature corresponding to the increase in NaCl concentration further confirms that NaCl has a significant effect on AAV stability.

表1 隨不同NaCl濃度而變之熱穩定性篩選 緩衝物質 溶液之 pH 值 NaCl 濃度 ( mM ) T _起始 ( ℃ ) T 最大 ( ℃ ) T 最大 ( ℃ ) 磷酸鈉 7.5 50 46.2 75.0 88.8 磷酸鈉 7 50 46.3 75.0 88.8 磷酸鈉 7.4 100 48.8 72.5 88.8 磷酸鈉 7.4 150 50 72.5 91.2 磷酸鈉 7.4 300 51.2 75.0 93.1 Table 1 Screening of thermal stability as a function of different NaCl concentrations buffer substance The pH of the solution NaCl concentration ( mM ) T _start ( ℃ ) T max (℃) T max (℃) Sodium Phosphate 7.5 50 46.2 75.0 88.8 Sodium Phosphate 7 50 46.3 75.0 88.8 Sodium Phosphate 7.4 100 48.8 72.5 88.8 Sodium Phosphate 7.4 150 50 72.5 91.2 Sodium Phosphate 7.4 300 51.2 75.0 93.1

測試作為冷凍保存劑或冷凍保護劑之多種增積劑在冷凍溫度條件下維持液體調配物之穩定性的能力。諸如具有多元醇(諸如甘露醇)之海藻糖之糖的比較顯示，海藻糖在維持穩定性方面優良。74 mM二水合海藻糖測定為在120 mM NaCl存在下之最少量之海藻糖冷凍保護劑。最終調配物之所得溶液Tg'為大約-52℃且將使可以在長期儲存條件下發生之少量溫度偏移之作用降至最低。選擇74 mM海藻糖(2.8%海藻糖)之濃度以達成穩定作用，同時維持等張溶液。在各種儲存條件下進行在2%甘露醇或2.8%海藻糖中調配之本發明rAAV顆粒之比較研究：≤-60℃、2至8℃、25℃/60% RH及37℃。Various bulking agents as cryopreservatives or cryoprotectants were tested for their ability to maintain the stability of liquid formulations under freezing temperature conditions. Comparison of sugars such as trehalose with polyols such as mannitol shows that trehalose is excellent in maintaining stability. 74 mM trehalose dihydrate was determined as the minimum amount of trehalose cryoprotectant in the presence of 120 mM NaCl. The resulting solution Tg' of the final formulation is about -52°C and will minimize the effects of small temperature excursions that can occur under long term storage conditions. A concentration of 74 mM trehalose (2.8% trehalose) was chosen to achieve stabilization while maintaining an isotonic solution. Comparative studies of rAAV particles of the invention formulated in 2% mannitol or 2.8% trehalose were performed under various storage conditions: ≤ -60°C, 2 to 8°C, 25°C/60% RH and 37°C.

衣殼蛋白聚集藉由尺寸排阻層析高效液相層析法(SEC-HPLC)監測。衣殼蛋白顆粒藉由UV在280 nm下監測且根據尺寸以三聚體、二聚體及單體之次序溶離。樣品緩衝液中之賦形劑及鹽，諸如泊洛沙姆，在單體峰之後溶離。聚集含量以多聚體%報告，其中多聚體%為二聚體及三聚體峰面積相對於總峰面積(單體、二聚體及三聚體)之總和。參考在每個分析下運行以確認分析效能。Capsid protein aggregation was monitored by size exclusion chromatography high performance liquid chromatography (SEC-HPLC). Capsid protein particles were monitored by UV at 280 nm and eluted in the order of trimers, dimers and monomers according to size. Excipients and salts in the sample buffer, such as poloxamers, elute after the monomer peak. Aggregate content is reported as % multimer, where % multimer is the sum of the dimer and trimer peak areas relative to the total peak area (monomer, dimer, and trimer). Reference runs under each assay to confirm assay performance.

結果分別以聚集含量自初始時間點之變化百分比形式描繪於圖2A至2D中。兩種調配物之穩定性概況在≤-60℃及2至8℃下相當(圖2A及2B)，研究持續時間內之聚集含量無顯著變化。然而，在25℃/60% RH及37℃之升高儲存條件下，當使用海藻糖代替甘露醇調配時，AAV顯示優異的穩定性(圖2C及2D)。The results are depicted in Figures 2A-2D, respectively, as percent change in aggregate content from the initial time point. The stability profiles of the two formulations were comparable at ≤ -60°C and 2 to 8°C (Figures 2A and 2B), with no significant changes in aggregate content over the duration of the study. However, AAV showed excellent stability when formulated using trehalose instead of mannitol at elevated storage conditions of 25°C/60% RH and 37°C (Figures 2C and 2D).

界面活性劑減少rAAV顆粒吸附至接觸表面，且因此減少沈澱且增加調配物穩定性。雖然先前測定0.2% w/v泊洛沙姆，例如泊洛沙姆188對於其他rAAV顆粒調配物為合乎需要的，但當使用少量時，含有編碼功能性PAH之核酸的本發明之rAAV顆粒已顯示為穩定的。以使用8E13 vg/mL rAAV5顆粒之濃度分析不同含量之泊洛沙姆濃度(0、0.05%、0.1%及0.2% (w/v))。觀測到泊洛沙姆之吸附特性會緩和吸附損失。少至0.05%泊洛沙姆顯示單體之保持且防止在無泊洛沙姆存在下檢測到之損失。0.1% w/v下泊洛沙姆188之濃度適合於在所有測試條件下維持液體調配物之穩定性。Surfactants reduce adsorption of rAAV particles to contact surfaces, and thus reduce precipitation and increase formulation stability. While it was previously determined that 0.2% w/v poloxamers, such as poloxamer 188, are desirable for other rAAV particle formulations, rAAV particles of the invention containing nucleic acids encoding functional PAHs have appears to be stable. Different levels of poloxamer concentrations (0, 0.05%, 0.1% and 0.2% (w/v)) were analyzed at concentrations using 8E13 vg/mL rAAV5 particles. The adsorption properties of poloxamers were observed to moderate adsorption losses. As little as 0.05% poloxamer showed retention of monomer and prevented loss detected in the absence of poloxamer. The concentration of Poloxamer 188 at 0.1% w/v was suitable to maintain the stability of the liquid formulation under all conditions tested.

調配物能夠維持6E13 vg/mL之相對較高AAV顆粒濃度(每mL 6×10¹³ 載體基因體)的穩定性。以6E13 vg/mL之濃度之rAAV顆粒的最終水性調配物，其具有10 mM磷酸鈉(2.47 mM或約0.4 mg/ml磷酸二氫鈉一元二水合物及7.53 mM或2.7 mg/ml磷酸鈉十二水合物)、120 mM或約7 mg/ml氯化鈉、74 mM或28 mg/ml二水合海藻糖及0.1% w/v或1 mg/ml泊洛沙姆188，使得最終溶液保持澄清至稍微乳白色，無色至淡黃色，且該最終溶液基本上不含顆粒，同時在預期用途及儲存條件下維持整體產品穩定性。測試顯示，預期調配物在約-60℃ (零下60)或更低溫度下穩定高達2年。藥品之平均滲透重量莫耳濃度係325 mOsm/kg，其略微大於人類血清之大約290 mOsm/L值，但大大低於450 mOsm/L，其經陳述為在經由周邊靜脈投與時攜帶最低的靜脈炎風險。產物之稍微超滲透壓性質並非關注點。Formulations capable of maintaining a relatively high concentration of AAV particles (6 × per mL 10 ¹³ vector genomes) 6E13 vg / mL of stability. Final aqueous formulation of rAAV particles at a concentration of 6E13 vg/mL with 10 mM sodium phosphate (2.47 mM or about 0.4 mg/ml sodium dihydrogen phosphate monobasic dihydrate and 7.53 mM or 2.7 mg/ml sodium phosphate ten dihydrate), 120 mM or about 7 mg/ml sodium chloride, 74 mM or 28 mg/ml trehalose dihydrate and 0.1% w/v or 1 mg/ml poloxamer 188 so that the final solution remains clear to slightly opalescent, colorless to pale yellow, and the final solution is substantially free of particles while maintaining overall product stability under the intended use and storage conditions. Testing has shown that the formulations are expected to be stable for up to 2 years at temperatures of about -60°C (minus 60) or lower. The mean osmotic weight molar concentration of the drug product is 325 mOsm/kg, which is slightly greater than the approximately 290 mOsm/L value of human serum, but well below 450 mOsm/L, which is stated to carry the lowest Phlebitis risk. The slightly hyperosmotic nature of the product is not a concern.

rAAV顆粒之6E13 vg/mL濃度使得能夠在合理體積之液體下進行臨床給藥。舉例而言，可以向70 kg患者投與2E13 vg/kg之劑量(每公斤個體體重2×10¹³ 個載體基因體)，其中23.3 mL液體，同時可以向70 kg患者投與6E13 vg/kg之劑量(每公斤個體體重6×10¹³ 個載體基因體)，其中70 mL液體。 6.3 實例3：向人類個體投與AAV顆粒The 6E13 vg/mL concentration of rAAV particles enables clinical administration with a reasonable volume of fluid. By way of example, can be administered with 2E13 vg / kg of the dose (per kg of body weight of 2 × 10 ¹³ earner genes thereof) to a 70 kg patient, wherein the liquid 23.3 mL, while the 70 kg patient administered with 6E13 vg / kg of Dose (6 x 10 ¹³ vector gene bodies per kg body weight), of which 70 mL of liquid. 6.3 Example 3: Administration of AAV particles to human subjects

以每公斤體重2E13、6E13或2E14載體基因體之劑量(vg/kg)向人類個體投與rAAV顆粒，以評估rAAV顆粒之功效、安全性及耐受性，該rAAV顆粒包含AAV5型衣殼及本文所描述之重組載體建構體(SEQ ID NO: 15至23或52中之一者)。可研究不超過2E14 vg/kg之額外劑量含量。To assess the efficacy, safety and tolerability of rAAV particles comprising AAV5-type capsids and The recombinant vector constructs described herein (one of SEQ ID NOs: 15 to 23 or 52). Additional dose levels up to 2E14 vg/kg may be investigated.

目標為表明在單次靜脈內投與rAAV製品之後，患有PKU之個體之血漿Phe的臨床上有意義的減少。基線平均血漿Phe含量大於600 μmol/L之個體在單次靜脈內輸注中以所需劑量投與rAAV顆粒，且追蹤5年以評估反應之持久性。至少一個劑量群組中之一部分個體將在輸注後第8週、第24週或第48週實現血漿Phe臨床上顯著減少(舉例而言，個體可實現小於或等於360 μmol/L之血漿Phe，或甚至小於或等於120 μmol/L之Phe正常化)。持久反應將持續至少6個月、1年、1.5年、2年、3年、4年或5年或更久。The goal was to demonstrate a clinically meaningful reduction in plasma Phe in individuals with PKU following a single intravenous administration of the rAAV preparation. Individuals with baseline mean plasma Phe levels greater than 600 μmol/L were administered rAAV particles at the desired dose in a single intravenous infusion and followed for 5 years to assess the durability of response. A subset of individuals in at least one dose cohort will achieve a clinically significant reduction in plasma Phe at Week 8, Week 24, or Week 48 after infusion (for example, an individual can achieve less than or equal to 360 μmol/L of plasma Phe, or even less than or equal to 120 μmol/L Phe normalization). Durable responses will last at least 6 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, or 5 years or more.

向具有大於1200 μ mol/L之基線平均血漿Phe含量的更嚴重PKU的額外個體投與rAAV顆粒且在輸注後第8週、第24週或第48週，將達成血漿Phe臨床上顯著降低(例如，個體可實現小於或等於600 μmol/L，或小於或等於360 μmol/L，或小於或等於120 μmol/L之血漿Phe)。升高Phe之神經毒性為過量Phe之直接效果。Phe含量之代謝控制已展示與較高執行功能及較佳認知效能相關。Administration of rAAV particles to additional individuals with more severe PKU with baseline mean plasma Phe levels greater than 1200 μmol/L and at weeks 8, 24 or 48 post-infusion, will achieve a clinically significant reduction in plasma Phe ( For example, an individual may achieve less than or equal to 600 μmol/L, or less than or equal to 360 μmol/L, or less than or equal to 120 μmol/L of plasma Phe). Elevated neurotoxicity of Phe is a direct effect of excess Phe. Metabolic control of Phe content has been shown to be associated with higher executive function and better cognitive performance.

此外，至少一個劑量群組中之個體的一部分將展現注意力不集中及/或執行功能之量測改良。至少一個劑量群組中的一部分個體將實現健康相關生活品質的改善。舉例而言，改善可藉由第24週、第32週、第48週、第96週或更久實現。In addition, a portion of individuals in at least one dose cohort will exhibit a measure of improvement in inattention and/or executive function. A subset of individuals in at least one dose cohort will achieve an improvement in health-related quality of life. For example, improvement can be achieved by week 24, week 32, week 48, week 96 or more.

另外，在投與rAAV顆粒之後第48週或更久，至少一個劑量群組中的一部分個體將在投與rAAV顆粒之後實現來自完整食品之膳食蛋白質攝入的增加(及伴隨來自醫療食品之膳食蛋白質攝入的減少)。可較早，例如在第24週或第32週或更久(例如在第96週或更久)發現改善。至少一個劑量組中之一部分個體將能夠在投與rAAV顆粒之後第48週或更久自完整食物消耗至少0.8 g/kg蛋白質攝入，同時將平均血漿Phe維持在小於或等於360 μmol/L。至少一個劑量群組中之個體比例將能夠在投與rAAV顆粒之後中斷醫療食品。Additionally, at week 48 or more following administration of rAAV particles, a subset of individuals in at least one dose cohort will achieve an increase in dietary protein intake from whole foods (and concomitant diets from medical foods) following administration of rAAV particles reduction in protein intake). Improvement can be seen earlier, eg, at week 24 or week 32 or longer (eg, week 96 or longer). A subset of individuals in at least one dose group will be able to consume at least 0.8 g/kg protein intake from complete food at week 48 or beyond following administration of rAAV particles, while maintaining mean plasma Phe less than or equal to 360 μmol/L. A proportion of individuals in at least one dose cohort will be able to discontinue the medical food after administration of the rAAV particles.

此外，rAAV顆粒之投與對於大部分患者而言將證明為安全的，例如治療引發之嚴重不良事件之發生率低、血漿低***酸血症之發生率低(Phe含量小於30 μmol/L)及肝轉胺酶升高或皮質類固醇療法後分解的短暫升高。納入及排除標準 In addition, administration of rAAV particles will prove to be safe for most patients, such as low incidence of treatment-emergent serious adverse events, low incidence of plasma hypophenylalanineemia (Phe content less than 30 μmol/L) and transient elevation of hepatic transaminases or decomposition following corticosteroid therapy. Inclusion and exclusion criteria

臨床研究納入標準包括以下各者：(1)年齡為15歲或更大，或18歲或更大；(2)在投與rAAV顆粒之前診斷苯酮尿症(PKU)及兩個血漿Phe含量之平均值大於600 μmol/L；(3)目前未接受藥物療法以治療PKU(例如，在rAAV顆粒投與之前至少30天的最後一次劑量之培格瓦力或大型中性胺基酸(LNAA)，或在rAAV顆粒投與之前至少7天的最後一次劑量之沙丙蝶呤)。額外標準包括意願在投與rAAV顆粒後至少第52週避免酒精、草藥及天然補藥、膳食增補劑及肝毒性藥物。Clinical study inclusion criteria included the following: (1) age 15 years or older, or 18 years or older; (2) diagnosis of phenylketonuria (PKU) and two plasma Phe levels prior to administration of rAAV particles (3) not currently receiving drug therapy to treat PKU (eg, last dose of pegvar or large neutral amino acids (LNAA) at least 30 days prior to rAAV particle administration) ), or the last dose of sapropterin at least 7 days prior to rAAV particle administration. Additional criteria included a willingness to avoid alcohol, herbal and natural tonics, dietary supplements, and hepatotoxic drugs for at least 52 weeks after administration of rAAV particles.

臨床研究排除標準包括以下後者：(1)活性感染或免疫抑止病症之證據；(2)癌症病史；(3)物質使用病症、重度抑鬱症、精神病或躁鬱症；(4)對皮質類固醇之禁忌；(5)AAV5衣殼之可檢測抗體(即血清陽性)；(6)展示如按0至4之等級評級之3或4之顯著纖維化的先前肝切片；(7)臨床上顯著之肝病，其包括丙胺酸轉胺酶(ALT)、天冬胺酸轉胺酶(AST)、γ-麩胺醯轉化酶(GGT)或膽紅素中之任一者的升高不超過正常值上限(ULN) 1.25倍，或其國際標準化比率等於或大於1.2；(8)先前未感染B型或C型肝炎或肺結核；(9)血清肌酐大於或等於1.5 mg/dL。監測安全性及功效 Exclusion criteria for clinical studies included the latter: (1) evidence of active infection or immunosuppressive disorder; (2) history of cancer; (3) substance use disorder, major depressive disorder, psychosis, or bipolar disorder; (4) contraindication to corticosteroids (5) Detectable antibody to AAV5 capsid (ie seropositivity); (6) previous liver section showing significant fibrosis as 3 or 4 on a scale of 0 to 4; (7) clinically significant liver disease , which includes elevations in any of alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamine converting enzyme (GGT), or bilirubin not exceeding the upper limit of normal (ULN) 1.25 times, or its international normalized ratio equal to or greater than 1.2; (8) not previously infected with hepatitis B or C or tuberculosis; (9) serum creatinine greater than or equal to 1.5 mg/dL. Monitoring safety and efficacy

在輸注rAAV顆粒之前，對個體評估以下各者：(1)基線物理檢驗；(2)基線臨床實驗室測試，其包括(a)血漿Phe含量、(b)血漿酪胺酸(Tyr)含量及(c)肝酶測試，其包括ALT、AST、GGT及膽紅素；(d)及基線AAV5抗體檢測；(3)自完整食物及自醫療食品攝入之基線蛋白；(4)量測注意力不集中及/或執行功能，例如，注意力不足過動症等級量表(ADHD-RS IV)，其為調查員分級之注意力不集中得分，劍橋神經心理學測試自動電池(CANTAB)得分(包括快速視覺處理、停止信號及空間工作記憶)；(5)量測健康相關生活品質(HRQoL)，例如，苯酮尿症影響及治療生活品質調查表(PKU-QOL)得分或生活品質享受及滿意調查表(Q-LES-Q-SF)得分；(6)在研究期間監測之其他參數的基線含量；及(7)PAH基因分型，若允許。Prior to infusion of rAAV particles, subjects were assessed for (1) baseline physical tests; (2) baseline clinical laboratory tests including (a) plasma Phe levels, (b) plasma tyrosine (Tyr) levels and (c) Liver enzyme tests, including ALT, AST, GGT, and bilirubin; (d) and baseline AAV5 antibody tests; (3) baseline protein intake from whole foods and from medical foods; (4) measurement cautions Inattention and/or executive functioning, e.g., Attention Deficit Hyperactivity Disorder Rating Scale (ADHD-RS IV), which is an investigator-rated inattention score, Cambridge Neuropsychological Test Automated Battery (CANTAB) score (including rapid visual processing, stop signals, and spatial working memory); (5) measure health-related quality of life (HRQoL), e.g., phenylketonuria impact and treatment quality of life questionnaire (PKU-QOL) score or quality of life enjoyment and Satisfaction Questionnaire (Q-LES-Q-SF) scores; (6) baseline levels of other parameters monitored during the study; and (7) PAH genotyping, if allowed.

在輸注rAAV顆粒之前，經由第24週、第48週、第96週或更久監測之參數包括：(1)每週之血漿Phe含量，自基線檢測之在輸注後第8週、第12週及第24週的平均血漿Phe含量之變化；(2)每週之神經傳遞質或神經傳遞質代謝物含量，例如血漿Tyr含量，Phe/Tyr比率；(3)在第24週之後，耐受膳食蛋白質攝入之增加及/或醫療食品攝入之減少(Phe降低或無Phe之食物)的能力，例如在輸注後第48週個體之一部分可以消耗來自完整食物之至少0.8 g/kg蛋白質攝入，同時在輸注後第48週維持血漿Phe小於或等於360 μmol/L，及/或不消耗醫療食品；(4)注意力不集中之症狀及執行功能之量測，例如藉由ADHD-RS IV(調查員分級之注意力不集中得分)、CANTAB得分(快速視覺處理、停止信號及空間工作記憶)；(5)健康相關生活品質(HRQoL)，例如，如藉由PKU-QOL得分或Q-LES-Q-SF得分所量測。觀測到此等參數中之任一者之臨床上顯著改善。Before infusion of rAAV particles, parameters monitored through Week 24, Week 48, Week 96 or beyond include: (1) Weekly plasma Phe levels, measured from baseline at Week 8, Week 12 after infusion and changes in mean plasma Phe levels at week 24; (2) weekly neurotransmitter or neurotransmitter metabolite levels, such as plasma Tyr levels, Phe/Tyr ratio; (3) after week 24, tolerance The ability to increase dietary protein intake and/or decrease medical food intake (Phe-reduced or Phe-free food), eg, at week 48 post-infusion, a portion of the individual can consume at least 0.8 g/kg of protein intake from whole foods while maintaining plasma Phe less than or equal to 360 μmol/L at week 48 after infusion, and/or not consuming medical food; (4) inattention symptoms and measures of executive function, such as by ADHD-RS IV (Investigator-rated inattention score), CANTAB score (rapid visual processing, stopping signals, and spatial working memory); (5) Health-related quality of life (HRQoL), e.g., as measured by PKU-QOL score or QoL -Measured by LES-Q-SF score. Clinically significant improvements in any of these parameters were observed.

額外參數包括：神經傳遞質代謝物含量(例如，苯乙醯基麩醯胺酸[PAG]、高香草酸[HVA]、3-甲氧基-4-羥苯基二醇[MOPEG]及5-羥基吲哚乙酸[5HIAA])；Phe氧化速率；營養標記物；空腹血脂。觀測到此等參數中之任一者之臨床上顯著改善。Additional parameters include: neurotransmitter metabolite content (eg, phenylacetylglutamic acid [PAG], homovanillic acid [HVA], 3-methoxy-4-hydroxyphenyldiol [MOPEG], and 5 -hydroxyindoleacetic acid [5HIAA]); Phe oxidation rate; nutritional markers; fasting lipids. Clinically significant improvements in any of these parameters were observed.

PAH催化Phe轉化成酪胺酸，因此未經治療之PKU在血液中產生低於正常酪胺酸濃度。PKU患者已展示缺乏數種營養標記物，且包括：25-羥基(OH)維生素D、甲基丙二酸(B-12缺乏之指示符)、血清鐵蛋白(鐵缺乏之指示符)、硒及鋅。PAH catalyzes the conversion of Phe to tyrosine, so untreated PKU produces lower than normal tyrosine concentrations in the blood. PKU patients have been shown to be deficient in several nutritional markers and include: 25-hydroxy(OH) vitamin D, methylmalonic acid (an indicator of B-12 deficiency), serum ferritin (an indicator of iron deficiency), selenium and zinc.

如使用Phe呼吸測試所量測之Phe氧化速率用作PAH活性之量測。空腹Phe呼吸測試允許藉由經口投與***酸同位素(非放射性示蹤劑，L-[1-¹³ C]-***酸)之後在呼吸中出現¹³ CO₂ 來定量評估Phe代謝。在120分鐘時段內¹³ CO₂ 之累積恢復為評估在研究過程中Phe氧化速率相對於基線之變化的終點。The rate of Phe oxidation as measured using the Phe breath test was used as a measure of PAH activity. Fasting Phe allow breath test by oral administration phenylalanine isotope (non-radioactive tracers, L- [1- ¹³ C] - phenylalanine) occurs after the ¹³ CO ₂ to quantitatively assess the metabolic Phe in respiration. Over 120 minutes period of ¹³ CO ₂ recovery is accumulated in the course of the study assessment endpoints Phe oxidation rate of the change from baseline.

以安全方式，例如無臨床上顯著治療引發之嚴重不良事件，不繼續發生低***酸血症(血漿Phe發生率在2次連續量測時小於30 μmol/L)，及在標準臨床實驗室值或肝毒性標記物(諸如AST及/或ALT)中無臨床顯著變化(或若發生變化，則大部分在用全身性免疫抑制劑治療之後為短暫的或分解)之方式投與rAAV顆粒。監測針對AAV衣殼及PAH轉基因之免疫反應，如同血液生物分佈，且尿液、大便、***及唾液載體排出。Phe 受限膳食之變化 In a safe manner, such as without clinically significant treatment-induced serious adverse events, without continued hypophenylalanineemia (the incidence of plasma Phe less than 30 μmol/L in 2 consecutive measurements), and at standard clinical laboratory values rAAV particles were administered in such a way that there were no clinically significant changes (or, if changes occurred, were mostly transient or disintegrated after treatment with systemic immunosuppressants) in markers of hepatotoxicity, such as AST and/or ALT. Immune responses to AAV capsids and PAH transgenes were monitored, as were blood biodistribution, and urine, stool, semen and salivary carriers were excreted. Changes in Phe Restricted Meals

在輸注rAAV顆粒後第24週之前，指導個體維持一致的膳食攝入，例如來自完整食品之膳食蛋白質攝入自基線改變小於25%且醫療食品蛋白質攝入自基線改變小於25%。在輸注後第24週之後，使在相隔至少一週的兩次連續評估中達成血漿Phe小於或等於360 μmol/L之個體如下增加完整蛋白質攝入：Subjects are instructed to maintain a consistent dietary intake, such as less than 25% change from baseline in dietary protein intake from whole foods and less than 25% change from baseline in medical food protein intake, prior to week 24 after infusion of rAAV particles. After Week 24 post-infusion, subjects who achieved a plasma Phe less than or equal to 360 μmol/L in two consecutive assessments at least one week apart were allowed to increase their intact protein intake as follows:

若膳食調節不足以達成目標血漿Phe含量或不可行，則可在第24週評估之後考慮經口療法之藥物療法，且可根據標準照護在第48週評估之後考慮用可注射劑之藥物療法。If dietary adjustment is insufficient or infeasible to achieve target plasma Phe levels, drug therapy with oral therapy may be considered after the Week 24 assessment, and drug therapy with injectables may be considered after the Week 48 assessment according to standard of care.

預防性皮質類固醇療法prophylactic corticosteroid therapy

可藉由預防性皮質類固醇療法減少或避免瞬時肝轉胺酶升高。16週預防性皮質類固醇療程以40毫克/天之普賴松等效物起始劑量投與，在第1天開始以40毫克/天給藥預輸注持續13週之時段，接著在第14週開始3週之劑量遞減(至30毫克/天之普賴松等效物劑量持續一週，20毫克/天持續一週，及10毫克/天持續一週)。在輸注當天，應在rAAV顆粒輸注之前最少3小時投與預防性皮質類固醇。每週監測ALT及AST含量。若ALT升高至大於正常值上限(ULN)或大於2倍基線ALT值，則在前12週期間，對皮質類固醇給藥之調節係基於臨床判斷，且可更頻繁地監測肝臟酶。Transient hepatic transaminase elevations can be reduced or avoided by prophylactic corticosteroid therapy. A 16-week course of prophylactic corticosteroids was administered at a starting dose of 40 mg/day of prisone equivalent, followed by a 40 mg/day pre-infusion on Day 1 for a 13-week period, followed by a 14-week period Dose escalation was initiated at 3 weeks (to 30 mg/day of prisone equivalent dose for one week, 20 mg/day for one week, and 10 mg/day for one week). On the day of infusion, prophylactic corticosteroids should be administered a minimum of 3 hours prior to rAAV particle infusion. ALT and AST levels were monitored weekly. If ALT rises to greater than the upper limit of normal (ULN) or greater than 2 times the baseline ALT value, during the first 12 weeks, adjustments to corticosteroid dosing are based on clinical judgment and liver enzymes may be monitored more frequently.

瞬時肝酶升高之反應性皮質類固醇療法Responsive corticosteroid therapy for transient liver enzyme elevations

反應性皮質類固醇療法可在預防性方案完成之後開始，或回應於符合預先指定標準之輕微ALT升高，或基於臨床判斷。若ALT在72小時內之兩次連續評估中大於ULN或大於2倍基線，或在48小時內之兩次連續評估中為3倍ULN，則開始。若ALT小於或等於ULN且小於或等於2倍基線值二者，推薦之反應性CS方案之總持續時間為8週，其中5週為40毫克/天普賴松等效物給藥，隨後為3週劑量遞減。在中斷反應性皮質類固醇療法之後的期間，歷經4週每週監測肝臟酶，或若ALT值高於ULN，則更頻繁地監測肝臟酶。Reactive corticosteroid therapy may be initiated after completion of the prophylactic regimen, or in response to mild ALT elevations meeting prespecified criteria, or based on clinical judgment. Start if ALT is greater than ULN or greater than 2 times baseline in two consecutive assessments within 72 hours, or 3 times ULN in two consecutive assessments within 48 hours. If ALT is both less than or equal to ULN and less than or equal to 2 times the baseline value, the recommended total duration of the reactive CS regimen is 8 weeks, with 5 weeks of 40 mg/day prisone equivalent administration followed by 3-week dose escalation. During the period following discontinuation of reactive corticosteroid therapy, monitor liver enzymes weekly for 4 weeks, or more frequently if ALT values are above ULN.

若ALT升高與rAAV顆粒之治療性干預明顯無關(例如由於運動密集或病毒肝炎而同時增加之ALT與肌酸磷酸激酶(CPK))，則不投與反應性皮質類固醇。 6.4 實例4：向人類個體投與AAV顆粒Reactive corticosteroids were not administered if ALT elevations were significantly unrelated to therapeutic intervention with rAAV particles (eg, concurrent increases in ALT and creatine phosphokinase (CPK) due to exercise intensity or viral hepatitis). 6.4 Example 4: Administration of AAV particles to human subjects

以每公斤體重2E13、6E13或＜2E14載體基因體之劑量(vg/kg)向人類個體投與rAAV顆粒，以評估rAAV顆粒之功效、安全性及耐受性，該rAAV顆粒包含AAV5型衣殼及本文所描述之重組載體建構體(SEQ ID NO: 15至23或52中之一者)。可研究不超過2E14 vg/kg之額外劑量含量。To evaluate the efficacy, safety, and tolerability of rAAV particles comprising AAV5-type capsids by administering rAAV particles per kilogram body weight of 2E13, 6E13, or <2E14 vector genomes (vg/kg) to human subjects and the recombinant vector constructs described herein (one of SEQ ID NOs: 15 to 23 or 52). Additional dose levels up to 2E14 vg/kg may be investigated.

此外，至少一個劑量群組中之個體的一部分將展現注意力不集中及/或執行功能之量測改良。舉例而言，至少一個劑量群組中之個體比例將看到如藉由ADHD-RS IV及/或CANTAB(包括快速可視處理、停止信號及空間記憶)評估自基線輸注後之改善。至少一個劑量群組中的一部分個體將實現健康相關生活品質的改善。舉例而言，如藉由PKU-QOL得分及/或Q-LEG-SF得分所評估，在至少一個劑量群組中，個體之比例將實現自基線輸注後之改善。此類改善可藉由第24週、第32週、第48週、第96週或更久實現。In addition, a portion of individuals in at least one dose cohort will exhibit a measure of improvement in inattention and/or executive function. For example, the proportion of individuals in at least one dose cohort will see improvement from baseline infusion as assessed by ADHD-RS IV and/or CANTAB (including rapid visual processing, stop signals, and spatial memory). A subset of individuals in at least one dose cohort will achieve an improvement in health-related quality of life. For example, the proportion of individuals in at least one dose cohort will achieve improvement from baseline infusion as assessed by PKU-QOL score and/or Q-LEG-SF score. Such improvement can be achieved by week 24, week 32, week 48, week 96 or more.

此外，rAAV顆粒之投與對於大部分患者而言將證明為安全的，例如治療引發之嚴重不良事件之發生率低、血漿低***酸血症之發生率低(Phe含量小於30 μmol/L)及肝轉胺酶升高或皮質類固醇療法(CS)後分解的短暫升高。In addition, administration of rAAV particles will prove to be safe for most patients, such as low incidence of treatment-emergent serious adverse events, low incidence of plasma hypophenylalanineemia (Phe content less than 30 μmol/L) and transient elevation of hepatic transaminase elevation or decomposition following corticosteroid therapy (CS).

納入及排除標準 臨床研究納入標準包括以下各者：(1)年齡為15歲或更大，或18歲或更大；(2)在投與rAAV顆粒之前診斷苯酮尿症(PKU)及兩個血漿Phe含量之平均值大於600 μmol/L，苯酮尿症表徵為PAH不足；(3)未接受藥物療法以治療PKU或若此前基於藥物療法以治療PKU，由於缺乏耐受性或實現目標功效之不可能性，藥物療法必須經中止；個體不應中止有效治療以參與臨床研究；在投與rAAV顆粒之前必須中止此前基於藥物療法之個體(例如，在rAAV顆粒投與之前至少30天的最後一次劑量之培格瓦力或大型中性胺基酸(LNAA)，或在rAAV顆粒投與之前至少7天的最後一次劑量之沙丙蝶呤)。額外標準包括意願在投與rAAV顆粒之前至投與rAAV顆粒之後至少第52週避免酒精、草藥及天然補藥、膳食增補劑及肝毒性藥物；(ii)意願避免***或血液捐獻直至3次連續***或血液樣本低於載體檢測之極限；(iii)意願避免卵母細胞捐獻；(iv)意願避免器官捐獻及組織捐獻；(v)在rAAV顆粒投與後至少12週男性意願使用有效避孕之方法；(vi)女性在第-28天及第-7天進行陰性血清妊娠；(vii)女性意願在研究期間使用有效避孕之方法；及(viii)新式接種疫苗。 Inclusion and Exclusion Criteria Clinical study inclusion criteria included the following: (1) age 15 years or older, or 18 years or older; (2) diagnosis of phenylketonuria (PKU) prior to administration of rAAV particles and two The average plasma Phe content of each individual is greater than 600 μmol/L, and phenylketonuria is characterized by PAH deficiency; (3) not receiving drug therapy for the treatment of PKU or if previously based on drug therapy for the treatment of PKU, due to lack of tolerance or achievement of goals Impossibility of efficacy, drug therapy must be discontinued; individuals should not discontinue effective treatment to participate in clinical studies; prior drug-based therapy must be discontinued for individuals prior to administration of rAAV particles (eg, at least 30 days prior to rAAV particle administration) The last dose of pegvar or large neutral amino acids (LNAA), or the last dose of sapropterin at least 7 days prior to rAAV particle administration). Additional criteria included willingness to avoid alcohol, herbal and natural tonics, dietary supplements, and hepatotoxic drugs prior to administration of rAAV particles to at least 52 weeks after administration of rAAV particles; (ii) willingness to avoid semen or blood donation until 3 consecutive semen or blood samples below the limit of vector detection; (iii) willingness to avoid oocyte donation; (iv) willingness to avoid organ donation and tissue donation; (v) willingness to use effective contraception for at least 12 weeks after rAAV particle administration ; (vi) women with negative seropregnancy on days -28 and -7; (vii) women's willingness to use effective contraception during the study period; and (viii) novel vaccinations.

臨床研究排除標準包括以下各者：(1)患有原生BH4不足或其他形式之BH4代謝不足之個體；(2)活性感染(包括SARS-CoV-2)或免疫抑止病症(包括HIV)之證據；(3)投與rAAV顆粒5年內之惡性病病史；(4)在rAAV顆粒投與之前1年的物質使用病症或重度抑鬱症；(5)精神病或躁鬱症之任何病史；(6)對皮質類固醇之禁忌或用皮質類固醇療法而惡化之病況的病史；(7)在rAAV顆粒投與之前的AAV5衣殼之可檢測抗體(即血清陽性)；(8)在rAAV顆粒投與之前，如藉由肝纖維化掃描或增強型肝纖維化(ELF)檢測得分大於9.8評估之3級或4級纖維化；(9)展示如按0至4之等級評級之3或4之顯著纖維化的先前肝切片，其基於Batts-Ludwig (Batts等人, The American journal of surgical pathology. DEC 1995;19(12):1409-17)或METAVIR(Bedossa及Poynard, Hepatology. 1996年8月;24(2):289-93)或若使用替代等級，則等效等級之纖維化；(10)用基因療法之先前治療； (10)如在rAAV顆粒投與之前藉由超音波所評估之臨床上顯著肝病；(11)在投與rAAV顆粒之前顯著肝功能障礙，如藉由丙胺酸轉胺酶(ALT)、天冬胺酸轉胺酶(AST)、γ-麩胺醯轉化酶(GGT)或膽紅素中之任一者的升高超過正常值上限(ULN) 1.25倍，或其國際標準化比率等於或大於1.2； (11)如藉由例如血清學分析或PCR評估之先前感染B型或C型肝炎；(12)如藉由在rAAV顆粒投與之前的陽性QuantiFERON所測定之活性肺結核或未經治療之潛在肺結核感染；(13)在rAAV顆粒投與之前30天內使用包括皮質類固醇之全身性藥劑；(14)在rAAV顆粒投與之前30天內使用全身性抑制劑；(15)在rAAV顆粒投與之前30天內免疫接種活疫苗或減毒活疫苗或在rAAV顆粒投與之前14天內免疫接種其他疫苗；(16)在rAAV顆粒投與之前診斷出青光眼；(17)在rAAV顆粒投與之前大於或等於1.5 mg/dL之血清肌酐；(18)血紅素A1C≥8.0%或葡萄糖≥250 mg/dL；及(19)在rAAV顆粒投與之前的顯著血小板減少症(例如血小板計數＜100×10⁹ /L)。Exclusion criteria for clinical studies include the following: (1) individuals with native BH4 insufficiency or other forms of BH4 insufficiency; (2) evidence of active infection (including SARS-CoV-2) or immunosuppressive disorders (including HIV) (3) History of malignancy within 5 years of administration of rAAV particles; (4) Substance use disorder or major depressive disorder 1 year prior to administration of rAAV particles; (5) Any history of psychosis or bipolar disorder; (6) History of contraindication to corticosteroids or conditions exacerbated by corticosteroid therapy; (7) detectable antibodies to the AAV5 capsid prior to rAAV particle administration (ie, seropositivity); (8) prior to rAAV particle administration, If grade 3 or 4 fibrosis assessed by liver fibrosis scan or enhanced liver fibrosis (ELF) test score greater than 9.8; 1996 Aug;24( 2): 289-93) or equivalent grade of fibrosis if surrogate grade is used; (10) prior treatment with gene therapy; (10) as clinically assessed by ultrasound prior to rAAV particle administration Significant liver disease; (11) significant liver dysfunction prior to administration of rAAV particles, such as by alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamine converting enzyme (GGT) or an increase in any of bilirubin more than 1.25 times the upper limit of normal (ULN), or its international normalized ratio equal to or greater than 1.2; (11) as assessed by, for example, serological analysis or PCR prior infection with type B or hepatitis C; (12) active tuberculosis or untreated latent tuberculosis infection as determined by positive QuantiFERON prior to rAAV particle administration; (13) use including corticosteroids within 30 days prior to rAAV particle administration (14) use of a systemic inhibitor within 30 days prior to rAAV particle administration; (15) immunization with live or live attenuated vaccines within 30 days prior to rAAV particle administration or within 30 days prior to rAAV particle administration Immunization with other vaccines within the previous 14 days; (16) Diagnosed glaucoma prior to rAAV particle administration; (17) Serum creatinine greater than or equal to 1.5 mg/dL prior to rAAV particle administration; (18) Heme A1C ≥ 8.0 % or glucose > 250 mg/dL; and (19) significant thrombocytopenia (eg, platelet count < 100 x 10 ⁹ /L) prior to rAAV particle administration.

監測安全性及功效Monitoring safety and efficacy

在臨床中常規地監測rAAV顆粒之免疫原性且將包括抗AAV5衣殼及抗PAH總結合抗體以及PAH特異性及衣殼特異性細胞免疫之評估。在投與rAAV顆粒之後的任何時間發生輸注相關反應的情況下或在ALT升高高於某一臨限值(ALT≥3倍正常值上限)的情況下，亦監測免疫原性。The immunogenicity of rAAV particles is routinely monitored in the clinic and will include assessment of anti-AAV5 capsid and anti-PAH total binding antibodies as well as PAH-specific and capsid-specific cellular immunity. Immunogenicity was also monitored in the event of infusion-related reactions occurring at any time after administration of rAAV particles or in the event of ALT elevations above a certain threshold (ALT > 3 times the upper limit of normal).

額外參數包括：神經傳遞質代謝物含量(例如，苯乙醯基麩醯胺酸[PAG]、高香草酸[HVA]、3-甲氧基-4-羥苯基二醇[MOPEG]及5-羥基吲哚乙酸[5HIAA])；Phe氧化速率；營養標記物；空腹血脂。觀測到此等參數中之任一者之臨床上顯著改善。將在第20週時開始獲得視情況選用之(一或多個)治療後肝切片。Additional parameters include: neurotransmitter metabolite content (eg, phenylacetylglutamic acid [PAG], homovanillic acid [HVA], 3-methoxy-4-hydroxyphenyldiol [MOPEG], and 5 -hydroxyindoleacetic acid [5HIAA]); Phe oxidation rate; nutritional markers; fasting lipids. Clinically significant improvements in any of these parameters were observed. Optional post-treatment liver slice(s) will be obtained starting at week 20.

如使用Phe呼吸測試所量測之Phe氧化速率用作PAH活性之量測。空腹Phe呼吸測試允許藉由經口投與***酸同位素(非放射性示蹤劑，L-[1-¹³ C]-***酸)之後在呼吸中出現¹³ CO₂ 來定量評估Phe代謝。在120分鐘時段內¹³ CO₂ 之累積恢復為評估在研究過程中Phe氧化速率相對於基線之變化的終點。個體在任何血漿Phe量測之前禁食至少2.5小時，個體在肝臟超音波及纖維化掃描之前禁食至少4小時，及個體針對任何13C-***酸呼吸測試(PBT)禁食至少8小時。The rate of Phe oxidation as measured using the Phe breath test was used as a measure of PAH activity. Fasting Phe allow breath test by oral administration phenylalanine isotope (non-radioactive tracers, L- [1- ¹³ C] - phenylalanine) occurs after the ¹³ CO ₂ to quantitatively assess the metabolic Phe in respiration. Over 120 minutes period of ¹³ CO ₂ recovery is accumulated in the course of the study assessment endpoints Phe oxidation rate of the change from baseline. Subjects fasted for at least 2.5 hours prior to any plasma Phe measurement, subjects fasted for at least 4 hours prior to liver ultrasound and fibrosis scans, and subjects fasted for at least 8 hours for any 13C-phenylalanine breath test (PBT).

以安全方式，例如無臨床上顯著治療引發之嚴重不良事件，不繼續發生低***酸血症(血漿Phe發生率在2次連續量測時小於30 μmol/L)，及在標準臨床實驗室值或肝毒性標記物(諸如AST及/或ALT)中無臨床顯著變化(或若發生變化，則大部分在用全身性免疫抑制劑治療之後為短暫的或分解)之方式投與rAAV顆粒。監測針對AAV衣殼及PAH轉基因之免疫反應，如同血液生物分佈，且尿液、大便、***及唾液載體排出。In a safe manner, such as without clinically significant treatment-induced serious adverse events, without continued hypophenylalanineemia (the incidence of plasma Phe less than 30 μmol/L in 2 consecutive measurements), and at standard clinical laboratory values rAAV particles were administered in such a way that there were no clinically significant changes (or, if changes occurred, were mostly transient or disintegrated after treatment with systemic immunosuppressants) in markers of hepatotoxicity, such as AST and/or ALT. Immune responses to AAV capsids and PAH transgenes were monitored, as were blood biodistribution, and urine, stool, semen and salivary carriers were excreted.

PhePhe 受限膳食之變化Changes to Restricted Meals

在輸注rAAV顆粒後第24週之前，指導個體維持一致的膳食攝入，例如來自完整食品之膳食蛋白質攝入自基線改變小於25%，且醫療食品蛋白質攝入自基線改變小於25%，除非：(1)血漿Phe含量在研究上之任何時間處＜30 µmol/L；(2)在第24週之前的兩個最近連續評估中的血漿Phe≤360 µmol/L；或(3)如藉由研究調查員或營養學家指導以支撐充分的營養。在輸注後第24週之後，使在相隔至少一週的兩次連續評估中達成血漿Phe小於或等於360 μmol/L之個體如下增加完整蛋白質攝入： (a)若個體已自完整來源之蛋白質攝入大於2倍膳食參考攝入DRI(大於1.6 g/kg)，則個體可以維持來自完整來源之蛋白質攝入且中斷來自醫療食品之蛋白質攝入。 (b)若個體之蛋白質攝入為0.5倍至2倍DRI(0.4至1.6 g/kg)，則個體可將來自完整來源之蛋白質攝入增加10 g/天，且減少來自醫療食品之蛋白質攝入10 g/天。 (c)若個體之蛋白質攝入小於0.5倍DRI(小於0.4 g/kg)，則個體可將來自完整來源之蛋白質攝入增加20 g/天且減少來自醫療食物之蛋白質攝入20 g/天。Prior to Week 24 after rAAV particle infusion, instruct individuals to maintain consistent dietary intake, such as less than 25% change from baseline in dietary protein intake from whole foods and less than 25% change from baseline in protein intake from medical foods, unless: (1) Plasma Phe levels <30 µmol/L at any time on the study; (2) Plasma Phe ≤ 360 µmol/L in the two most recent consecutive assessments prior to Week 24; or (3) as determined by Study investigator or nutritionist guidance to support adequate nutrition. After Week 24 post-infusion, subjects who achieved a plasma Phe less than or equal to 360 μmol/L in two consecutive assessments at least one week apart were allowed to increase their intact protein intake as follows: (a) If the subject has consumed more than 2 times the dietary reference intake DRI (greater than 1.6 g/kg) of protein from intact sources, then the subject can maintain protein intake from intact sources and discontinue protein intake from medical foods. (b) If the subject's protein intake is 0.5 to 2 times the DRI (0.4 to 1.6 g/kg), the subject may increase protein intake from intact sources by 10 g/day and decrease protein intake from medical foods 10 g/day. (c) If the subject's protein intake is less than 0.5 times the DRI (less than 0.4 g/kg), the subject may increase protein intake from intact sources by 20 g/day and decrease protein intake from medical foods by 20 g/day .

若血漿Phe含量小於30 μmol/L且在重複血漿Phe量測(在大約2週內進行)之後得到證實，則應對個體之膳食進行修改。若個體消耗小於2倍DRI(1.6 g/kg/天)，則營養學家可指導個體將其完整蛋白質增加20 g/天且使其醫療食品蛋白質減少20 g/天，(b)若個體消耗2倍或更多DRI，則營養學家可指導個體將其完整蛋白質增加10 g/天且使其醫療食品蛋白質減少10 g/天。若血漿Phe含量＜30 µmol/L，則允許如上所述之蛋白質攝入進一步增加，以便實現完整蛋白質攝入之2倍DRI準則(1.6 g/kg/天)。If the plasma Phe level is less than 30 μmol/L and confirmed after repeated plasma Phe measurements (performed within approximately 2 weeks), the individual's diet should be modified. If the individual consumes less than 2 times the DRI (1.6 g/kg/day), the nutritionist may instruct the individual to increase their complete protein by 20 g/day and decrease their medical food protein by 20 g/day, (b) if the individual consumes 2 times or more DRI, the nutritionist can instruct the individual to increase their complete protein by 10 g/day and decrease their medical food protein by 10 g/day. If plasma Phe levels are <30 µmol/L, a further increase in protein intake as described above is allowed to achieve the 2-fold DRI guideline for intact protein intake (1.6 g/kg/day).

預防性皮質類固醇療法prophylactic corticosteroid therapy

在完成預防性皮質類固醇療程之後，個體之後持續4週每兩週一次及之後每4週一次總共24週經歷引發不良作用、短暫體檢(對於臨床問診)、生命體徵及臨床實驗室小組之檢查。此期間之後，由家庭保健專家於家訪期間進行上述評估。在皮質類固醇治療週期及皮質類固醇後週期期間分別檢查個體中與皮質類固醇及下丘腦-垂體-腎上腺(HPA)軸抑制相關之不良作用以促使鑑別引發相關不良作用。After completing a course of prophylactic corticosteroids, subjects experienced elicited adverse effects, brief physical examinations (for clinical interviews), vital signs, and clinical laboratory panel examinations every two weeks for 4 weeks thereafter and every 4 weeks thereafter for a total of 24 weeks. After this period, the above assessments are performed by the family health professional during the home visit. Adverse effects associated with corticosteroid and hypothalamic-pituitary-adrenal (HPA) axis inhibition are examined in individuals during corticosteroid treatment cycles and post-corticosteroid cycles, respectively, to facilitate identification of trigger-related adverse effects.

若ALT升高與rAAV顆粒之治療性干預明顯無關(例如由於運動密集或病毒肝炎而同時增加之ALT與肌酸磷酸激酶(CPK))，則不投與反應性皮質類固醇。Reactive corticosteroids were not administered if ALT elevations were significantly unrelated to therapeutic intervention with rAAV particles (eg, concurrent increases in ALT and creatine phosphokinase (CPK) due to exercise intensity or viral hepatitis).

在反應性CS治療期間，個體在臨床問診時或經由家庭保健專家問診(在定期或不定期問診時)每4週經歷引發不良作用、短暫體檢(對於臨床問診)、生命體徵及臨床實驗室測試小組之檢查。檢查個體之與CS治療相關之不良作用，以評估出現之不良作用。During reactive CS treatment, subjects experience elicited adverse effects, brief physical examinations (for clinical visits), vital signs, and clinical laboratory tests every 4 weeks at clinical visits or through home health professional visits (at regular or irregular visits) Group inspection. Individuals were examined for adverse effects associated with CS treatment to assess the occurrence of adverse effects.

在完成反應性CS之後，個體之後持續4週每兩週一次及之後每4週一次總共24週經歷引發不良作用、短暫體檢(對於臨床問診)、生命體徵及臨床實驗室小組之檢查。在此時段之後，如所安排進行評估。將由家庭保健專家進行在家庭就診期間發生的評估。將檢查個體之與HPA軸線抑制相關之不良作用以在皮質類固醇後期間評估個體，以促使鑑別引發相關不良作用。Following completion of reactive CS, subjects experienced elicited adverse effects, brief physical examinations (for clinical interviews), vital signs, and examination by the clinical laboratory panel once every two weeks for 4 weeks thereafter and every 4 weeks thereafter for a total of 24 weeks. After this period, assessments are conducted as scheduled. Assessments that occur during the home visit will be performed by a home health professional. Individuals will be examined for adverse effects associated with inhibition of the HPA axis to assess individuals during the post-corticosteroid period to facilitate identification of trigger-related adverse effects.

在開始rAAV顆粒投與前30天及至研究結束前開始禁止以下藥物：培格瓦力(除在允許救援療法時之外)；大型中性胺基酸(LNAA) (除在允許救援療法時之外)；及全身性免疫抑制劑，包括CS(超過根據方案所允許之CS以降低轉胺酶升高風險)。若臨床上認為皮質類固醇投與無效、不耐受及/或禁忌，則可使用全身性免疫抑制劑(例如CS激發劑，諸如肌苷單磷酸去氫酶抑制劑及鈣調神經磷酸酶抑制劑)。 7. 實施例 1.一種降低有需要之人類個體中之血漿***酸(Phe)含量的方法，其包含向該個體投與治療有效量之重組腺相關病毒(rAAV)顆粒，該重組腺相關病毒(rAAV)顆粒包含AAV衣殼及重組載體建構體，該重組載體建構體包含編碼功能性***酸羥化酶(PAH)之核酸及視情況選用之異源肝特異性轉錄調節區。 2.一種治療患有苯酮尿症之人類個體之方法，其包含向該個體投與治療有效量之包含AAV衣殼之重組腺相關病毒(rAAV)顆粒，及重組載體建構體，該重組載體建構體包含編碼功能性***酸羥化酶(PAH)之核酸及視情況選用之異源肝特異性轉錄調節區。 3.如實施例1至2之方法，其中該rAAV顆粒之該重組載體建構體包含：(a)以下中之一或兩者：(i) AAV 5'反向末端重複序列(ITR)及(ii) AAV 3' ITR；(b)異源肝特異性轉錄調節區；及(c)編碼功能性人類***酸羥化酶(hPAH)之核酸，視情況其中該等AAV ITR為AAV2 ITR。 4.如實施例3之方法，其中該編碼功能性hPAH之核酸編碼與SEQ ID NO: 2至少95%一致之胺基酸序列。 5.如實施例1至4中任一者之方法，編碼功能性hPAH之核酸包含與SEQ ID NO: 1或7至13至少90%一致的核苷酸序列。 6.如實施例1至5中任一者之方法，其中該編碼PAH之核酸可操作地連接至包含hAAT啟動子之片段的啟動子及HCR增強子/ApoE增強子之片段。 7.如實施例1至5中任一者之方法，其中該肝特異性轉錄調節區包含與SEQ ID NO: 3、4或24中之任一者至少90%一致或可替代地與SEQ ID NO: 25或26中之任一者至少90%一致的核苷酸序列。 8.如實施例1至5中任一者之方法，其中該重組載體建構體包含與SEQ ID NO: 6至少90%一致之核苷酸序列。 9.如實施例1至8中任一者之方法，其中該重組載體建構體進一步包含內含子。 10.如實施例9之方法，其中該內含子包含與SEQ ID NO: 14或27或29或34至少90%一致之核苷酸序列。 11.如實施例1至10中任一者之方法，其中該重組載體建構體進一步包含多腺苷酸化信號。 12.如實施例11之方法，其中該重組載體建構體包含牛生長激素(bGH)多腺苷酸化信號。 13.如實施例1至12中任一者之方法，其中該rAAV顆粒包含與SEQ NO: 15至23或52中之任一者至少90%一致的重組載體建構體。 14.如實施例1至13中任一者之方法，其中該AAV衣殼包含與SEQ ID NO: 35至51中之任一者至少85%一致之胺基酸序列。 15.如實施例1至13中任一者之方法，其中該AAV衣殼為具有肝趨向性之AAV衣殼。 16.如實施例15之方法，其中具有肝趨向性之該AAV衣殼不包括AAV8及/或AAVHSC15。 17.如實施例15之方法，其中具有肝趨向性之該AAV衣殼為AAV5型衣殼，視情況與SEQ ID NO: 44至少85%、90%或95%一致。 18.如前述實施例中任一者之方法，其中該個體患有苯酮尿症(PKU)。 19.如前述實施例中任一者之方法，其中該個體患有典型PKU或重度PKU。 20.如前述實施例中任一者之方法，其中在該投與之前該個體之血漿Phe含量為600 μmol/L或更高。 21.如前述實施例中任一者之方法，其中在該投與之前該個體之血漿Phe含量為1200 μmol/L或更高。 22.如前述實施例中任一者之方法，其中該個體為15歲或更大。 23.如前述實施例中任一者之方法，其中該個體為成人。 24.如前述實施例中任一者之方法，其中該個體為女性。 25.如前述實施例中任一者之方法，其中該個體在編碼PAH之內在基因中具有突變，視情況為突變F39L、L48S、I65T、R68S、A104D、S110C、D129G、E178G、V190A、P211T、R241C、R261Q、A300S、L308F、A313T、K320N、A373T、V388M E390G、A395P、P407S及Y414C。 26.如前述實施例中任一者之方法，其中該個體為非妊娠女性。 27.如前述實施例中任一項之方法，其中該個體未接受治療PKU之藥物療法。 28.如實施例27之方法，其中該個體在該投與之前至少30天未接受培格瓦力。 29.如實施例27之方法，其中該個體在該投與之前至少30天未接受大型中性胺基酸。 30.如實施例27之方法，其中該個體在該投與之前至少7天未接受沙丙蝶呤。 31.如前述實施例中任一者之方法，其中該個體在該投與之前至少30天未接受類固醇。 32.如前述實施例中任一者之方法，其中在該投與之前，該個體在血液中不具有可檢測之抗AAV衣殼抗體。 33.如前述實施例中任一項之方法，其中該個體在該投與之前不具有臨床上顯著之肝病。 34.如前述實施例中任一者之方法，其中該rAAV顆粒以單次投與進行靜脈內投與。 35.如前述實施例中任一者之方法，其中該rAAV顆粒以在每公斤個體體重約2E13至約2E14載體基因體(vg/kg)範圍內之劑量投與。 36.如實施例35之方法，其中該rAAV顆粒以約2E13 vg/kg之劑量投與。 37.如實施例35之方法，其中該rAAV顆粒以約6E13 vg/kg之劑量投與。 38.如實施例35之方法，其中該rAAV顆粒以約2E14 vg/kg之劑量投與。 39.如前述實施例中任一者之方法，其進一步包含向該個體投與預防性免疫抑制治療。 40.如實施例39之方法，其中該預防性免疫抑制治療包含投與預防有效量之全身性免疫抑制劑以預防肝毒性，視情況與rAAV顆粒之該投與同時進行。 41.如實施例40之方法，其中該全身性免疫抑制劑為皮質類固醇，視情況為***、普賴松、普賴蘇穠、氟可體松、氫化可體松或布***。 42.如實施例40之方法，其中該全身性免疫抑制劑為皮質類固醇且該預防有效量為10毫克/天至40毫克/天之普賴松等效物劑量，視情況持續至少約13週之時段，接著遞減量之該皮質類固醇持續約3週之時段。 43.如前述實施例中任一者之方法，其進一步包含以下步驟：(a)在該投與之前，視情況在該投與之前約一個月測定該個體之血液中的肝毒性標記之基線含量，及(b)在該投與之後，每週測定該個體之血液中的投與後該肝毒性標記之含量。 44.如實施例43之方法，其進一步包含向該個體投與治療性免疫抑制治療。 45如實施例44之方法，其中該治療性免疫抑制治療包含以下步驟：(c)在生物化學或臨床症狀檢測到肝毒性後，向該個體投與治療有效量之全身性免疫抑制劑以減少肝毒性。 46.如實施例44之方法，其中肝毒性之檢測係藉由(i)投與後該肝毒性標記之含量大於正常值上限(ULN)或(ii)投與後該肝毒性標記之含量大於或等於該肝毒性標記之基線含量兩倍。 47.如實施例44至46中任一項之方法，其中該全身性免疫抑制劑為皮質類固醇，視情況為***、普賴松、普賴蘇穠、氟可體松、氫化可體松或布***。 48.如實施例44至46中任一者之方法，其中該全身性免疫抑制劑為皮質類固醇且該治療有效量為10毫克/天至40毫克/天之普賴松等效物劑量，視情況持續至少約5週之時段，接著遞減量之該皮質類固醇持續約3週之時段。 49.如實施例43至48中任一者之方法，其中該肝毒性標記為ALT及/或AST。 50.如實施例43至48中任一者之方法，其中該肝毒性標記為ALT。 51.如前述實施例中任一項之方法，其進一步包含藉由包含自該個體之肝細胞提取DNA及檢測環形載體基因體之步驟，視情況藉由PCR或南方墨點法監測游離基因體形成。 52.如前述實施例中任一者之方法，其進一步包含每週量測該個體之血漿Phe含量的步驟。 53.如前述實施例中任一者之方法，其進一步包含實施Phe呼吸測試。 54.如前述實施例中任一者之方法，其進一步包含每週量測該個體之一或多種神經傳遞質或神經傳遞質代謝物之血漿含量的步驟。 55.如實施例54之方法，其中該一或多種神經傳遞質或神經傳遞質代謝物為苯乙胺、苯乙醇胺、酪胺、多巴胺、正腎上腺素、腎上腺素、色胺、羥基色胺、苯乙酸、苯乙醯基麩醯胺酸、杏仁酸、羥基苯乙酸、DOPAC、高香草酸、DOMA、MOPEG、香草基杏仁酸、吲哚乙酸及5-羥基吲哚乙酸。 56.如前述實施例中任一項之方法，其中在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與之後8週為360 μmol/L或更低。 57. 如前述實施例中任一項之方法，其中在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與之後2、3或4年為360 μmol/L或更低。 58.如前述實施例中任一項之方法，其中在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與之後8週在120及360 μmol/L之間。 59.如前述實施例中任一項之方法，其中在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與之後8週為120 μmol/L或更低。 60.如前述實施例中任一項之方法，其中在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與之後2、3或4年為120 μmol/L或更低。 61.如前述實施例中任一者之方法，其中與基線處之Phe受限膳食相比，該個體耐受Phe攝入增加。 62.如前述實施例中任一者之方法，其中在該投與之後，該個體之神經傳遞質或神經傳遞質代謝物之血液含量降低。 63.如實施例62之方法，其中該一或多種神經傳遞質或神經傳遞質代謝物為苯乙胺、苯乙醇胺、酪胺、多巴胺、正腎上腺素、腎上腺素、色胺、羥基色胺、苯乙酸、苯乙醯基麩醯胺酸、杏仁酸、羥基苯乙酸、DOPAC、高香草酸、DOMA、MOPEG、香草基杏仁酸、吲哚乙酸及5-羥基吲哚乙酸。 64.如前述實施例中任一者之方法，其中該個體之生活品質在該投與之後改善，視情況如藉由PKU-QOL或Q-LES-Q-SF調查表所量測。 65.如前述實施例中任一者之方法，其中該個體之一或多種神經認知症狀或量測在該投與之後改善。 66.如前述實施例中任一者之方法，其中該個體在該投與之後未經歷持續性的低***酸血症。 67.一種醫藥組合物，其包含濃度為至少1E13 vg/ml至約1E14 vg/ml之rAAV顆粒、緩衝劑、等張劑、冷凍保存劑及界面活性劑，該醫藥組合物在約-60℃(攝氏零下六十度)或更低溫度下儲存期間穩定持續至少約1年、1.5年或2年。 68.如實施例67之醫藥組合物，其中該界面活性劑之濃度為小於0.2% w/v或小於0.15% w/v。 69.如實施例67之醫藥組合物，其中該界面活性劑之濃度為約0.1% w/v。 70.如實施例67至69中任一者之醫藥組合物，其中該冷凍保存劑為糖。 71.如實施例67至69中任一者之醫藥組合物，其中該冷凍保存劑為海藻糖。 72.一種醫藥組合物，其包含濃度為至少1E13 vg/ml至約1E14 vg/ml之rAAV顆粒，該醫藥組合物包含濃度為約5至約15 mM之磷酸鈉；濃度為約100 mM至約165 mM之氯化鈉；為糖，視情況為海藻糖之冷凍保存劑；及濃度為低於0.2% w/v之泊洛沙姆或聚山梨醇酯。 73.如實施例72之醫藥組合物，其包含磷酸氫二鈉及磷酸二氫鈉。 74.如實施例72至73中任一者之醫藥組合物，其中該糖為濃度為約60 mM至約80 mM之海藻糖。 75.如實施例72至74中任一者之醫藥組合物，其中該泊洛沙姆為濃度為約0.05% w/v至0.15% w/v之泊洛沙姆188。 76.如實施例72之醫藥組合物，其包含磷酸鈉、氯化鈉、海藻糖及泊洛沙姆188。 77.如實施例72或76之醫藥組合物，其包含濃度為約5至約15 mM之磷酸鈉、濃度為約100至約140 mM之氯化鈉、濃度為約60至約90 mM之糖及濃度為約0.05% w/v至約0.15% w/v之泊洛沙姆。 78.如實施例72或76之醫藥組合物，其包含濃度為約8至約12 mM之磷酸鈉、濃度為約110至約130 mM之氯化鈉、濃度為約70至約80 mM之糖及濃度為約0.08% w/v至約0.12% w/v之泊洛沙姆。 79.如實施例72或76中任一者之醫藥組合物，其中該磷酸二氫鈉之濃度為大於0.1 mg/mL且小於0.5 mg/mL，視情況約0.3 mg/mL，且該磷酸氫二鈉之濃度為大於2.5 mg/ml且小於3 mg/ml，視情況約2.7 mg/mL。 80.如實施例72或76或79中任一者之醫藥組合物，其中該氯化鈉之濃度為大於5 mg/ml且小於8 mg/ml，視情況約7 mg/ml。 81.如實施例72或76或79至80中任一者之醫藥組合物，其中該糖為濃度為大於20 mg/ml至小於40 mg/ml，或約25 mg/ml至約35 mg/ml，或約28 mg/ml之二水合海藻糖。 82.如實施例72或76或79至81中任一者之醫藥組合物，其中該泊洛沙姆188之濃度為小於1.5 mg/ml或約1 mg/ml。 83.如實施例72至82中任一者之醫藥組合物，其中該rAAV顆粒之濃度為約6E13 vg/ml。 84.一種醫藥組合物，其包含濃度為約6E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM二水合海藻糖及0.1% w/v泊洛沙姆188。 85.如實施例72至84中任一者之醫藥組合物，其為液體。 86.如實施例72至84中任一者之醫藥組合物，其經凍乾。 87.如實施例72至84中任一者之醫藥組合物，其用於向患有苯酮尿症之患者靜脈內投與rAAV顆粒。 88.一種產生rAAV顆粒之方法，其包含(a)為允許AAV複製之細胞(例如昆蟲細胞或哺乳動物細胞) 提供一或多種核酸建構體，該一或多種核酸建構體包含：(i)重組載體建構體，其包含(1)至少一種AAV ITR、(2)異源肝特異性轉譯調節區及(3)編碼功能性人類***酸羥化酶(hPAH)之核酸；(ii)編碼一或多種Rep蛋白之核苷酸序列，該序列可操作地連接至能夠驅動該細胞中之該一或多種Rep蛋白之表現的啟動子；及(iii)編碼一或多種AAV衣殼蛋白之核苷酸序列，該序列可操作地連接至能夠驅動該細胞中之該一或多種衣殼蛋白之表現的啟動子；(b)在允許表現Rep及衣殼蛋白之條件下培養該細胞；及視情況選用之(c)回收該AAV顆粒。 89.如實施例88之方法，其中該細胞為昆蟲細胞。 90.如實施例88之方法，其中該細胞為哺乳動物細胞。 91.如實施例88至90中任一者之方法，其中該編碼功能性hPAH之核酸編碼與SEQ ID NO: 2至少95%一致之胺基酸序列。 92.如實施例88至90中任一者之方法，其中該編碼功能性hPAH之核酸包含與SEQ ID NO: 1或7至13至少90%一致的核苷酸序列。 93.如實施例88至90中任一者之方法，其中該編碼PAH之核酸可操作地連接至包含hAAT啟動子之片段的啟動子及HCR增強子/ApoE增強子之片段。 94.如實施例88至93中任一者之方法，其中該肝特異性轉錄調節區包含與SEQ ID NO: 3、4或24中之任一者至少90%一致或可替代地與SEQ ID NO: 25或26中之任一者至少90%一致的核苷酸序列。 95.如實施例88至93中任一者之方法，其中該重組載體建構體包含與SEQ ID NO: 6至少90%一致之核苷酸序列。 96.如實施例88至95中任一者之方法，其中該重組載體建構體進一步包含內含子。 97.如實施例96之方法，其中該內含子包含與SEQ ID NO: 14或27或29或34至少90%一致之核苷酸序列。 98.如實施例88至97中任一者之方法，其中該重組載體建構體進一步包含多腺苷酸化信號。 99.如實施例98之方法，其中該重組載體建構體包含牛生長激素(bGH)多腺苷酸化信號。 100.如實施例88至90中任一者之方法，其中該重組載體建構體包含與SEQ ID NO: 15至23或52至少90%一致之核苷酸序列。 101.如實施例88至100中任一者之方法，其中該AAV衣殼包含與SEQ ID NO: 35至51中之任一者至少85%一致之胺基酸序列。 102.如實施例88至100中任一者之方法，其中該AAV衣殼為具有肝趨向性之AAV衣殼。 103.如實施例102之方法，其中具有肝趨向性之該AAV衣殼不包括AAV8及/或AAVHSC15。 104.如實施例102之方法，其中具有肝趨向性之該AAV衣殼為AAV5型衣殼，視情況與SEQ ID NO: 44至少85%、90%或95%一致。 105.一種藉由如實施例88至104中任一者之方法產生的rAAV顆粒群，視情況藉由減少空衣殼之數目的步驟使包含全長或幾乎全長載體基因體之顆粒富集。 106.一種減少有需要之人類個體中之血漿Phe含量的方法，其包含投與如實施例105之rAAV顆粒群。 107.一種治療有需要之人類個體之PKU的方法，其包含投與如實施例105之rAAV顆粒群。 8. 精選序列 SEQ ID NO: 序列 18 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tgcggccgca cgcgtaggct cagaggcaca caggagtttc tgggctcacc ctgccccctt ccaacccctc agttcccatc ctccagcagc tgtttgtgtg ctgcctctga agtccacact gaacaaactt cagcctactc atgtccctaa aatgggcaaa cattgcaagc agcaaacagc aaacacacag ccctccctgc ctgctgacct tggagctggg gcagaggtca gagacctctc tgggcccatg ccacctccaa catccactcg accccttgga atttcggtgg agaggagcag aggttgtcct ggcgtggttt aggtagtgtg agaggggtcg acgatcttgc taccagtgga acagccacta aggattctgc agtgagagca gagggccagc taagtggtac tctcccagag actgtctgac tcacgccacc ccctccacct tggacacagg acgctgtggt ttctgagcca ggtacaatga ctcctttcgg taagtgcagt ggaagctgta cactgcccag gcaaagcgtc cgggcagcgt aggcgggcga ctcagatccc agccagtgga cttagcccct gtttgctcct ccgataactg gggtgacctt ggttaatatt caccagcagc ctcccccgtt gcccctctgg atccactgct taaatacgga cgaggacagg gccctgtctc ctcagcttca ggcaccacca ctgacctggg acagtgaatc gtaagtatgc ctttcactgc gaggggttct ggagaggctt ctgagctccc catggcccag gcaggcagca ggtctggggc aggagggggg ttgtggagtg ggtatccgcc tgctgaggtg cagggcagat ggagaggctg cagctgagct cctattttca taataacagc agccatgagg gttgtgtcct gtttcccagt cctgcccggt cccccctcgg tacctcctgg tggatacact ggttcctgta agcagaagtg gatgagggtg tctaggtctg cagtcctggc accccaggat gggggacacc agccaagata cagcaacagc aacaaagcgc agccatttct ttctgtttgc acagctcctc tgtctgtcgg gggctcctgt ctgttgtctc ctataagcct caccacctct cctactgctt gggcatgcat ctttctcccc ttctatagat gaggaggtta aggtccagag aggggtgggg aggaacgccg gctcacattc tccatcccct ccagatatga ccaggaacag acctgtgcca ggcctcagcc ttacatcaaa atgggcctcc ccatgcaccg tggacctctg ggccctcctg tcccagtgga ggacaggaag ctatgagggg cactgtcacc cagggctcaa gctggcattc ctgaataatc gctctgcacc aggccacggc taagctcagt gcgtgattaa gcctcataac cctccaaggc agttactagt gtgattccca ttttacagat gaggaagatg gggacagaga ggtgaataac tggccccaaa tcacacacca tccataattc gggctcaggc acctggctcc agtccccaaa ctcttgaacc tggccctagt gtcactgttt ctcttgggtc tcaggcgctg gatggggaac aggaaacctg ggctggactt gaggcctctc tgatgctcgg tgacttcaga cagttgctca acctctctgt tctcttgggc aaaacatgat aacctttgac ttctgtcccc tcccctcacc ccacccgacc ttgatctctg aagtgttgga aggatttaat ttttcctgca ctgagttttg gagacaggtc aaaaagatga ccaaggccaa ggtggccagt ttcctataga acgcctctaa aagacctgca gcaatagcag caagaactgg tattctcgag aacttgctgc gcagcaggca cttcttggca ttttatgtgt atttaatttc acaatagctc tatgacaaag tccacctttc tcatctccag gaaactgagg ttcagagagg ttaagtaact tgtccaaggt cacacagcta atagcaagtt gacgtggagc aatctggcct cagagccttt aattttagcc acagactgat gctcccctct tcatttagcc aggctgcctc tgaagttttc tgattcaaga cttctggctt cagctttgta cacagagatg attcaatgtc aggttttgga gtgaaatctg tttaatccca gacaaaacat ttaggattac atctcagttt tgtaagcaag tagctctgtg atttttagtg agttatttaa tgctctttgg ggctcaattt ttctatctat aaaatagggc taataatttg caccttatag ggtaagcttt gaggacagat tagatgatac ggtgcctgta aaacaccagg tgttagtaag tgtggcaatg atggtgacgc tgaggctgat gtttgcttag catagggtta ggcagctggc aggcagtaaa cagttggata atttaatgga aaatttgcca aactcagatg ctagcagcta caatccagct accattctgc ttttatttta tggttgggat aaggctggat tattctgagt ccaagctagg cccttttgct aatcatgttc atacctctta tcttcctccc acagctcctg ggcaacgtgc tggtctgtgt gctggcccat cactttggca aagaattgcg atcgccacca tgagcactgc tgtgctggag aaccctggcc tgggcagaaa gctgtctgac tttggccagg agaccagcta cattgaggac aactgcaacc agaatggagc catcagcctg atcttcagcc tgaaggagga ggtgggagcc ctggccaagg tgctgagact gtttgaggag aatgatgtga acctgaccca cattgagagc agacccagca gactgaagaa ggatgagtat gagttcttca cccacctgga caagagaagc ctgcctgccc tgaccaacat catcaagatc ctgagacacg atattggagc cactgtgcac gagctgagca gagacaagaa gaaggacact gtgccctggt tccccagaac tatccaggag ctggacagat ttgccaacca gatcctgagc tatggagctg agctggatgc tgaccaccct ggcttcaagg accctgtgta cagagccaga agaaagcagt ttgctgacat tgcctacaac tacagacacg gccagcccat ccccagagtg gagtacatgg aggaggagaa gaagacctgg ggcactgtgt tcaagaccct gaagagcctg tacaagaccc acgcctgcta tgagtacaac cacatcttcc ccctgctgga gaagtactgt ggcttccacg aggacaacat cccccagctg gaggatgtga gccagttcct gcagacctgc actggcttca gactgagacc tgtggctggc ctgctgagca gcagagactt cctggggggc ctggccttca gagtgttcca ctgcacccag tacatcagac acggcagcaa gcccatgtac acccctgagc ctgatatctg ccacgagctg ctgggccacg tgcccctgtt ctctgacaga agctttgccc agttcagcca ggagattggc ctggccagcc tgggagcccc tgatgagtat attgagaagc tggccactat ctactggttc actgtggagt ttggcctgtg caagcagggg gacagcatca aggcctatgg agctggcctg ctgagcagct ttggggagct gcagtactgc ctgtctgaga agcccaagct gctgcccctg gagctggaga agactgccat ccagaactac actgtgactg agttccagcc cctgtactat gtggctgaga gcttcaatga tgccaaggag aaggtgagaa actttgctgc caccatcccc agacccttct ctgtgagata tgacccctac acccagagaa ttgaggtgct ggacaacacc cagcagctga agatcctggc tgacagcatc aactctgaga ttggcatcct gtgctctgcc ctgcagaaga tcaagtaacc tcgagctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg accggtgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcagg 44 Met Ser Phe Val Asp His Pro Pro Asp Trp Leu Glu Glu Val Gly Glu Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys Asn Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu 52 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgcggc cgcacgcgta ggctcagagg cacacaggag tttctgggct caccctgccc ccttccaacc cctcagttcc catcctccag cagctgtttg tgtgctgcct ctgaagtcca cactgaacaa acttcagcct actcatgtcc ctaaaatggg caaacattgc aagcagcaaa cagcaaacac acagccctcc ctgcctgctg accttggagc tggggcagag gtcagagacc tctctgggcc catgccacct ccaacatcca ctcgacccct tggaatttcg gtggagagga gcagaggttg tcctggcgtg gtttaggtag tgtgagaggg gtcgacgatc ttgctaccag tggaacagcc actaaggatt ctgcagtgag agcagagggc cagctaagtg gtactctccc agagactgtc tgactcacgc caccccctcc accttggaca caggacgctg tggtttctga gccaggtaca atgactcctt tcggtaagtg cagtggaagc tgtacactgc ccaggcaaag cgtccgggca gcgtaggcgg gcgactcaga tcccagccag tggacttagc ccctgtttgc tcctccgata actggggtga ccttggttaa tattcaccag cagcctcccc cgttgcccct ctggatccac tgcttaaata cggacgagga cagggccctg tctcctcagc ttcaggcacc accactgacc tgggacagtg aatcgtaagt atgcctttca ctgcgagggg ttctggagag gcttctgagc tccccatggc ccaggcaggc agcaggtctg gggcaggagg ggggttgtgg agtgggtatc cgcctgctga ggtgcagggc agatggagag gctgcagctg agctcctatt ttcataataa cagcagccat gagggttgtg tcctgtttcc cagtcctgcc cggtcccccc tcggtacctc ctggtggata cactggttcc tgtaagcaga agtggatgag ggtgtctagg tctgcagtcc tggcacccca ggatggggga caccagccaa gatacagcaa cagcaacaaa gcgcagccat ttctttctgt ttgcacagct cctctgtctg tcgggggctc ctgtctgttg tctcctataa gcctcaccac ctctcctact gcttgggcat gcatctttct ccccttctat agatgaggag gttaaggtcc agagaggggt ggggaggaac gccggctcac attctccatc ccctccagat atgaccagga acagacctgt gccaggcctc agccttacat caaaatgggc ctccccatgc accgtggacc tctgggccct cctgtcccag tggaggacag gaagctatga ggggcactgt cacccagggc tcaagctggc attcctgaat aatcgctctg caccaggcca cggctaagct cagtgcgtga ttaagcctca taaccctcca aggcagttac tagtgtgatt cccattttac agatgaggaa gatggggaca gagaggtgaa taactggccc caaatcacac accatccata attcgggctc aggcacctgg ctccagtccc caaactcttg aacctggccc tagtgtcact gtttctcttg ggtctcaggc gctggatggg gaacaggaaa cctgggctgg acttgaggcc tctctgatgc tcggtgactt cagacagttg ctcaacctct ctgttctctt gggcaaaaca tgataacctt tgacttctgt cccctcccct caccccaccc gaccttgatc tctgaagtgt tggaaggatt taatttttcc tgcactgagt tttggagaca ggtcaaaaag atgaccaagg ccaaggtggc cagtttccta tagaacgcct ctaaaagacc tgcagcaata gcagcaagaa ctggtattct cgagaacttg ctgcgcagca ggcacttctt ggcattttat gtgtatttaa tttcacaata gctctatgac aaagtccacc tttctcatct ccaggaaact gaggttcaga gaggttaagt aacttgtcca aggtcacaca gctaatagca agttgacgtg gagcaatctg gcctcagagc ctttaatttt agccacagac tgatgctccc ctcttcattt agccaggctg cctctgaagt tttctgattc aagacttctg gcttcagctt tgtacacaga gatgattcaa tgtcaggttt tggagtgaaa tctgtttaat cccagacaaa acatttagga ttacatctca gttttgtaag caagtagctc tgtgattttt agtgagttat ttaatgctct ttggggctca atttttctat ctataaaata gggctaataa tttgcacctt atagggtaag ctttgaggac agattagatg atacggtgcc tgtaaaacac caggtgttag taagtgtggc aatgatggtg acgctgaggc tgatgtttgc ttagcatagg gttaggcagc tggcaggcag taaacagttg gataatttaa tggaaaattt gccaaactca gatgctagca gctacaatcc agctaccatt ctgcttttat tttatggttg ggataaggct ggattattct gagtccaagc taggcccttt tgctaatcat gttcatacct cttatcttcc tcccacagct cctgggcaac gtgctggtct gtgtgctggc ccatcacttt ggcaaagaat tgcgatcgcc accatgagca ctgctgtgct ggagaaccct ggcctgggca gaaagctgtc tgactttggc caggagacca gctacattga ggacaactgc aaccagaatg gagccatcag cctgatcttc agcctgaagg aggaggtggg agccctggcc aaggtgctga gactgtttga ggagaatgat gtgaacctga cccacattga gagcagaccc agcagactga agaaggatga gtatgagttc ttcacccacc tggacaagag aagcctgcct gccctgacca acatcatcaa gatcctgaga cacgatattg gagccactgt gcacgagctg agcagagaca agaagaagga cactgtgccc tggttcccca gaactatcca ggagctggac agatttgcca accagatcct gagctatgga gctgagctgg atgctgacca ccctggcttc aaggaccctg tgtacagagc cagaagaaag cagtttgctg acattgccta caactacaga cacggccagc ccatccccag agtggagtac atggaggagg agaagaagac ctggggcact gtgttcaaga ccctgaagag cctgtacaag acccacgcct gctatgagta caaccacatc ttccccctgc tggagaagta ctgtggcttc cacgaggaca acatccccca gctggaggat gtgagccagt tcctgcagac ctgcactggc ttcagactga gacctgtggc tggcctgctg agcagcagag acttcctggg gggcctggcc ttcagagtgt tccactgcac ccagtacatc agacacggca gcaagcccat gtacacccct gagcctgata tctgccacga gctgctgggc cacgtgcccc tgttctctga cagaagcttt gcccagttca gccaggagat tggcctggcc agcctgggag cccctgatga gtatattgag aagctggcca ctatctactg gttcactgtg gagtttggcc tgtgcaagca gggggacagc atcaaggcct atggagctgg cctgctgagc agctttgggg agctgcagta ctgcctgtct gagaagccca agctgctgcc cctggagctg gagaagactg ccatccagaa ctacactgtg actgagttcc agcccctgta ctatgtggct gagagcttca atgatgccaa ggagaaggtg agaaactttg ctgccaccat ccccagaccc ttctctgtga gatatgaccc ctacacccag agaattgagg tgctggacaa cacccagcag ctgaagatcc tggctgacag catcaactct gagattggca tcctgtgctc tgccctgcag aagatcaagt aacctcgagc tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggaccggt gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagag agggagtggc caa The following drugs were prohibited starting 30 days prior to starting rAAV particle administration and until the end of the study: pegvar (except when rescue therapy was allowed); large neutral amino acid (LNAA) (except when rescue therapy was allowed) and systemic immunosuppressants, including CS (in excess of what is allowed under the protocol to reduce the risk of elevated transaminases). If corticosteroid administration is considered clinically ineffective, intolerable, and/or contraindicated, systemic immunosuppressive agents (eg, CS-stimulators such as inosine monophosphate dehydrogenase inhibitors and calcineurin inhibitors may be used) ). 7. Embodiment 1. A method of reducing plasma phenylalanine (Phe) levels in a human individual in need, comprising administering to the individual a therapeutically effective amount of recombinant adeno-associated virus (rAAV) particles, the recombinant adeno-associated virus (rAAV) particles comprise an AAV capsid and a recombinant vector construct comprising a nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcriptional regulatory region. 2. A method of treating a human individual suffering from phenylketonuria, comprising administering to the individual a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) particle comprising an AAV capsid, and a recombinant vector construct, the recombinant vector The construct contains nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcriptional regulatory region. 3. The method of embodiments 1-2, wherein the recombinant vector construct of the rAAV particle comprises: (a) one or both of the following: (i) AAV 5' inverted terminal repeats (ITR) and ( ii) AAV 3'ITRs; (b) heterologous liver-specific transcriptional regulatory regions; and (c) nucleic acids encoding functional human phenylalanine hydroxylase (hPAH), optionally wherein the AAV ITRs are AAV2 ITRs. 4. The method of embodiment 3, wherein the nucleic acid encoding a functional hPAH encodes an amino acid sequence that is at least 95% identical to SEQ ID NO: 2. 5. The method of any one of embodiments 1-4, the nucleic acid encoding a functional hPAH comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 1 or 7-13. 6. The method of any one of embodiments 1 to 5, wherein the PAH-encoding nucleic acid is operably linked to a promoter comprising a fragment of the hAAT promoter and a fragment of the HCR enhancer/ApoE enhancer. 7. The method of any one of embodiments 1 to 5, wherein the liver-specific transcriptional regulatory region comprises at least 90% identical to any one of SEQ ID NOs: 3, 4 or 24 or alternatively SEQ ID NO: Nucleotide sequences that are at least 90% identical to either of 25 or 26. 8. The method of any one of embodiments 1 to 5, wherein the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO:6. 9. The method of any one of embodiments 1-8, wherein the recombinant vector construct further comprises an intron. 10. The method of embodiment 9, wherein the intron comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14 or 27 or 29 or 34. 11. The method of any one of embodiments 1-10, wherein the recombinant vector construct further comprises a polyadenylation signal. 12. The method of embodiment 11, wherein the recombinant vector construct comprises a bovine growth hormone (bGH) polyadenylation signal. 13. The method of any one of embodiments 1 to 12, wherein the rAAV particle comprises a recombinant vector construct that is at least 90% identical to any one of SEQ NOs: 15 to 23 or 52. 14. The method of any one of embodiments 1-13, wherein the AAV capsid comprises an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 35-51. 15. The method of any one of embodiments 1 to 13, wherein the AAV capsid is an AAV capsid having liver tropism. 16. The method of embodiment 15, wherein the AAV capsid having liver tropism does not include AAV8 and/or AAVHSC15. 17. The method of embodiment 15, wherein the AAV capsid having liver tropism is an AAV5-type capsid that is at least 85%, 90% or 95% identical to SEQ ID NO: 44 as appropriate. 18. The method of any of the preceding embodiments, wherein the individual suffers from phenylketonuria (PKU). 19. The method of any of the preceding embodiments, wherein the individual has classic PKU or severe PKU. 20. The method of any of the preceding embodiments, wherein the subject has a plasma Phe level of 600 μmol/L or higher prior to the administration. 21. The method of any of the preceding embodiments, wherein the subject's plasma Phe level prior to the administration is 1200 μmol/L or higher. 22. The method of any of the preceding embodiments, wherein the individual is 15 years old or older. 23. The method of any preceding embodiment, wherein the individual is an adult. 24. The method of any preceding embodiment, wherein the individual is female. 25. The method of any one of the preceding embodiments, wherein the individual has a mutation in a gene encoding PAH, optionally a mutation F39L, L48S, I65T, R68S, A104D, S110C, D129G, E178G, V190A, P211T, R241C, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G, A395P, P407S and Y414C. 26. The method of any preceding embodiment, wherein the individual is a non-pregnant female. 27. The method of any of the preceding embodiments, wherein the individual is not receiving drug therapy for the treatment of PKU. 28. The method of embodiment 27, wherein the subject has not received Pegwari for at least 30 days prior to the administration. 29. The method of embodiment 27, wherein the subject has not received a large neutral amino acid for at least 30 days prior to the administering. 30. The method of embodiment 27, wherein the subject has not received sapropterin for at least 7 days prior to the administration. 31. The method of any of the preceding embodiments, wherein the subject has not received steroids for at least 30 days prior to the administering. 32. The method of any of the preceding embodiments, wherein prior to the administering, the individual has no detectable anti-AAV capsid antibodies in blood. 33. The method of any preceding embodiment, wherein the subject does not have clinically significant liver disease prior to the administration. 34. The method of any of the preceding embodiments, wherein the rAAV particles are administered intravenously in a single administration. 35. The method of any one of the preceding embodiments, wherein the rAAV particles are administered at a dose ranging from about 2E13 to about 2E14 vector gene bodies per kilogram of body weight of the subject (vg/kg). 36. The method of embodiment 35, wherein the rAAV particles are administered at a dose of about 2E13 vg/kg. 37. The method of embodiment 35, wherein the rAAV particles are administered at a dose of about 6E13 vg/kg. 38. The method of embodiment 35, wherein the rAAV particles are administered at a dose of about 2E14 vg/kg. 39. The method of any preceding embodiment, further comprising administering to the individual prophylactic immunosuppressive therapy. 40. The method of embodiment 39, wherein the prophylactic immunosuppressive treatment comprises administering a prophylactically effective amount of a systemic immunosuppressive agent to prevent liver toxicity, optionally concurrently with the administration of rAAV particles. 41. The method of embodiment 40, wherein the systemic immunosuppressant is a corticosteroid, optionally dexamethasone, prisone, prisulol, flucortisone, hydrocortisone, or budesonide. 42. The method of embodiment 40, wherein the systemic immunosuppressant is a corticosteroid and the prophylactically effective amount is a prisone equivalent dose of 10 mg/day to 40 mg/day, optionally for at least about 13 weeks period, followed by a decreasing amount of the corticosteroid for a period of about 3 weeks. 43. The method of any one of the preceding embodiments, further comprising the steps of: (a) before the administration, optionally a baseline of hepatotoxicity markers in the blood of the individual about one month prior to the administration levels, and (b) the post-administration level of the hepatotoxic marker in the subject's blood is determined weekly after the administration. 44. The method of embodiment 43, further comprising administering to the individual a therapeutic immunosuppressive treatment. 45. The method of embodiment 44, wherein the therapeutic immunosuppressive treatment comprises the steps of: (c) administering to the individual a therapeutically effective amount of a systemic immunosuppressive agent to reduce hepatotoxicity after biochemical or clinical symptoms are detected Hepatotoxicity. 46. The method of embodiment 44, wherein hepatotoxicity is detected by (i) the content of the hepatotoxic marker after administration is greater than the upper limit of normal (ULN) or (ii) the content of the hepatotoxic marker after administration is greater than or equal to twice the baseline level of the hepatotoxicity marker. 47. The method of any one of embodiments 44 to 46, wherein the systemic immunosuppressant is a corticosteroid, optionally dexamethasone, prisone, prisulol, flucortisone, hydrocortisone pine or budesonide. 48. The method of any one of embodiments 44 to 46, wherein the systemic immunosuppressant is a corticosteroid and the therapeutically effective amount is a prisone equivalent dose of 10 mg/day to 40 mg/day, depending on The condition persists for a period of at least about 5 weeks, followed by a tapering amount of the corticosteroid for a period of about 3 weeks. 49. The method of any one of embodiments 43-48, wherein the hepatotoxicity marker is ALT and/or AST. 50. The method of any one of embodiments 43-48, wherein the hepatotoxicity marker is ALT. 51. The method of any one of the preceding embodiments, further comprising by comprising the steps of extracting DNA from hepatocytes of the individual and detecting the circular vector gene body, monitoring the episomal body by PCR or Southern blotting as appropriate form. 52. The method of any of the preceding embodiments, further comprising the step of measuring the subject's plasma Phe levels weekly. 53. The method of any preceding embodiment, further comprising performing a Phe breath test. 54. The method of any preceding embodiment, further comprising the step of measuring weekly plasma levels of one or more neurotransmitters or neurotransmitter metabolites in the individual. 55. The method of embodiment 54, wherein the one or more neurotransmitters or neurotransmitter metabolites are phenethylamine, phenethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, Phenylacetic acid, phenacetylglutamic acid, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillyl mandelic acid, indoleacetic acid and 5-hydroxyindoleacetic acid. 56. The method of any preceding embodiment, wherein the subject's plasma Phe level is 360 μmol/L or less 8 weeks after the administration without concomitant drug therapy. 57. The method of any one of the preceding embodiments, wherein the subject's plasma Phe level in the absence of concomitant drug therapy is 360 μmol/L or less 2, 3 or 4 years after the administration. 58. The method of any of the preceding embodiments, wherein the subject's plasma Phe level, without concomitant drug therapy, is between 120 and 360 μmol/L 8 weeks after the administration. 59. The method of any one of the preceding embodiments, wherein the subject's plasma Phe level is 120 μmol/L or less 8 weeks after the administration without concomitant drug therapy. 60. The method of any one of the preceding embodiments, wherein the subject's plasma Phe level, without concomitant drug therapy, is 120 μmol/L or less 2, 3 or 4 years after the administration. 61. The method of any of the preceding embodiments, wherein the individual tolerates increased Phe intake compared to a Phe-restricted diet at baseline. 62. The method of any one of the preceding embodiments, wherein following the administration, blood levels of neurotransmitters or neurotransmitter metabolites in the individual are reduced. 63. The method of embodiment 62, wherein the one or more neurotransmitters or neurotransmitter metabolites are phenethylamine, phenethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, Phenylacetic acid, phenacetylglutamic acid, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillyl mandelic acid, indoleacetic acid and 5-hydroxyindoleacetic acid. 64. The method of any of the preceding embodiments, wherein the subject's quality of life improves after the administration, as measured by the PKU-QOL or Q-LES-Q-SF questionnaire, as appropriate. 65. The method of any of the preceding embodiments, wherein one or more neurocognitive symptoms or measures of the individual improve after the administration. 66. The method of any one of the preceding embodiments, wherein the individual does not experience persistent hypophenylalaninemia after the administration. 67. A pharmaceutical composition comprising rAAV particles at a concentration of at least 1E13 vg/ml to about 1E14 vg/ml, a buffer, an isotonicity agent, a cryopreservative and a surfactant, the pharmaceutical composition being at about -60°C Stable for at least about 1 year, 1.5 years or 2 years during storage at (minus sixty degrees Celsius) or lower temperatures. 68. The pharmaceutical composition of embodiment 67, wherein the concentration of the surfactant is less than 0.2% w/v or less than 0.15% w/v. 69. The pharmaceutical composition of embodiment 67, wherein the concentration of the surfactant is about 0.1% w/v. 70. The pharmaceutical composition of any one of embodiments 67-69, wherein the cryopreservative is a sugar. 71. The pharmaceutical composition of any one of embodiments 67 to 69, wherein the cryopreservative is trehalose. 72. A pharmaceutical composition comprising rAAV particles at a concentration of at least 1E13 vg/ml to about 1E14 vg/ml, the pharmaceutical composition comprising sodium phosphate at a concentration of about 5 to about 15 mM; a concentration of about 100 mM to about Sodium chloride at 165 mM; as a sugar, as the case may be, a cryopreservative for trehalose; and poloxamers or polysorbates at a concentration of less than 0.2% w/v. 73. The pharmaceutical composition of embodiment 72, comprising disodium hydrogen phosphate and sodium dihydrogen phosphate. 74. The pharmaceutical composition of any one of embodiments 72-73, wherein the sugar is trehalose at a concentration of about 60 mM to about 80 mM. 75. The pharmaceutical composition of any one of embodiments 72-74, wherein the poloxamer is Poloxamer 188 at a concentration of about 0.05% w/v to 0.15% w/v. 76. The pharmaceutical composition of embodiment 72, comprising sodium phosphate, sodium chloride, trehalose and poloxamer 188. 77. The pharmaceutical composition of embodiment 72 or 76, comprising sodium phosphate at a concentration of about 5 to about 15 mM, sodium chloride at a concentration of about 100 to about 140 mM, sugar at a concentration of about 60 to about 90 mM and a poloxamer at a concentration of from about 0.05% w/v to about 0.15% w/v. 78. The pharmaceutical composition of embodiment 72 or 76, comprising sodium phosphate at a concentration of about 8 to about 12 mM, sodium chloride at a concentration of about 110 to about 130 mM, sugar at a concentration of about 70 to about 80 mM and poloxamer at a concentration of from about 0.08% w/v to about 0.12% w/v. 79. The pharmaceutical composition of any one of embodiments 72 or 76, wherein the concentration of the sodium dihydrogen phosphate is greater than 0.1 mg/mL and less than 0.5 mg/mL, optionally about 0.3 mg/mL, and the hydrogen phosphate The concentration of disodium is greater than 2.5 mg/ml and less than 3 mg/ml, and about 2.7 mg/ml as appropriate. 80. The pharmaceutical composition of any one of embodiments 72 or 76 or 79, wherein the concentration of the sodium chloride is greater than 5 mg/ml and less than 8 mg/ml, optionally about 7 mg/ml. 81. The pharmaceutical composition of any one of embodiments 72 or 76 or 79 to 80, wherein the sugar is a concentration of greater than 20 mg/ml to less than 40 mg/ml, or about 25 mg/ml to about 35 mg/ml ml, or about 28 mg/ml of trehalose dihydrate. 82. The pharmaceutical composition of any one of embodiments 72 or 76 or 79 to 81, wherein the concentration of the poloxamer 188 is less than 1.5 mg/ml or about 1 mg/ml. 83. The pharmaceutical composition of any one of embodiments 72-82, wherein the concentration of the rAAV particles is about 6E13 vg/ml. 84. A pharmaceutical composition comprising rAAV particles at a concentration of about 6E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. 85. The pharmaceutical composition of any one of embodiments 72-84, which is a liquid. 86. The pharmaceutical composition of any one of embodiments 72-84, which is lyophilized. 87. The pharmaceutical composition of any one of embodiments 72-84 for intravenous administration of rAAV particles to a patient suffering from phenylketonuria. 88. A method of producing rAAV particles, comprising (a) providing one or more nucleic acid constructs for cells (such as insect cells or mammalian cells) that allow AAV replication, the one or more nucleic acid constructs comprising: (i) recombination A vector construct comprising (1) at least one AAV ITR, (2) a heterologous liver-specific translation regulatory region, and (3) a nucleic acid encoding a functional human phenylalanine hydroxylase (hPAH); (ii) a nucleic acid encoding one or Nucleotide sequences of various Rep proteins operably linked to a promoter capable of driving expression of the one or more Rep proteins in the cell; and (iii) nucleotides encoding one or more AAV capsid proteins a sequence operably linked to a promoter capable of driving the expression of the one or more capsid proteins in the cell; (b) culturing the cell under conditions that allow the expression of the Rep and capsid proteins; and optionally In (c) the AAV particles are recovered. 89. The method of embodiment 88, wherein the cell is an insect cell. 90. The method of embodiment 88, wherein the cell is a mammalian cell. 91. The method of any one of embodiments 88-90, wherein the nucleic acid encoding a functional hPAH encodes an amino acid sequence that is at least 95% identical to SEQ ID NO:2. 92. The method of any one of embodiments 88-90, wherein the nucleic acid encoding a functional hPAH comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 1 or 7-13. 93. The method of any one of embodiments 88-90, wherein the PAH-encoding nucleic acid is operably linked to a promoter comprising a fragment of the hAAT promoter and a fragment of the HCR enhancer/ApoE enhancer. 94. The method of any one of embodiments 88 to 93, wherein the liver-specific transcriptional regulatory region comprises at least 90% identical to any one of SEQ ID NOs: 3, 4 or 24 or alternatively SEQ ID NO: Nucleotide sequences that are at least 90% identical to either of 25 or 26. 95. The method of any one of embodiments 88-93, wherein the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO:6. 96. The method of any one of embodiments 88-95, wherein the recombinant vector construct further comprises an intron. 97. The method of embodiment 96, wherein the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 14 or 27 or 29 or 34. 98. The method of any one of embodiments 88-97, wherein the recombinant vector construct further comprises a polyadenylation signal. 99. The method of embodiment 98, wherein the recombinant vector construct comprises a bovine growth hormone (bGH) polyadenylation signal. 100. The method of any one of embodiments 88 to 90, wherein the recombinant vector construct comprises a nucleotide sequence that is at least 90% identical to SEQ ID NOs: 15 to 23 or 52. 101. The method of any one of embodiments 88-100, wherein the AAV capsid comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 35-51. 102. The method of any one of embodiments 88 to 100, wherein the AAV capsid is an AAV capsid having liver tropism. 103. The method of embodiment 102, wherein the AAV capsid having liver tropism does not include AAV8 and/or AAVHSC15. 104. The method of embodiment 102, wherein the AAV capsid having liver tropism is an AAV5-type capsid that is at least 85%, 90% or 95% identical to SEQ ID NO: 44 as appropriate. 105. A population of rAAV particles produced by the method of any one of embodiments 88 to 104, optionally enriched for particles comprising full-length or nearly full-length vector gene bodies by a step of reducing the number of empty capsids. 106. A method of reducing plasma Phe levels in a human subject in need thereof, comprising administering the population of rAAV particles of embodiment 105. 107. A method of treating PKU in a human subject in need thereof, comprising administering the population of rAAV particles of embodiment 105. 8. Featured Sequences SEQ ID NO: sequence 18 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tgcggccgca cgcgtaggct cagaggcaca caggagtttc tgggctcacc ctgccccctt ccaacccctc agttcccatc ctccagcagc tgtttgtgtg ctgcctctga agtccacact gaacaaactt cagcctactc atgtccctaa aatgggcaaa cattgcaagc agcaaacagc aaacacacag ccctccctgc ctgctgacct tggagctggg gcagaggtca gagacctctc tgggcccatg ccacctccaa catccactcg accccttgga atttcggtgg agaggagcag aggttgtcct ggcgtggttt aggtagtgtg agaggggtcg acgatcttgc taccagtgga acagccacta aggattctgc agtgagagca gagggccagc taagtggtac tctcccagag actgtctgac tcacgccacc ccctccacct tggacacagg acgctgtggt ttctgagcca ggtacaatga ctcctttcgg taagtgcagt ggaagctgta cactgcccag gcaaagcgtc cgggcagcgt aggcgggcga ctcagatccc agccagtgga cttagcccct gtttgctcct ccgataactg gggtgacctt ggttaatatt caccagcagc ctcccccgtt gcccctctgg atccactgct taaatacgga cgaggacagg gccctgtctc ctcagcttca ggcaccacca ctgacctggg acagtgaatc gtaagtatgc ctttcactgc gaggggttct ggagaggctt ctgagctccc catggcccag gcaggcagca ggtctggggc aggagggggg ttgtggagtg ggtatccgcc tgctgaggtg cagggcagat ggagaggctg cagctgagct cctattttca taataacagc agccatgagg gttgtgtcct gtttcccagt cctgcccggt cccccctcgg tacctcctgg tggatacact ggttcctgta agcagaagtg gatgagggtg tctaggtctg cagtcctggc accccaggat gggggacacc agccaagata cagcaacagc aacaaagcgc agccatttct ttctgtttgc acagctcctc tgtctgtcgg gggctcctgt ctgttgtctc ctataagcct caccacctct cctactgctt gggcatgcat ctttctcccc ttctatagat gaggaggtta aggtccagag aggggtgggg aggaacgccg gctcacattc tccatcccct ccagatatga ccaggaacag acctgtgcca ggcctcagcc ttacatcaaa atgggcctcc ccatgcaccg tggacctctg ggccctcctg tcccagtgga ggacaggaag ctatgagggg cactgtcacc cagggctcaa gctggcattc ctgaataatc gctctgcacc aggccacggc taagctcagt gcgtgattaa gcctcataac cctccaaggc agttactagt gtgattccca ttttacagat gaggaagatg gggacagaga ggtgaataac tggccccaaa tcacacacca tccataattc gggctcaggc acctggctcc agtccccaaa ctcttgaacc tggccctagt gtcactgttt ctcttgggtc tcaggcgctg gatggggaac aggaaacctg ggctggact t gaggcctctc tgatgctcgg tgacttcaga cagttgctca acctctctgt tctcttgggc aaaacatgat aacctttgac ttctgtcccc tcccctcacc ccacccgacc ttgatctctg aagtgttgga aggatttaat ttttcctgca ctgagttttg gagacaggtc aaaaagatga ccaaggccaa ggtggccagt ttcctataga acgcctctaa aagacctgca gcaatagcag caagaactgg tattctcgag aacttgctgc gcagcaggca cttcttggca ttttatgtgt atttaatttc acaatagctc tatgacaaag tccacctttc tcatctccag gaaactgagg ttcagagagg ttaagtaact tgtccaaggt cacacagcta atagcaagtt gacgtggagc aatctggcct cagagccttt aattttagcc acagactgat gctcccctct tcatttagcc aggctgcctc tgaagttttc tgattcaaga cttctggctt cagctttgta cacagagatg attcaatgtc aggttttgga gtgaaatctg tttaatccca gacaaaacat ttaggattac atctcagttt tgtaagcaag tagctctgtg atttttagtg agttatttaa tgctctttgg ggctcaattt ttctatctat aaaatagggc taataatttg caccttatag ggtaagcttt gaggacagat tagatgatac ggtgcctgta aaacaccagg tgttagtaag tgtggcaatg atggtgacgc tgaggctgat gtttgcttag catagggtta ggcagctggc aggcagtaaa cagttggata atttaatgga aaatttgcca aactcagatg ctagcagcta caatccagct accattct gc ttttatttta tggttgggat aaggctggat tattctgagt ccaagctagg cccttttgct aatcatgttc atacctctta tcttcctccc acagctcctg ggcaacgtgc tggtctgtgt gctggcccat cactttggca aagaattgcg atcgccacca tgagcactgc tgtgctggag aaccctggcc tgggcagaaa gctgtctgac tttggccagg agaccagcta cattgaggac aactgcaacc agaatggagc catcagcctg atcttcagcc tgaaggagga ggtgggagcc ctggccaagg tgctgagact gtttgaggag aatgatgtga acctgaccca cattgagagc agacccagca gactgaagaa ggatgagtat gagttcttca cccacctgga caagagaagc ctgcctgccc tgaccaacat catcaagatc ctgagacacg atattggagc cactgtgcac gagctgagca gagacaagaa gaaggacact gtgccctggt tatccaggag ctggacagat ttgccaacca gatcctgagc tatggagctg agctggatgc tgaccaccct ggcttcaagg accctgtgta cagagccaga agaaagcagt ttgctgacat tgcctacaac tacagacacg gccagcccat ccccagagtg gagtacatgg aggaggagaa gaagacctgg ggcactgtgt tcaagaccct gaagagcctg tacaagaccc acgcctgcta tgagtacaac cacatcttcc ccctgctgga gaagtactgt ggcttccacg aggacaacat cccccagctg gaggatgtga gccagttcct gcagacctgc actggcttca gactgagacc tgtggctggc ctgctga tccccagaac gca gcagagactt cctggggggc ctggccttca gagtgttcca ctgcacccag tacatcagac acggcagcaa gcccatgtac acccctgagc ctgatatctg ccacgagctg ctgggccacg tgcccctgtt ctctgacaga agctttgccc agttcagcca ggagattggc ctggccagcc tgggagcccc tgatgagtat attgagaagc tggccactat ctactggttc actgtggagt ttggcctgtg caagcagggg gacagcatca aggcctatgg agctggcctg ctgagcagct ttggggagct gcagtactgc ctgtctgaga agcccaagct gctgcccctg gagctggaga agactgccat ccagaactac actgtgactg agttccagcc cctgtactat gtggctgaga gcttcaatga tgccaaggag aaggtgagaa actttgctgc caccatcccc agacccttct ctgtgagata tgacccctac acccagagaa ttgaggtgct ggacaacacc cagcagctga agatcctggc tgacagcatc aactctgaga ttggcatcct gtgctctgcc ctgcagaaga tcaagtaacc tcgagctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg accggtgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgct cact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcagg 44 Met Ser Phe Val Asp His Pro Asp Trp Leu Glu Glu Val Gly Glu Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys Asn Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu 52 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgcggc cgcacgcgta ggctcagagg cacacaggag tttctgggct caccctgccc ccttccaacc cctcagttcc catcctccag cagctgtttg tgtgctgcct ctgaagtcca cactgaacaa acttcagcct actcatgtcc ctaaaatggg caaacattgc aagcagcaaa cagcaaacac acagccctcc ctgcctgctg accttggagc tggggcagag gtcagagacc tctctgggcc catgccacct ccaacatcca ctcgacccct tggaatttcg gtggagagga gcagaggttg tcctggcgtg gtttaggtag tgtgagaggg gtcgacgatc ttgctaccag tggaacagcc actaaggatt ctgcagtgag agcagagggc cagctaagtg gtactctccc agagactgtc tgactcacgc caccccctcc accttggaca caggacgctg tggtttctga gccaggtaca atgactcctt tcggtaagtg cagtggaagc tgtacactgc ccaggcaaag cgtccgggca gcgtaggcgg gcgactcaga tcccagccag tggacttagc ccctgtttgc tcctccgata actggggtga ccttggttaa tattcaccag cagcctcccc cgttgcccct ctggatccac tgcttaaata cggacgagga cagggccctg tctcctcagc ttcaggcacc accactgacc tgggacagtg aatcgtaagt atgcctttca ctgcgagggg ttctggagag gcttctgagc tccccatggc ccaggcaggc agcaggtctg gggcaggagg ggggttgtgg agtgggtatc cgcctgctga ggtgcagggc agatggagag gctgcagctg agctcctatt ttcataataa cagcagccat gagggttgtg tcctgtttcc cagtcctgcc cggtcccccc tcggtacctc ctggtggata cactggttcc tgtaagcaga agtggatgag ggtgtctagg tctgcagtcc tggcacccca ggatggggga caccagccaa gatacagcaa cagcaacaaa gcgcagccat ttctttctgt ttgcacagct cctctgtctg tcgggggctc ctgtctgttg tctcctataa gcctcaccac ctctcctact gcttgggcat gcatctttct ccccttctat agatgaggag gttaaggtcc agagaggggt ggggaggaac gccggctcac attctccatc ccctccagat atgaccagga acagacctgt gccaggcctc agccttacat caaaatgggc ctccccatgc accgtggacc tctgggccct cctgtcccag tggaggacag gaagctatga ggggcactgt cacccagggc tcaagctggc attcctgaat aatcgctctg caccaggcca cggctaagct cagtgcgtga ttaagcctca taaccctcca aggcagttac tagtgtgatt cccattttac agatgaggaa gatggggaca gagaggtgaa taactggccc caaatcacac accatccata attcgggctc aggcacctgg ctccagtccc caaactcttg aacctggccc tagtgtcact gtttctcttg ggtctcaggc gctggatggg gaacaggaaa cctgggctg g acttgaggcc tctctgatgc tcggtgactt cagacagttg ctcaacctct ctgttctctt gggcaaaaca tgataacctt cccctcccct caccccaccc gaccttgatc tctgaagtgt tggaaggatt taatttttcc tgcactgagt ggtcaaaaag atgaccaagg ccaaggtggc cagtttccta tagaacgcct ctaaaagacc tgcagcaata gcagcaagaa ctggtattct cgagaacttg ctgcgcagca ggcacttctt ggcattttat gtgtatttaa tttcacaata gctctatgac aaagtccacc tttctcatct ccaggaaact gaggttcaga gaggttaagt aacttgtcca aggtcacaca gctaatagca agttgacgtg gagcaatctg gcctcagagc ctttaatttt agccacagac tgatgctccc ctcttcattt agccaggctg tgacttctgt tttggagaca cctctgaagt tttctgattc aagacttctg gcttcagctt tgtacacaga gatgattcaa tgtcaggttt tggagtgaaa tctgtttaat cccagacaaa acatttagga ttacatctca gttttgtaag caagtagctc tgtgattttt agtgagttat ttaatgctct ttggggctca atttttctat ctataaaata gggctaataa tttgcacctt atagggtaag ctttgaggac agattagatg atacggtgcc tgtaaaacac caggtgttag taagtgtggc aatgatggtg acgctgaggc tgatgtttgc ttagcatagg gttaggcagc tggcaggcag taaacagttg gataatttaa tggaaaattt gccaaactca gatgctagca gctacaatcc agctacca tt ctgcttttat tttatggttg ggataaggct ggattattct gagtccaagc taggcccttt tgctaatcat gttcatacct cttatcttcc tcccacagct cctgggcaac gtgctggtct gtgtgctggc ccatcacttt ggcaaagaat tgcgatcgcc agcagactga accatgagca ctgctgtgct ggagaaccct ggcctgggca gaaagctgtc tgactttggc caggagacca gctacattga ggacaactgc aaccagaatg gagccatcag cctgatcttc agcctgaagg aggaggtggg agccctggcc aaggtgctga gactgtttga ggagaatgat gtgaacctga cccacattga gagcagaccc acatcatcaa gatcctgaga cacgatattg agaaggatga gtatgagttc ttcacccacc tggacaagag aagcctgcct gccctgacca gagccactgt gcacgagctg agcagagaca agaagaagga cactgtgccc tggttcccca gaactatcca ggagctggac agatttgcca accagatcct gagctatgga gctgagctgg atgctgacca ccctggcttc aaggaccctg tgtacagagc cagaagaaag cagtttgctg acattgccta caactacaga cacggccagc ccatccccag agtggagtac atggaggagg agaagaagac ctggggcact gtgttcaaga ccctgaagag cctgtacaag acccacgcct gctatgagta caaccacatc ttccccctgc tggagaagta ctgtggcttc cacgaggaca acatccccca gctggaggat gtgagccagt tcctgcagac ctgcactggc ttcagactga gacctgtggc tggcctg ctg agcagcagag acttcctggg gggcctggcc ttcagagtgt tccactgcac ccagtacatc agacacggca gcaagcccat gtacacccct gagcctgata tctgccacga gctgctgggc cacgtgcccc tgttctctga cagaagcttt gcccagttca gccaggagat tggcctggcc agcctgggag cccctgatga gtatattgag aagctggcca ctatctactg gttcactgtg gagtttggcc tgtgcaagca gggggacagc atcaaggcct atggagctgg cctgctgagc agctttgggg agctgcagta ctgcctgtct gagaagccca agctgctgcc cctggagctg gagaagactg ccatccagaa ctacactgtg actgagttcc agcccctgta ctatgtggct gagagcttca atgatgccaa ggagaaggtg agaaactttg ctgccaccat ccccagaccc ttctctgtga gatatgaccc ctacacccag agaattgagg tgctggacaa cacccagcag ctgaagatcc tggctgacag catcaactct gagattggca tcctgtgctc tgccctgcag aagatcaagt aacctcgagc tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggaccggt gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgct cgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagag agggagtggc caa

本文所描述之實施例僅意欲為例示性的，且熟習此項技術者將認識到，或將能夠僅使用常規實驗確定特定化合物、材料及程序之許多等效物。所有此類等效物視為在本發明之範疇內。The embodiments described herein are intended to be illustrative only, and those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to specific compounds, materials and procedures. All such equivalents are considered to be within the scope of this invention.

本文所提及之所有專利、專利申請案及公開案以全文引用之方式併入本文中。本申請案中任何參考文獻之引用或認同並非承認該參考文獻可用作本申請案之先前技術。參看所附申請專利範圍更好地理解本發明之完整範疇。All patents, patent applications, and publications mentioned herein are incorporated by reference in their entirety. Citation or identification of any reference in this application is not an admission that such reference is available as prior art to this application. See the appended claims for a better understanding of the full scope of the invention.

圖1A展示當在pH 7.1、7.5或7.9下在4℃下儲存於緩衝液中時，隨時間推移之AAV5顆粒之脫醯胺含量(脫醯胺百分比)。圖1B展示當在pH 7.1、7.5或7.9下在室溫下儲存於緩衝液中時，隨時間推移之AAV5顆粒之脫醯胺含量(脫醯胺百分比)。Figure 1A shows the deamidation content (percent deamidation) of AAV5 particles over time when stored in buffer at pH 7.1, 7.5 or 7.9 at 4°C. Figure IB shows the deamidation content (percent deamidation) of AAV5 particles over time when stored in buffer at pH 7.1, 7.5 or 7.9 at room temperature.

圖2A至2D展示AAV5顆粒之穩定性(聚集含量自初始時間點之變化百分比)，其分別在以下各種儲存條件下調配於2%甘露醇或2.8%海藻糖中：≤-60℃、2至8℃、25℃/60% RH及37℃。圖2A：儲存於≤-60℃下之rAAV5顆粒隨時間推移之聚集變化。圖2B：儲存於2至8℃下之rAAV5顆粒隨時間推移之聚集變化。圖2C：儲存於25℃/60% RH下之rAAV5顆粒隨時間推移之聚集變化。圖2D：儲存於37℃下之rAAV5顆粒隨時間推移之聚集變化。Figures 2A to 2D show the stability of AAV5 particles (percent change in aggregate content from initial time point) formulated in 2% mannitol or 2.8% trehalose under various storage conditions: ≤-60°C, 2 to 8°C, 25°C/60% RH and 37°C. Figure 2A: Aggregation changes over time of rAAV5 particles stored at <-60°C. Figure 2B: Aggregation changes over time of rAAV5 particles stored at 2 to 8°C. Figure 2C: Aggregation changes over time of rAAV5 particles stored at 25°C/60% RH. Figure 2D: Aggregation changes over time of rAAV5 particles stored at 37°C.

Claims

一種包含濃度為至少1E13 vg/ml至約1E14 vg/ml之rAAV顆粒之醫藥組合物，該醫藥組合物包含以下：濃度約5至約15 mM之磷酸鈉；濃度約100 mM至約165 mM之氯化鈉；做為冷凍保存劑之糖(視情況為海藻糖)；及濃度低於0.2% w/v之泊洛沙姆(poloxamer)或聚山梨醇酯。A pharmaceutical composition comprising rAAV particles at a concentration of at least 1E13 vg/ml to about 1E14 vg/ml, the pharmaceutical composition comprising the following: sodium phosphate at a concentration of about 5 to about 15 mM; sodium phosphate at a concentration of about 100 mM to about 165 mM Sodium chloride; sugar (trehalose as appropriate) as cryopreservative; and poloxamer or polysorbate at a concentration of less than 0.2% w/v.

如請求項1之醫藥組合物，其包含磷酸氫二鈉及磷酸二氫鈉。The pharmaceutical composition of claim 1, comprising disodium hydrogen phosphate and sodium dihydrogen phosphate.

如請求項1或2之醫藥組合物，其中該糖為濃度約60 mM至約80 mM之海藻糖。The pharmaceutical composition of claim 1 or 2, wherein the sugar is trehalose at a concentration of about 60 mM to about 80 mM.

如請求項1至3中任一項之醫藥組合物，其中該泊洛沙姆為濃度約0.05% w/v至0.15% w/v之泊洛沙姆188。The pharmaceutical composition of any one of claims 1 to 3, wherein the poloxamer is Poloxamer 188 at a concentration of about 0.05% w/v to 0.15% w/v.

如請求項1之醫藥組合物，其包含磷酸鈉、氯化鈉、海藻糖及泊洛沙姆188。The pharmaceutical composition of claim 1, comprising sodium phosphate, sodium chloride, trehalose and poloxamer 188.

如請求項1或5之醫藥組合物，其包含濃度約5至約15 mM之磷酸鈉、濃度約100至約140 mM之氯化鈉、濃度約60至約90 mM之糖及濃度約0.05% w/v至約0.15% w/v之泊洛沙姆。The pharmaceutical composition of claim 1 or 5, comprising sodium phosphate at a concentration of about 5 to about 15 mM, sodium chloride at a concentration of about 100 to about 140 mM, sugar at a concentration of about 60 to about 90 mM, and at a concentration of about 0.05% w/v to about 0.15% w/v Poloxamer.

如請求項1或5之醫藥組合物，其包含濃度約8至約12 mM之磷酸鈉、濃度約110至約130 mM之氯化鈉、濃度約70至約80 mM之糖及濃度約0.08% w/v至約0.12% w/v之泊洛沙姆。The pharmaceutical composition of claim 1 or 5, comprising sodium phosphate at a concentration of about 8 to about 12 mM, sodium chloride at a concentration of about 110 to about 130 mM, sugar at a concentration of about 70 to about 80 mM, and a concentration of about 0.08% w/v to about 0.12% w/v Poloxamer.

如請求項1或5之醫藥組合物，其中該磷酸二氫鈉之濃度大於0.1 mg/mL且小於0.5 mg/mL，視情況約0.3 mg/mL，且該磷酸氫二鈉之濃度大於2.5 mg/ml且小於3 mg/ml，視情況約2.7 mg/mL。The pharmaceutical composition of claim 1 or 5, wherein the concentration of the sodium dihydrogen phosphate is greater than 0.1 mg/mL and less than 0.5 mg/mL, depending on the situation, about 0.3 mg/mL, and the concentration of the disodium hydrogen phosphate is greater than 2.5 mg /ml and less than 3 mg/ml, about 2.7 mg/mL as appropriate.

5或8之醫藥組合物，其中該氯化鈉之濃度大於5 mg/ml且小於8 mg/ml，視情況約7 mg/ml。The pharmaceutical composition of 5 or 8, wherein the concentration of the sodium chloride is greater than 5 mg/ml and less than 8 mg/ml, and optionally about 7 mg/ml.

5、8或9之醫藥組合物，其中該糖為濃度大於20 mg/ml至小於40 mg/ml，或約25 mg/ml至約35 mg/ml，或約28 mg/ml之二水合海藻糖。The pharmaceutical composition of 5, 8 or 9, wherein the sugar is a dihydrate seaweed in a concentration of greater than 20 mg/ml to less than 40 mg/ml, or about 25 mg/ml to about 35 mg/ml, or about 28 mg/ml sugar.

5或8至10中任一項之醫藥組合物，其中該泊洛沙姆188之濃度小於1.5 mg/ml或約1 mg/ml。5 or the pharmaceutical composition of any one of 8 to 10, wherein the concentration of the poloxamer 188 is less than 1.5 mg/ml or about 1 mg/ml.

如請求項1第11中任一項之醫藥組合物，其中該rAAV顆粒之濃度約6E13 vg/ml。The pharmaceutical composition of any one of claims 1 to 11, wherein the concentration of the rAAV particles is about 6E13 vg/ml.

一種醫藥組合物，其包含濃度至少1E13 vg/ml至約1E14 vg/ml之重組AAV (recombinant AAV；rAAV)顆粒、緩衝劑、等張劑、冷凍保存劑及界面活性劑，該醫藥組合物在約-60℃(攝氏零下六十度)或更低溫度下儲存期間穩定持續至少約1年、1.5年或2年。A pharmaceutical composition comprising recombinant AAV (recombinant AAV; rAAV) particles at a concentration of at least 1E13 vg/ml to about 1E14 vg/ml, a buffer, an isotonic agent, a cryopreservation agent and a surfactant, the pharmaceutical composition is in Stable for at least about 1 year, 1.5 years or 2 years during storage at about -60°C (minus sixty degrees Celsius) or below.

如請求項13之醫藥組合物，其中該界面活性劑之濃度小於0.2% w/v或小於0.15% w/v。The pharmaceutical composition of claim 13, wherein the concentration of the surfactant is less than 0.2% w/v or less than 0.15% w/v.

如請求項13之醫藥組合物，其中該界面活性劑之濃度約0.1% w/v。The pharmaceutical composition of claim 13, wherein the concentration of the surfactant is about 0.1% w/v.

如請求項13至15中任一項之醫藥組合物，其中該冷凍保存劑為糖。The pharmaceutical composition of any one of claims 13 to 15, wherein the cryopreservative is a sugar.

如請求項1至15中任一項之醫藥組合物，其中該冷凍保存劑為海藻糖。The pharmaceutical composition of any one of claims 1 to 15, wherein the cryopreservative is trehalose.

一種醫藥組合物，其包含濃度約6E13 vg/ml之rAAV顆粒、10 mM磷酸鈉、120 mM氯化鈉、74 mM二水合海藻糖及0.1% w/v泊洛沙姆188。A pharmaceutical composition comprising rAAV particles at a concentration of about 6E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188.

如請求項6至18中任一項之醫藥組合物，其為液體。The pharmaceutical composition of any one of claims 6 to 18, which is a liquid.

如請求項6至18中任一項之醫藥組合物，其經凍乾。The pharmaceutical composition of any one of claims 6 to 18, which is lyophilized.

如請求項6至18中任一項之醫藥組合物，其用於向患有苯酮尿症之患者靜脈內投與rAAV顆粒。The pharmaceutical composition of any one of claims 6 to 18 for the intravenous administration of rAAV particles to a patient suffering from phenylketonuria.

如請求項1至21中任一項之醫藥組合物，其中該rAAV顆粒包含AAV衣殼及重組載體建構體，該重組載體建構體包含編碼功能性***酸羥化酶(phenylalanine hydroxylase；PAH)之核酸及視情況選用之異源肝特異性轉錄調節區。The pharmaceutical composition of any one of claims 1 to 21, wherein the rAAV particle comprises an AAV capsid and a recombinant vector construct comprising a protein encoding a functional phenylalanine hydroxylase (PAH) Nucleic acids and optionally heterologous liver-specific transcriptional regulatory regions.

如請求項22之醫藥組合物，其中該重組AAV載體，且其中該重組AAV載體包含：(a)以下中之一或兩者：(i) AAV 5'反向末端重複序列(inverted terminal repeat；ITR)及(ii) AAV 3' ITR；(b)異源肝特異性轉錄調節區；及(c)編碼功能性人類***酸羥化酶(human phenylalanine hydroxylase；hPAH)之核酸，視情況其中該等AAV ITR為AAV2 ITR。The pharmaceutical composition of claim 22, wherein the recombinant AAV vector, and wherein the recombinant AAV vector comprises: (a) one or both of the following: (i) AAV 5' inverted terminal repeats; ITR) and (ii) AAV 3' ITR; (b) a heterologous liver-specific transcriptional regulatory region; and (c) a nucleic acid encoding a functional human phenylalanine hydroxylase (hPAH), where appropriate etc. AAV ITR is AAV2 ITR.

如請求項22或23之醫藥組合物，其中該編碼功能性hPAH之核酸編碼與SEQ ID NO: 2至少95%一致的胺基酸序列。The pharmaceutical composition of claim 22 or 23, wherein the nucleic acid encoding a functional hPAH encodes an amino acid sequence that is at least 95% identical to SEQ ID NO: 2.

如請求項22或23中任一項之醫藥組合物，其中該編碼功能性hPAH之核酸包含與SEQ ID NO: 1或7至13至少90%一致的核苷酸序列。The pharmaceutical composition of any one of claims 22 or 23, wherein the nucleic acid encoding a functional hPAH comprises a nucleotide sequence that is at least 90% identical to SEQ ID NOs: 1 or 7 to 13.

如請求項22至26中任一項之醫藥組合物，其中該編碼PAH之核酸可操作地連接至包含hAAT啟動子之片段的啟動子及HCR增強子/ApoE增強子之片段。The pharmaceutical composition of any one of claims 22 to 26, wherein the PAH-encoding nucleic acid is operably linked to a promoter comprising a fragment of the hAAT promoter and a fragment of the HCR enhancer/ApoE enhancer.

如請求項22至26中任一項之醫藥組合物，其中該肝特異性轉錄調節區包含與SEQ ID NO: 3、4或24中之任一者至少90%一致或可替代地與SEQ ID NO: 25或26中之任一者至少90%一致的核苷酸序列。The pharmaceutical composition of any one of claims 22 to 26, wherein the liver-specific transcriptional regulatory region comprises at least 90% identical to any one of SEQ ID NOs: 3, 4 or 24 or alternatively SEQ ID NO: Nucleotide sequences that are at least 90% identical to either of 25 or 26.

如請求項22至27中任一項之醫藥組合物，其中該重組載體建構體包含與SEQ ID NO: 6至少90%一致之核苷酸序列。The pharmaceutical composition of any one of claims 22 to 27, wherein the recombinant vector construct comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:6.

如請求項22至26中任一項之醫藥組合物，其中該重組載體建構體進一步包含內含子。The pharmaceutical composition of any one of claims 22 to 26, wherein the recombinant vector construct further comprises an intron.

如請求項29之醫藥組合物，其中該內含子包含與SEQ ID NO: 14或27或29或34至少90%一致之核苷酸序列。The pharmaceutical composition of claim 29, wherein the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 14 or 27 or 29 or 34.

如請求項22至29中任一項之醫藥組合物，其中該重組載體建構體進一步包含多腺苷酸化信號。The pharmaceutical composition of any one of claims 22 to 29, wherein the recombinant vector construct further comprises a polyadenylation signal.

如請求項31之醫藥組合物，其中該重組載體建構體包含牛生長激素(bovine growth hormone；bGH)多腺苷酸化信號。The pharmaceutical composition of claim 31, wherein the recombinant vector construct comprises a bovine growth hormone (bGH) polyadenylation signal.

如請求項22至32中任一項之醫藥組合物，其中該rAAV顆粒包含與SEQ NO: 15至23或52中之任一者至少90%一致的重組載體建構體。The pharmaceutical composition of any one of claims 22 to 32, wherein the rAAV particle comprises a recombinant vector construct that is at least 90% identical to any one of SEQ NOs: 15 to 23 or 52.

如請求項22至33中任一項之醫藥組合物，其中該AAV衣殼包含與SEQ ID NO: 35至51中之任一者至少85%一致的胺基酸序列。The pharmaceutical composition of any one of claims 22 to 33, wherein the AAV capsid comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 35 to 51.

如請求項1至34中任一項之醫藥組合物，其中該AAV衣殼為具有肝趨向性之AAV衣殼。The pharmaceutical composition of any one of claims 1 to 34, wherein the AAV capsid is an AAV capsid with liver tropism.

如請求項35之醫藥組合物，其中具有肝趨向性之該AAV衣殼不包括AAV8及/或AAVHSC15。The pharmaceutical composition of claim 35, wherein the AAV capsid having liver tropism does not include AAV8 and/or AAVHSC15.

如請求項35之醫藥組合物，其中具有肝趨向性之該AAV衣殼為AAV5型衣殼，視情況與SEQ ID NO: 44至少85%、90%或95%一致。The pharmaceutical composition of claim 35, wherein the AAV capsid having liver tropism is an AAV5-type capsid that is at least 85%, 90% or 95% identical to SEQ ID NO: 44 as appropriate.

一種降低有需要之人類個體中血漿***酸(phenylalanine；Phe)含量之方法，其包含向該個體投與治療有效量之如請求項22至38中任一項之醫藥組合物。A method of reducing plasma phenylalanine (Phe) levels in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 22-38.

如請求項38之方法，其中該個體患有苯酮尿症(phenylketonuria；PKU)。The method of claim 38, wherein the individual suffers from phenylketonuria (PKU).

一種治療患有苯酮尿症(PKU)之人類個體之方法，其包含向該個體投與治療有效量之如請求項22至37中任一項之醫藥組合物。A method of treating a human subject suffering from phenylketonuria (PKU) comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of any one of claims 22-37.

如請求項39或40之方法，其中該個體患有典型PKU或重度PKU。The method of claim 39 or 40, wherein the individual has classic PKU or severe PKU.

如請求項38至41中任一項之方法，其中該治療有效量係該個體每公斤為2E13載體基因體之劑量。The method of any one of claims 38 to 41, wherein the therapeutically effective amount is the dose of 2E13 vector gene body per kilogram of the individual.

如請求項38至41中任一項之方法，其中該治療有效量係該個體每公斤為6E13載體基因體之劑量。The method of any one of claims 38 to 41, wherein the therapeutically effective amount is a dose of 6E13 vector gene body per kilogram of the individual.

如請求項38至41中任一項之方法，其中該治療有效量係該個體每公斤為2E14載體基因體之劑量。The method of any one of claims 38 to 41, wherein the therapeutically effective amount is the dose of 2E14 vector gene body per kilogram of the individual.

如請求項38至41中任一項之方法，其中該治療有效量係該個體每公斤為約2E13至約9E13載體基因體範圍內之劑量。The method of any one of claims 38 to 41, wherein the therapeutically effective amount is a dose in the range of about 2E13 to about 9E13 vector genomes per kilogram of the subject.

一種降低有需要之人類個體中血漿***酸(Phe)含量之方法，其包含向該個體投與約2E13至約2E14的範圍內之劑量之重組腺相關病毒(rAAV)顆粒，該重組腺相關病毒(rAAV)顆粒包含AAV衣殼及重組載體建構體，該重組載體建構體包含編碼功能性***酸羥化酶(PAH)之核酸及視情況選用之異源肝特異性轉錄調節區。A method of reducing plasma phenylalanine (Phe) levels in a human individual in need, comprising administering to the individual a recombinant adeno-associated virus (rAAV) particle in a dose ranging from about 2E13 to about 2E14, the recombinant adeno-associated virus (rAAV) particles comprise an AAV capsid and a recombinant vector construct comprising a nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcriptional regulatory region.

如請求項45之方法，其中該個體患有苯酮尿症(PKU)。The method of claim 45, wherein the individual suffers from phenylketonuria (PKU).

一種治療患有苯酮尿症之人類個體之方法，其包含向該個體投與約2E13至約2E14的範圍內之劑量之重組腺相關病毒(rAAV)顆粒，該重組腺相關病毒(rAAV)顆粒包含AAV衣殼及重組載體建構體，該重組載體建構體包含編碼功能性***酸羥化酶(PAH)之核酸及視情況選用之異源肝特異性轉錄調節區。A method of treating a human subject with phenylketonuria, comprising administering to the subject a recombinant adeno-associated virus (rAAV) particle in a dose ranging from about 2E13 to about 2E14, the recombinant adeno-associated virus (rAAV) particle Comprising an AAV capsid and a recombinant vector construct comprising a nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcriptional regulatory region.

如請求項47或48之方法，其中該個體患有典型PKU或重度PKU。The method of claim 47 or 48, wherein the individual has classic PKU or severe PKU.

如請求項46至49中任一項之方法，其中該rAAV顆粒之該重組載體建構體包含：(a)以下中之一或兩者：(i) AAV 5'反向末端重複序列(ITR)及(ii) AAV 3' ITR；(b)異源肝特異性轉錄調節區；及(c)編碼功能性人類***酸羥化酶(hPAH)之核酸，視情況其中該等AAV ITR為AAV2 ITR。The method of any one of claims 46 to 49, wherein the recombinant vector construct of the rAAV particle comprises: (a) one or both of the following: (i) AAV 5' inverted terminal repeats (ITRs) and (ii) AAV 3' ITRs; (b) heterologous liver-specific transcriptional regulatory regions; and (c) nucleic acids encoding functional human phenylalanine hydroxylase (hPAH), as the case may be wherein these AAV ITRs are AAV2 ITRs .

如請求項50之方法，其中該編碼功能性hPAH之核酸編碼與SEQ ID NO: 2至少95%一致的胺基酸序列。The method of claim 50, wherein the nucleic acid encoding a functional hPAH encodes an amino acid sequence that is at least 95% identical to SEQ ID NO:2.

如請求項46至50中任一項之方法，其中該編碼功能性hPAH之核酸包含與SEQ ID NO: 1或7至13至少90%一致之核苷酸序列。The method of any one of claims 46 to 50, wherein the nucleic acid encoding a functional hPAH comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1 or 7 to 13.

如請求項46至52中任一項之方法，其中該編碼PAH之核酸可操作地連接至包含hAAT啟動子之片段的啟動子及HCR增強子/ApoE增強子之片段。The method of any one of claims 46 to 52, wherein the PAH-encoding nucleic acid is operably linked to a promoter comprising a fragment of the hAAT promoter and a fragment of the HCR enhancer/ApoE enhancer.

如請求項46至53中任一項之方法，其中該肝特異性轉錄調節區包含與SEQ ID NO: 3、4或24中之任一者至少90%一致或可替代地與SEQ ID NO: 25或26中之任一者至少90%一致的核苷酸序列。The method of any one of claims 46 to 53, wherein the liver-specific transcriptional regulatory region comprises at least 90% identical to any one of SEQ ID NO: 3, 4 or 24 or alternatively to SEQ ID NO: Any of 25 or 26 nucleotide sequences that are at least 90% identical.

如請求項46至54中任一項之方法，其中該重組載體建構體包含與SEQ ID NO: 6至少90%一致之核苷酸序列。The method of any one of claims 46 to 54, wherein the recombinant vector construct comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:6.

如請求項46至53中任一項之方法，其中該重組載體建構體進一步包含內含子。The method of any one of claims 46 to 53, wherein the recombinant vector construct further comprises an intron.

如請求項56之方法，其中該內含子包含與SEQ ID NO: 14或27或29或34至少90%一致之核苷酸序列。The method of claim 56, wherein the intron comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14 or 27 or 29 or 34.

如請求項46至57中任一項之方法，其中該重組載體建構體進一步包含多腺苷酸化信號。The method of any one of claims 46 to 57, wherein the recombinant vector construct further comprises a polyadenylation signal.

如請求項58之方法，其中該重組載體建構體包含牛生長激素(bGH)多腺苷酸化信號。The method of claim 58, wherein the recombinant vector construct comprises a bovine growth hormone (bGH) polyadenylation signal.

如請求項46至59中任一項之方法，其中該rAAV顆粒包含與SEQ NO: 15至23或52中之任一者至少90%一致的重組載體建構體。The method of any one of claims 46 to 59, wherein the rAAV particle comprises a recombinant vector construct that is at least 90% identical to any one of SEQ NOs: 15 to 23 or 52.

如請求項46至59中任一項之方法，其中該AAV衣殼包含與SEQ ID NO: 35至51中之任一者至少85%一致的胺基酸序列。The method of any one of claims 46 to 59, wherein the AAV capsid comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 35 to 51.

如請求項46至61中任一項之方法，其中該AAV衣殼為具有肝趨向性之AAV衣殼。The method of any one of claims 46 to 61, wherein the AAV capsid is an AAV capsid having liver tropism.

如請求項62之方法，其中具有肝趨向性之該AAV衣殼不包括AAV8及/或AAVHSC15。The method of claim 62, wherein the AAV capsid having liver tropism does not include AAV8 and/or AAVHSC15.

如請求項62之方法，其中具有肝趨向性之該AAV衣殼為AAV5型衣殼，視情況與SEQ ID NO: 44至少85%、90%或95%一致。The method of claim 62, wherein the AAV capsid having liver tropism is an AAV5-type capsid that is at least 85%, 90% or 95% identical to SEQ ID NO: 44, as appropriate.

如請求項38至64中任一項之方法，其中該個體在該投與前之血漿Phe含量為600 μmol/L或更高。The method of any one of claims 38 to 64, wherein the subject has a plasma Phe level of 600 μmol/L or higher prior to the administration.

如請求項38至64中任一項之方法，其中該個體在該投與前之血漿Phe含量為1200 μmol/L或更高。The method of any one of claims 38 to 64, wherein the subject has a plasma Phe level of 1200 μmol/L or higher prior to the administration.

如請求項38至66中任一項之方法，其中該個體為15歲或更大。The method of any one of claims 38 to 66, wherein the individual is 15 years old or older.

如請求項38至66中任一項之方法，其中該個體為成人。The method of any one of claims 38 to 66, wherein the individual is an adult.

如請求項38至68中任一項之方法，其中該個體為女性。The method of any one of claims 38 to 68, wherein the individual is female.

如請求項38至69中任一項之方法，其中該個體在編碼PAH之內在基因中具有突變，視情況為突變F39L、L48S、I65T、R68S、A104D、S110C、D129G、E178G、V190A、P211T、R241C、R261Q、A300S、L308F、A313T、K320N、A373T、V388M、E390G、A395P、P407S及Y414C。The method of any one of claims 38 to 69, wherein the individual has a mutation in the gene encoding PAH, optionally a mutation F39L, L48S, I65T, R68S, A104D, S110C, D129G, E178G, V190A, P211T, R241C, R261Q, A300S, L308F, A313T, K320N, A373T, V388M, E390G, A395P, P407S and Y414C.

如請求項38至70中任一項之方法，其中該個體為非妊娠女性。The method of any one of claims 38 to 70, wherein the individual is a non-pregnant female.

如請求項38至71中任一項之方法，其中該個體未接受治療PKU之藥物療法。The method of any one of claims 38 to 71, wherein the individual is not receiving drug therapy for the treatment of PKU.

如請求項72之方法，其中該個體在該投與前至少30天未接受培格瓦力(pegvaliase)。The method of claim 72, wherein the subject has not received pegvaliase for at least 30 days prior to the administration.

如請求項72之方法，其中該個體在該投與前至少30天未接受大型中性胺基酸。The method of claim 72, wherein the subject has not received a large neutral amino acid for at least 30 days prior to the administering.

如請求項72之方法，其中該個體在該投與前至少7天未接受沙丙蝶呤(sapropterin)。The method of claim 72, wherein the subject has not received sapropterin for at least 7 days prior to the administering.

如請求項38至75中任一項之方法，其中該個體在該投與前至少30天未接受類固醇。The method of any one of claims 38 to 75, wherein the subject has not received steroids for at least 30 days prior to the administration.

如請求項38至76中任一項之方法，其中在該投與前，該個體在血液中不具有可偵測到之抗AAV衣殼抗體。The method of any one of claims 38 to 76, wherein prior to the administering, the individual has no detectable anti-AAV capsid antibodies in blood.

如請求項38至77中任一項之方法，其中該個體在該投與前未患有臨床上顯著之肝病。The method of any one of claims 38 to 77, wherein the subject does not have clinically significant liver disease prior to the administration.

如請求項38至78中任一項之方法，其中該rAAV顆粒係以靜脈內投藥法單次投與。The method of any one of claims 38 to 78, wherein the rAAV particles are administered in a single dose intravenously.

如請求項38至79中任一項之方法，其進一步包含向該個體投與預防性免疫抑制治療。The method of any one of claims 38 to 79, further comprising administering to the individual prophylactic immunosuppressive therapy.

如請求項80之方法，其中該預防性免疫抑制治療包含投與預防有效量之全身性免疫抑制劑以預防肝毒性，視情況與rAAV顆粒之該投與同時進行。The method of claim 80, wherein the prophylactic immunosuppressive treatment comprises administering a prophylactically effective amount of a systemic immunosuppressive agent to prevent liver toxicity, optionally concurrently with the administration of rAAV particles.

如請求項81之方法，其中該全身性免疫抑制劑為皮質類固醇，視情況為***(dexamethasone)、普賴松(prednisone)、普賴蘇穠(prednisolone)、氟可體松(fludrocortisone)、氫化可體松(hydrocortisone)或布***(budesonide)。The method of claim 81, wherein the systemic immunosuppressant is a corticosteroid, optionally dexamethasone, prednisone, prednisolone, fludrocortisone , hydrocortisone (hydrocortisone) or budesonide (budesonide).

如請求項81之方法，其中該全身性免疫抑制劑為皮質類固醇且該預防有效量為10毫克/天至40毫克/天之普賴松等效物劑量，視情況持續至少約13週之時段，接著遞減量之該皮質類固醇持續約3週之時段。The method of claim 81, wherein the systemic immunosuppressant is a corticosteroid and the prophylactically effective amount is a prisone equivalent dose of 10 mg/day to 40 mg/day, as appropriate, for a period of at least about 13 weeks , followed by decreasing amounts of the corticosteroid for a period of about 3 weeks.

如請求項38至83中任一項之方法，其進一步包含以下步驟：(a)在該投與前，視情況在該投與前約一個月測定該個體之血液中的肝毒性標記之基線含量，及(b)在該投與後每週測定該個體之血液中的投與後該肝毒性標記之含量。The method of any one of claims 38 to 83, further comprising the step of: (a) determining a baseline of a marker of hepatotoxicity in the blood of the individual prior to the administration, optionally about one month prior to the administration levels, and (b) the post-administration level of the hepatotoxic marker in the subject's blood is determined weekly after the administration.

如請求項84之方法，其進一步包含向該個體投與治療性免疫抑制治療。The method of claim 84, further comprising administering to the individual a therapeutic immunosuppressive treatment.

如請求項85之方法，其中該治療性免疫抑制治療包含以下步驟：(c)當藉由生物化學或臨床症狀檢測到肝毒性後，向該個體投與治療有效量之全身性免疫抑制劑以減少肝毒性。The method of claim 85, wherein the therapeutic immunosuppressive treatment comprises the steps of: (c) upon detection of hepatotoxicity by biochemical or clinical symptoms, administering to the individual a therapeutically effective amount of a systemic immunosuppressive agent to Reduce liver toxicity.

如請求項86之方法，其中肝毒性之檢測係藉由(i)投與後該肝毒性標記之含量大於正常值上限(upper limit of normal；ULN)或(ii)投與後該肝毒性標記之含量大於或等於該肝毒性標記之基線含量的兩倍。The method of claim 86, wherein hepatotoxicity is detected by (i) the hepatotoxic marker level after administration is greater than the upper limit of normal (ULN) or (ii) the hepatotoxic marker after administration The level is greater than or equal to twice the baseline level of the hepatotoxicity marker.

如請求項85至87中任一項之方法，其中該全身性免疫抑制劑為皮質類固醇，視情況為***、普賴松、普賴蘇穠、氟可體松、氫化可體松或布***。The method of any one of claims 85 to 87, wherein the systemic immunosuppressant is a corticosteroid, optionally dexamethasone, prisone, prisulol, flucortisone, hydrocortisone, or Budesonide.

如請求項85至87中任一項之方法，其中該全身性免疫抑制劑為皮質類固醇且該治療有效量為10毫克/天至40毫克/天之普賴松等效物劑量，視情況持續至少約5週之時段，接著遞減量之該皮質類固醇持續約3週之時段。The method of any one of claims 85 to 87, wherein the systemic immunosuppressant is a corticosteroid and the therapeutically effective amount is a prisone equivalent dose of 10 mg/day to 40 mg/day, as the case may be A period of at least about 5 weeks followed by a decreasing amount of the corticosteroid for a period of about 3 weeks.

如請求項84至87中任一項之方法，其中該肝毒性標記為ALT及/或AST。The method of any one of claims 84 to 87, wherein the hepatotoxicity marker is ALT and/or AST.

如請求項84至87中任一項之方法，其中該肝毒性標記為ALT。The method of any one of claims 84 to 87, wherein the liver toxicity marker is ALT.

如請求項38至91中任一項之方法，其進一步包含藉由包含以下步驟監測游離基因體(episome)形成：自該個體之肝細胞提取DNA及檢測環形載體基因體，視情況藉由PCR或南方墨點法(southern blotting)進行。The method of any one of claims 38 to 91, further comprising monitoring episome formation by comprising the steps of: extracting DNA from hepatocytes of the individual and detecting circular vector gene bodies, optionally by PCR Or southern blotting method (southern blotting).

如請求項38至92中任一項之方法，其進一步包含每週量測該個體之血漿Phe含量的步驟。The method of any one of claims 38 to 92, further comprising the step of measuring the subject's plasma Phe levels on a weekly basis.

如請求項38至93中任一項之方法，其進一步包含實施Phe呼吸測試。The method of any one of claims 38 to 93, further comprising performing a Phe breath test.

如請求項38至94中任一項之方法，其進一步包含每週量測該個體血漿中一或多種神經傳遞質或神經傳遞質代謝物之含量的步驟。The method of any one of claims 38 to 94, further comprising the step of measuring weekly the level of one or more neurotransmitters or neurotransmitter metabolites in the individual's plasma.

如請求項95之方法，其中該一或多種神經傳遞質或神經傳遞質代謝物為苯乙胺、苯乙醇胺、酪胺、多巴胺、正腎上腺素、腎上腺素、色胺、羥基色胺、苯乙酸、苯乙醯基麩醯胺酸、杏仁酸(mandelic acid)、羥基苯乙酸、DOPAC、高香草酸(homovanillic acid)、DOMA、MOPEG、香草基杏仁酸(vanillylmandelic acid)、吲哚乙酸及5-羥基吲哚乙酸。The method of claim 95, wherein the one or more neurotransmitters or neurotransmitter metabolites are phenethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid , phenylacetylglutamic acid, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillylmandelic acid, indoleacetic acid and 5- Hydroxy indole acetic acid.

如請求項38至96中任一項之方法，其中在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與後8週為360 μmol/L或更低。The method of any one of claims 38 to 96, wherein in the absence of concomitant drug therapy, the subject's plasma Phe level is 360 μmol/L or less 8 weeks after the administration.

如請求項38至97中任一項之方法，其中在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與後2、3或4年為360 μmol/L或更低。The method of any one of claims 38 to 97, wherein in the absence of concomitant drug therapy, the subject's plasma Phe level is 360 μmol/L or less 2, 3 or 4 years after the administration.

如請求項38至98中任一項之方法，其中在未同時藥物療法之情況下，該個體之血漿Phe含量投與後8週為介於120與360 μmol/L之間。The method of any one of claims 38 to 98, wherein in the absence of concomitant drug therapy, the subject's plasma Phe level is between 120 and 360 μmol/L 8 weeks after administration.

如請求項38至98中任一項之方法，其中在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與後8週為120 μmol/L或更低。The method of any one of claims 38 to 98, wherein in the absence of concomitant drug therapy, the subject's plasma Phe level is 120 μmol/L or less 8 weeks after the administration.

如請求項38至100中任一項之方法，其中在未同時藥物療法之情況下，該個體之血漿Phe含量在該投與後2、3或4年為120 μmol/L或更低。The method of any one of claims 38 to 100, wherein in the absence of concomitant drug therapy, the subject's plasma Phe level is 120 μmol/L or less 2, 3 or 4 years after the administration.

如請求項38至101中任一項之方法，其中與限制Phe膳食之基線相比，該個體耐受Phe攝入量增加。The method of any one of claims 38 to 101, wherein the individual tolerates an increase in Phe intake compared to a baseline on a Phe-restricted diet.

如請求項38至102中任一項之方法，其中在該投與後，該個體血液中神經傳遞質或神經傳遞質代謝物之含量降低。The method of any one of claims 38 to 102, wherein following the administration, the level of neurotransmitter or neurotransmitter metabolites in the blood of the individual is reduced.

如請求項103之方法，其中該一或多種神經傳遞質或神經傳遞質代謝物為苯乙胺、苯乙醇胺、酪胺、多巴胺、正腎上腺素、腎上腺素、色胺、羥基色胺、苯乙酸、苯乙醯基麩醯胺酸、杏仁酸、羥基苯乙酸、DOPAC、高香草酸、DOMA、MOPEG、香草基杏仁酸、吲哚乙酸及5-羥基吲哚乙酸。The method of claim 103, wherein the one or more neurotransmitters or neurotransmitter metabolites are phenethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid , Phenylglutamic acid, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillyl mandelic acid, indoleacetic acid and 5-hydroxyindoleacetic acid.

如請求項38至104中任一項之方法，其中該個體之生活品質在該投與後改善，視情況如藉由PKU-QOL或Q-LES-Q-SF調查表所量測。The method of any one of claims 38 to 104, wherein the subject's quality of life improves after the administration, as measured by the PKU-QOL or Q-LES-Q-SF questionnaire, as appropriate.

如請求項38至105中任一項之方法，其中該個體之一或多種神經認知症狀或量測在該投與後改善。The method of any one of claims 38 to 105, wherein one or more neurocognitive symptoms or measures of the individual improve after the administration.

如請求項38至106中任一項之方法，其中該個體在該投與後未經歷持續性的低***酸血症。The method of any one of claims 38 to 106, wherein the subject does not experience persistent hypophenylalaninemia after the administration.