TW202340467A

TW202340467A - Compositions and methods useful for treatment of c9orf72-mediated disorders

Info

Publication number: TW202340467A
Application number: TW112101016A
Authority: TW
Inventors: 克里斯欽亨德勒; 詹姆士Ｍ威爾森; 艾倫沃克曼
Original assignee: 賓州大學委員會
Priority date: 2022-01-10
Filing date: 2023-01-10
Publication date: 2023-10-16
Also published as: AR128239A1; WO2023133574A1

Abstract

Provided herein are rAAV and other vectors and compositions useful for treating a patient having C9orf72 comprising an engineered hC9orf72 coding sequence and the at least one miRNA coding sequence, wherein the engineered human C9orf72 coding sequence has a sequence which differs from endogenous human C9orf72 in the patient in the target site of the encoded miRNA. Also provided are methods for treating C9orf72-associated ALS, FTD, and related disorders.

Description

有用於治療C9ORF72介導之病症之組成物及方法Compositions and methods for treating C9ORF72-mediated disorders

電子序列表引用Electronic Sequence Listing Reference

在此提交的電子序列表名稱為「UPN-18-8536TW.xml」(台灣申請案序列表的檔案名稱，中文本：PD1227626_Sequence.xml)；外文本：PD1227626_Foreign Sequence.xml)，大小為157,533位元組，創建於2023年1月5日，電子序列表的內容(例如，其中的序列和文字)在此全部藉由引用而併入。The name of the electronic sequence listing submitted here is "UPN-18-8536TW.xml" (the file name of the Taiwan application sequence listing, Chinese text: PD1227626_Sequence.xml); foreign text: PD1227626_Foreign Sequence.xml), with a size of 157,533 bits. Group, created on January 5, 2023. The contents of the electronic sequence listing (e.g., the sequences and text therein) are hereby incorporated by reference in their entirety.

本文提供有用於治療具有C9orf72的患者之rAAV及其他載體及組成物。亦提供用於治療與C9orf72相關的ALS、FTD及相關病症之方法。Provided herein are rAAV and other vectors and compositions for treating patients with C9orf72. Methods for treating ALS, FTD and related conditions associated with C9orf72 are also provided.

肌肉萎縮性脊髓側索硬化症(Amyotrophic lateral sclerosis (ALS))為一種慢性進行性且致命的神經退化疾病，由上下運動神經元的退化所引起。其特徵為進行性肌肉無力和萎縮，最終導致呼吸衰竭。約5-50%的ALS患者有額顳葉癡呆症(frontotemporal dementia (FTD))的臨床症狀(Hudson, 1981, Amyotrophic lateral sclerosis and its association with dementia, parkinsonism and other neurological disorders：a review. Brain 104, 217-247. doi：10.1093/brain/104.2.217；Lomen-Hoerth et al., 2003, Are amyotrophic lateral sclerosis patients cognitively normal? Neurology 60, 1094–1097. doi：10.1212/01.wnl.0000055861.95202.8d)。FTD為第二種最常見的早發性癡呆形式，表現為額葉及/或顳葉萎縮，伴隨有性格及行為改變以及語言功能障礙。事實上，一部分罹患FTD的患者亦會發展為ALS。除了臨床重疊外，泛蛋白陽性tau陰性包涵體(TDP-43)(ubiquitin-positive tau-negative inclusion bodies (TDP-43))被認為是ALS及FTD病理學研究中的主要病理蛋白質(Neumann et al., 2006, Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130-133. doi：10.1126/science.1134108)。於2011年，取得一項連結ALS和FTD的重大發現:C9orf72基因之擴展的GGGGCC六核苷酸重複序列為ALS/FTD的重要遺傳原因，約佔家族性ALS患者的40%，佔家族性FTD患者的25%，並且在家族性ALS/FTD患者中高達88%(DeJesus-Hernandez et al., 2011, Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256. doi：10.1016/j.neuron.2011.09.011)。ALS和FTD存在顯著的臨床、遺傳及組織病理學的重疊；因此，它們被視為同一疾病連續區域的兩個極端。Amyotrophic lateral sclerosis (ALS) is a chronic progressive and fatal neurodegenerative disease caused by the degeneration of upper and lower motor neurons. It is characterized by progressive muscle weakness and atrophy, eventually leading to respiratory failure. About 5-50% of ALS patients have clinical symptoms of frontotemporal dementia (FTD) (Hudson, 1981, Amyotrophic lateral sclerosis and its association with dementia, parkinsonism and other neurological disorders: a review. Brain 104, 217-247. doi: 10.1093/brain/104.2.217; Lomen-Hoerth et al., 2003, Are amyotrophic lateral sclerosis patients cognitively normal? Neurology 60, 1094–1097. doi: 10.1212/01.wnl.0000055861.95202.8d) . FTD is the second most common form of dementia praecox and manifests as frontal and/or temporal lobe atrophy, accompanied by personality and behavioral changes and language impairment. In fact, some patients with FTD will also develop ALS. In addition to clinical overlap, ubiquitin-positive tau-negative inclusion bodies (TDP-43) are considered to be the main pathological protein in ALS and FTD pathology studies (Neumann et al ., 2006, Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130-133. doi: 10.1126/science.1134108). In 2011, a major discovery was made linking ALS and FTD: the expanded GGGGCC hexanucleotide repeat sequence of the C9orf72 gene is an important genetic cause of ALS/FTD, accounting for approximately 40% of familial ALS patients and accounting for familial FTD. 25% of patients and up to 88% of patients with familial ALS/FTD (DeJesus-Hernandez et al., 2011, Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 chromosome causes 9p-linked FTD and ALS. Neuron 72, 245– 256. doi:10.1016/j.neuron.2011.09.011). ALS and FTD have significant clinical, genetic, and histopathological overlap; therefore, they are considered two extreme ends of the same disease continuum.

需要的是有用於減輕C9orf72相關的ALS、FTD及相關病症之症狀、嚴重性及/或進展的治療。What is needed are treatments for reducing the symptoms, severity and/or progression of C9orf72-related ALS, FTD and related conditions.

本文提供用於治療具有與人類C9ORF72表現缺陷相關的症狀的患者及/或患有ALS或FTD的患者之病毒及非病毒載體和組成物。Provided herein are viral and non-viral vectors and compositions for the treatment of patients with symptoms associated with defects in human C9ORF72 expression and/or patients with ALS or FTD.

於某些具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含AAV衣殼及載體基因體。該rAAV包含：(a)編碼人類C9orf72的工程化核酸序列；(b)位於(a)與(c)之間的間隔子序列；(c)編碼至少一個miRNA序列的核酸序列，該miRNA序列對ALS或FTD患者的內源性人類C9orf72具有特異性，且該miRNA序列位於(a)和(b)序列的3’端；其中(a)的工程化核酸序列缺少所編碼的至少一種miRNA的靶位點，從而防止所編碼的miRNA靶向工程化的人類C9orf72編碼序列；及(c)可操作連接至(a)和(c)之調節序列。於某些具體實施例中，該AAV衣殼選自AAV9、AAVhu68、AAV1或AAVrh91。於某些具體實施例中，該間隔子長度為75個核苷酸至約250個核苷酸。於一態樣，提供一種載體，其包含可操作連接至調節序列的工程化人類C9orf72編碼序列，該調節序列指導其在人類標靶細胞中的表現。於某些具體實施例中，提供一種載體，其包含編碼至少一個髮夾miRNA的核酸序列，其中該編碼的miRNA對人類受試者中的內源性人類C9orf72具有特異性且可操作連接至指導其在受試者中表現的調節序列。於某些具體實施例中，載體或其他組成物包含工程化人類C9orf72編碼序列及至少一個miRNA的編碼序列兩者。於此種具體實施例中，該工程化C9orf72編碼序列缺少對於至少一個miRNA的靶位點，從而防止miRNA靶向工程化人類C9orf72編碼序列。In certain embodiments, a recombinant adeno-associated virus (rAAV) is provided, which includes an AAV capsid and a vector genome. The rAAV contains: (a) an engineered nucleic acid sequence encoding human C9orf72; (b) a spacer sequence located between (a) and (c); (c) a nucleic acid sequence encoding at least one miRNA sequence, which is Endogenous human C9orf72 in patients with ALS or FTD is specific, and the miRNA sequence is located at the 3' end of the sequences (a) and (b); wherein the engineered nucleic acid sequence of (a) lacks the target of at least one of the encoded miRNAs site, thereby preventing the encoded miRNA from targeting the engineered human C9orf72 coding sequence; and (c) operably linked to the regulatory sequences of (a) and (c). In certain embodiments, the AAV capsid is selected from AAV9, AAVhu68, AAV1, or AAVrh91. In certain embodiments, the spacer is 75 nucleotides to about 250 nucleotides in length. In one aspect, a vector is provided that includes an engineered human C9orf72 coding sequence operably linked to regulatory sequences that direct its expression in human target cells. In certain embodiments, a vector is provided comprising a nucleic acid sequence encoding at least one hairpin miRNA, wherein the encoded miRNA is specific for endogenous human C9orf72 in a human subject and is operably linked to the guide The regulatory sequence in which it manifests itself in a subject. In certain embodiments, a vector or other composition includes both an engineered human C9orf72 coding sequence and a coding sequence for at least one miRNA. In such specific embodiments, the engineered C9orf72 coding sequence lacks a target site for at least one miRNA, thereby preventing the miRNA from targeting the engineered human C9orf72 coding sequence.

於某些具體實施例中，載體為一複製缺陷型病毒載體，其包含載體基因體，該載體基因體包含人類C9orf72編碼序列、至少一個miRNA的編碼序列及調節序列。於某些具體實施例中，該病毒載體為具有AAV衣殼的重組腺相關病毒(rAAV)粒，其具有包裝於其中的載體基因體。於某些具體實施例中，該AAV衣殼為AAVhu68、AAV1或AAVrh91。In certain embodiments, the vector is a replication-deficient viral vector that includes a vector genome that includes a human C9orf72 coding sequence, at least one coding sequence for a miRNA, and regulatory sequences. In certain embodiments, the viral vector is a recombinant adeno-associated virus (rAAV) particle having an AAV capsid with the vector genome packaged therein. In certain embodiments, the AAV capsid is AAVhu68, AAV1 or AAVrh91.

於某些具體實施例中，提供一種載體，其包含工程化C9orf72編碼序列，該序列具有SEQ ID NO：13的核酸序列或與其至少90%相同的序列，條件為藉由編碼的miRNA靶向的核酸序列係不同於內源性人類C9orf72序列。 In certain embodiments, a vector is provided that includes an engineered C9orf72 coding sequence having the nucleic acid sequence of SEQ ID NO: 13 or a sequence that is at least 90% identical thereto, provided that it is targeted by the encoded miRNA The nucleic acid sequence is different from the endogenous human C9orf72 sequence .

於某些具體實施例中，組成物包含可操作連接至調節序列之編碼工程化人類C9orf72編碼序列的重組核酸序列(該調節序列指導該核酸序列在人類標靶細胞中的表現)以及可操作連接至調節序列之編碼至少一個對患者內源性人類C9orf72特異的miRNA的核酸序列(該調節序列指導其在受試者中的表現)，其中該工程化C9orf72編碼序列缺少對於編碼的至少一個miRNA的靶位點，從而防止miRNA靶向工程化C9orf72編碼序列。In certain embodiments, the compositions comprise a recombinant nucleic acid sequence encoding an engineered human C9orf72 coding sequence operably linked to regulatory sequences that direct expression of the nucleic acid sequence in a human target cell and operably linked to to a nucleic acid sequence encoding at least one miRNA specific for endogenous human C9orf72 in a patient (the regulatory sequence directs its expression in the subject), wherein the engineered C9orf72 coding sequence lacks a nucleic acid sequence for the encoded at least one miRNA target site, thereby preventing the miRNA from targeting the engineered C9orf72 coding sequence.

於某些具體實施例中，一種醫藥組成物包含載體、rAAV、或組成物、及醫藥上可接受的水性懸浮液、賦形劑及/或稀釋劑。In certain embodiments, a pharmaceutical composition includes a vector, rAAV, or composition, and a pharmaceutically acceptable aqueous suspension, excipients, and/or diluents.

於某些具體實施例中，提供一種治療患有C9orf72相關病症(例如，ALS或FTD)的患者的方法，其包含將有效量的載體、重組AAV或組成物遞送至有需要的患者。In certain embodiments, a method of treating a patient suffering from a C9orf72-related disorder (eg, ALS or FTD) is provided, comprising delivering an effective amount of a vector, recombinant AAV, or composition to a patient in need thereof.

於某些具體實施例中，提供一種治療患有C9orf72相關病症的患者之組合給藥方案，其包含共同投予： (a)可操作連接至調節序列的編碼工程化人類C9orf72編碼序列之重組核酸序列，該調節序列指導其在人類標靶細胞中的表現，其中該人類c9orf72編碼序列具有SEQ ID NO：13的序列或與其至少95%相同的序列且該序列不同於患者中的內源性人類c9，由於(b)的miRNA標靶序列中有錯誤配對(mismatch)， (b)對人類受試者中內源性人類c9序列特異性的至少一個miRNA，其中該mRNA可操作連接至指導其在受試者中表現的調節序列。 In certain embodiments, a combination dosage regimen for treating a patient suffering from a C9orf72-related disorder is provided, comprising co-administering: (a) A recombinant nucleic acid sequence encoding an engineered human C9orf72 coding sequence operably linked to a regulatory sequence that directs its expression in a human target cell, wherein the human c9orf72 coding sequence has the sequence of SEQ ID NO: 13 or a sequence that is at least 95% identical to it and is different from endogenous human c9 in the patient due to a mismatch in the miRNA target sequence of (b), (b) At least one miRNA specific for an endogenous human c9 sequence in a human subject, wherein the mRNA is operably linked to a regulatory sequence that directs its expression in the subject.

此等及其他優點將由下列發明詳細說明中顯而易見。These and other advantages will be apparent from the following detailed description of the invention.

本文提供序列、載體及組成物，用於共同投予表現人類c9orf72蛋白質之核酸序列及編碼至少一個miRNA的核酸序列至患者，該miRNA特異性靶向人類C9orf72基因第一個內含子中內源性六核苷酸重複擴增的位點，該靶位點不存在於工程化C9orf72編碼序列上。適當地，工程化c9orf72編碼序列被工程化以移除miRNA的特定靶位點。本文提供新穎的工程化C9orf72及新穎的miRNA標靶序列。此等可單獨使用或彼此組合及/或與其他療法組合使用，用於治療C9orf72相關的ALS、FTD和相關病症。Provided herein are sequences, vectors, and compositions for co-administering to a patient a nucleic acid sequence expressing a human c9orf72 protein and a nucleic acid sequence encoding at least one miRNA that specifically targets endogenous content in the first intron of the human C9orf72 gene. The target site is a hexanucleotide repeat amplification site that does not exist on the engineered C9orf72 coding sequence. Appropriately, the engineered c9orf72 coding sequence is engineered to remove the specific target site of the miRNA. This article provides novel engineered C9orf72 and novel miRNA target sequences. These can be used alone or in combination with each other and/or with other therapies for the treatment of C9orf72-related ALS, FTD and related disorders.

如本文所使用，「內源性C9orf72」係指在人類中編碼C9蛋白質的C9orf72基因(染色體9開讀框72)。人類C9orf72基因位於染色體9開讀框72的短(p)臂，從鹼基對27,546,546至鹼基對27,573,866 (GRCh38)。其細胞遺傳學位置位於9p21. 2。此亦稱為C9orf72、染色體9開讀框72、ALSFTD、FTDALS、FTDALS1、DENNL72、C9orf72-SMCR8複合次單元、DENND9。C9orf72的功能異常與ALS、家族性FTD或相關病症有關。As used herein, "endogenous C9orf72" refers to the C9orf72 gene encoding the C9 protein in humans (chromosome 9 open reading frame 72). The human C9orf72 gene is located in the short (p) arm of chromosome 9 open reading frame 72, from base pair 27,546,546 to base pair 27,573,866 (GRCh38). Its cytogenetic location is at 9p21.2. This is also known as C9orf72, chromosome 9 open reading frame 72, ALSFTD, FTDALS, FTDALS1, DENNL72, C9orf72-SMCR8 complex subunit, DENND9. Functional abnormalities of C9orf72 are associated with ALS, familial FTD, or related conditions.

於某些具體實施例中，功能性C9蛋白質具有SEQ NO：14之序列。然而，於某些具體實施例中，與SEQ ID NO：14的胺基酸序列具有小於100%同一性的蛋白質可藉由本文提供的組成物遞送(例如，與SEQ ID NO：14具有97%至100%同一性的ORF蛋白質)。In certain embodiments, the functional C9 protein has the sequence of SEQ NO: 14. However, in certain embodiments, proteins having less than 100% identity to the amino acid sequence of SEQ ID NO: 14 can be delivered by the compositions provided herein (e.g., 97% to SEQ ID NO: 14 to 100% identity of the ORF protein).

於一具體實施例中，提供之工程化C9orf72編碼序列具有SEQ ID NO：13之核酸序列或與SEQ ID NO：13約90%、至少95%相同的、至少97%相同的、至少98%相同的、或99%至100%相同的序列，其表現在非C9orf72相關的ALS及FTD患者中發現的人類C9蛋白質。參見例如，SEQ ID NO：14。In a specific embodiment, the engineered C9orf72 coding sequence provided has the nucleic acid sequence of SEQ ID NO: 13 or is about 90%, at least 95%, at least 97% identical, or at least 98% identical to SEQ ID NO: 13. or 99% to 100% identical sequences to the human C9 protein found in non-C9orf72-related ALS and FTD patients. See, eg, SEQ ID NO: 14.

於某些具體實施例中，提供之工程化C9orf72編碼序列具有SEQ ID NO：13之核酸序列或與其至少90%相同的序列，當工程化編碼序列與miR487序列(包含至少5’側翼區、至少SEQ ID NO：15 (miR487)或與SEQ ID NO：15至少99%相同的序列、及3’側翼區)共同投予時，其中至少一個miRNA不與(a)之工程化C9orf72編碼序列或其編碼的傳訊RNA(mRNA)結合。於某些具體實施例中，5’側翼選自SEQ ID NO：5或SEQ ID NO：22之序列。適合地，具有與SEQ ID NO：13同一性的序列表現相同蛋白質。In certain embodiments, the engineered C9orf72 coding sequence provided has the nucleic acid sequence of SEQ ID NO: 13 or a sequence that is at least 90% identical to it, when the engineered coding sequence is identical to the miR487 sequence (including at least a 5' flanking region, at least SEQ ID NO: 15 (miR487) or a sequence at least 99% identical to SEQ ID NO: 15, and 3' flanking region) when co-administered, at least one of the miRNAs is not associated with the engineered C9orf72 coding sequence of (a) or its Encoded messenger RNA (mRNA) binding. In certain embodiments, the 5' flank is selected from the sequence of SEQ ID NO: 5 or SEQ ID NO: 22. Suitably, sequences having identity to SEQ ID NO: 13 represent the same protein.

「5’ UTR」位於基因產物編碼序列起始密碼子的上游。該5’ UTR通常比3’ UTR短。一般而言，5’ UTR長度為約3個核苷酸至約200個核苷酸，但可選擇為更長。The “5’ UTR” is located upstream of the start codon of the gene product coding sequence. The 5’ UTR is usually shorter than the 3’ UTR. Generally, the 5' UTR length ranges from about 3 nucleotides to about 200 nucleotides, but can be selected to be longer.

「3’ UTR」位於基因產物編碼序列的下游，通常比5’ UTR長。於某些具體實施例中，3’ UTR長度為約200個核苷酸至約800個核苷酸，但可選擇為較長或較短。The “3’ UTR” is located downstream of the gene product coding sequence and is usually longer than the 5’ UTR. In certain embodiments, the 3' UTR length ranges from about 200 nucleotides to about 800 nucleotides, but may be longer or shorter.

如本文所使用，「miRNA」係指微小RNA(microRNA)，其為小的非編碼RNA分子，其調節mRNA並停止mRNA被轉譯成蛋白質。一般而言，形成髮夾的RNA具有自互補(self-complementary)的「主幹-環圈(stem-loop)」結構，其包括編碼具有雙鏈體(duplex)的主幹部分的單一核酸，該雙鏈體包含藉由環圈序列連接至反義股(例如，引導股(guide strand))的正義股(例如，隨從股(passenger strand))。隨從股及引導股共有互補性。於一些具體實施例中，隨從股及引導股共有100%互補性。於一些具體實施例中，隨從股及引導股共有至少50%、至少60%、至少70%、至少80%、至少90%、至少95%、或至少99%互補性。隨從股及引導股可能由於鹼基對錯誤配對而缺少互補性。於一些具體實施例中，形成髮夾的RNA之隨從股及引導股具有至少1、至少2、至少3、至少4、至少5、至少6、至少7、至少8、至少9、或至少10個錯誤配對。一般而言，主幹的最初2-8個核苷酸(相對於環圈)被稱為「種子」殘基，在標靶識別及結合中扮演重要角色。主幹的第一個殘基(相對於環圈)被稱為「錨」殘基。於一些具體實施例中，形成髮夾的RNA在錨殘基處具有錯誤配對。如本文所使用，miRNA含有「種子序列」，其為藉由互補鹼基配對而與mRNA(例如，在內源性C9orf72中)特異性結合的核苷酸區域，導致mRNA的破壞或緘默化(silencing)。此種緘默化可能會造成內源性hC9orf72的向下調節而不是完全消除。除非另有指明，術語「miRNA」涵括人工微小RNA(amiRNA)，其為人工設計的。As used herein, "miRNA" refers to microRNA (microRNA), which are small non-coding RNA molecules that regulate mRNA and stop it from being translated into protein. In general, hairpin-forming RNAs have a self-complementary "stem-loop" structure, which consists of a single nucleic acid encoding a stem portion with a duplex. Strands include a sense strand (eg, a passenger strand) connected to an antisense strand (eg, a guide strand) by a loop sequence. Follower stocks and leading stocks share complementarity. In some embodiments, the following stocks and the leading stocks are 100% complementary. In some embodiments, the following stocks and the leading stocks are together at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% complementary. The follower and leader strands may lack complementarity due to base pair mispairing. In some embodiments, the follower and leader strands of the hairpin-forming RNA have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 Wrong pairing. Generally speaking, the first 2-8 nucleotides of the backbone (relative to the loop) are called "seed" residues and play an important role in target recognition and binding. The first residue of the backbone (relative to the loop) is called the "anchor" residue. In some embodiments, the hairpin-forming RNA has mispairing at anchor residues. As used herein, a miRNA contains a "seed sequence," which is a region of nucleotides that specifically binds to an mRNA (e.g., in endogenous C9orf72) via complementary base pairing, resulting in destruction or silencing of the mRNA ( silencing). Such silencing may result in downregulation of endogenous hC9orf72 rather than complete elimination. Unless otherwise specified, the term "miRNA" includes artificial microRNAs (amiRNAs), which are artificially designed.

「自互補核酸」係指由於核酸股中的核苷酸的互補性(例如鹼基配對)，而能夠與自身雜交(即，自身折疊)以形成單股雙鏈體結構的核酸。自互補核酸可形成多種二級結構，如髮夾環圈、環圈、凸起(bulge)、連接點(junction)及內部凸起。某些自互補核酸(例如，miRNA或AmiRNA)會進行調節功能，如基因緘默化。"Self-complementary nucleic acid" refers to a nucleic acid that is capable of hybridizing to itself (ie, folding on itself) to form a single-stranded duplex structure due to complementarity (eg, base pairing) of the nucleotides in the nucleic acid strands. Self-complementary nucleic acids can form a variety of secondary structures, such as hairpin loops, loops, bulges, junctions and internal bulges. Certain self-complementary nucleic acids (eg, miRNA or AmiRNA) perform regulatory functions such as gene silencing.

本文提供的經編碼的miRNA已設計為特異性地靶向患有與C9ORF72相關病症諸如ALS或FTD的患者中的內源性人類C9orf72基因。於某些具體實施例中，miRNA編碼序列包含反義序列。The encoded miRNAs provided herein have been designed to specifically target the endogenous human C9orf72 gene in patients with C9ORF72-related disorders such as ALS or FTD. In certain embodiments, the miRNA coding sequence includes an antisense sequence.

於某些具體實施例中，種子序列與表中描述的反義序列100%相同。於某些具體實施例中，種子序列位於成熟miRNA (5’至3’)且一般起始於miRNA之miRNA正義股的5'端起(正義(+)股的5'端起)的位置2至7、2至8、或約6個核苷酸，儘管其長度可能更長。於某些具體實施例中，種子序列的長度不少於miRNA序列的長度的約30%，其長度可為至少7個核苷酸至約28個核苷酸、長度可為至少8個核苷酸至約28個核苷酸、長度可為7個核苷酸至28個核苷酸、8個核苷酸至18個核苷酸、12個核苷酸至28個核苷酸、約20個至約26個核苷酸、約21個核苷酸、約24個核苷酸、或約26個核苷酸。於本文提供之例中，miRNA係以主幹-環圈miRNA前驅物序列的形式遞送，例如，長度為約50至約80個核苷酸、或約55個核苷酸至約70個核苷酸、或長度為60至65個核苷酸。於某些具體實施例中，此miRNA前驅物包含約5個核苷酸、約21個核苷酸種子序列、約19個核苷酸主幹環圈及約19個核苷酸正義序列，其中正義序列對應於具有一個或兩個錯誤配對的核苷酸的反義序列。適合的miRNA編碼序列之例為miR487序列(參見例如，於SEQ ID NO：17之載體基因體： 5’側翼(nt 3438)..(nt 3460)(SEQ ID NO：5之1-23)、miR487(nt 3461)..(nt 3524)、反義(nt 3466)..(nt 3486)、環圈(nt 3487)..(nt 3505)、正義(nt 3506)..(nt 3524)、及3’側翼(nt 3525)..(nt 3568)。亦參見SEQ ID NO：9。 In certain embodiments, the seed sequence is 100% identical to the antisense sequence described in the table. In certain embodiments, the seed sequence is located at position 2 of the mature miRNA (5' to 3') and generally starts from the 5' end of the sense strand of the miRNA (from the 5' end of the sense (+) strand) of the miRNA. to 7, 2 to 8, or about 6 nucleotides, although the length may be longer. In certain embodiments, the length of the seed sequence is no less than about 30% of the length of the miRNA sequence, and may be at least 7 nucleotides to about 28 nucleotides in length, and may be at least 8 nucleotides in length. Acid to about 28 nucleotides, length can be 7 nucleotides to 28 nucleotides, 8 nucleotides to 18 nucleotides, 12 nucleotides to 28 nucleotides, about 20 nucleotides to about 26 nucleotides, about 21 nucleotides, about 24 nucleotides, or about 26 nucleotides. In the examples provided herein, the miRNA is delivered in the form of a backbone-loop miRNA precursor sequence, e.g., from about 50 to about 80 nucleotides, or from about 55 to about 70 nucleotides in length. , or 60 to 65 nucleotides in length. In certain embodiments, the miRNA precursor includes about 5 nucleotides, about 21 nucleotide seed sequences, about 19 nucleotide backbone loops, and about 19 nucleotide sense sequences, wherein the sense sequence Sequences correspond to antisense sequences with one or two mispaired nucleotides. An example of a suitable miRNA coding sequence is the miR487 sequence (see, e.g., the vector genome in SEQ ID NO: 17: 5' flank (nt 3438)..(nt 3460) (SEQ ID NO: 1-23 of 5), miR487 (nt 3461)..(nt 3524), antisense (nt 3466)..(nt 3486), Loop (nt 3487)..(nt 3505), justice (nt 3506)..(nt 3524), and 3' flank (nt 3525)..(nt 3568). See also SEQ ID NO:9.

於某些具體實施例中，核酸分子(例如，表現匣或載體基因體)可含有至少一個或多於一個之miRNA編碼序列。於某些具體實施例中，核酸分子(例如，表現匣或載體基因體)可含有SEQ ID NO：15 (miR487)之一個、二個或以上之miRNA編碼序列、或進一步包含側翼區的miR487(例如，SEQ ID NO：16)。於某些具體實施例中，核酸分子(例如，表現匣或載體基因體)可含有SEQ ID NO：15 (miR487，64 nt)之一個、二個或以上miRNA編碼序列或SEQ ID NO：16。In certain embodiments, a nucleic acid molecule (eg, expression cassette or vector genome) may contain at least one or more than one miRNA coding sequence. In certain embodiments, the nucleic acid molecule (e.g., expression cassette or vector genome) may contain one, two or more miRNA coding sequences of SEQ ID NO: 15 (miR487), or further comprise flanking regions of miR487 (miR487). For example, SEQ ID NO: 16). In certain embodiments, the nucleic acid molecule (eg, expression cassette or vector genome) may contain one, two or more miRNA coding sequences of SEQ ID NO: 15 (miR487, 64 nt) or SEQ ID NO: 16.

如本文所使用，「miRNA標靶序列」為位於DNA正向股(5’至3’)(例如，C9orf72之正向股)的序列且與miRNA序列(包括miRNA種子序列)至少部分互補。miRNA標靶序列對於編碼的轉基因產物的非轉譯區為外源的(exogenous)，且被設計為在需要抑制轉基因表現的細胞中被miRNA所特異性地靶向。不欲受限於理論，因為hC9orf72為一種普遍存在的蛋白質，且過度表現可能與毒性及/或其他負向副作用有關，miRNA優先靶向內源性hC9orf72基因，同時避免靶向被遞送至患者的工程化hC9orf72基因。更特別地，經由載體遞送的編碼hC9orf72的序列被設計為在靶位點含有改變的密碼子序列。As used herein, a "miRNA target sequence" is a sequence located on the forward strand (5' to 3') of DNA (e.g., the forward strand of C9orf72) and is at least partially complementary to the miRNA sequence (including the miRNA seed sequence). The miRNA target sequence is exogenous to the untranslated region of the encoded transgene product and is designed to be specifically targeted by the miRNA in cells where inhibition of transgene expression is desired. Without wishing to be bound by theory, because hC9orf72 is a ubiquitous protein and overexpression may be associated with toxicity and/or other negative side effects, the miRNA preferentially targets the endogenous hC9orf72 gene while avoiding targeting that is delivered to the patient. Engineered hC9orf72 gene. More specifically, the hC9orf72-encoding sequence delivered via the vector is designed to contain altered codon sequences at the target site.

典型地，miRNA標靶序列係長度為至少7個核苷酸至約28個核苷酸，長度為至少8個核苷酸至約28個核苷酸，長度為7個核苷酸至28個核苷酸、8個核苷酸至18個核苷酸、12個核苷酸至28個核苷酸，約20至26個核苷酸、約22個核苷酸、約24個核苷酸、或約26個核苷酸，且其含有至少一個連續區域(例如，7或8個核苷酸)，其與miRNA種子序列互補。於某些具體實施例中，標靶序列包含與miRNA種子序列具有精確互補性(100%)或具有一些錯誤配對之部分互補性的序列。於某些具體實施例中，標靶序列包含至少7至8個核苷酸，其與miRNA種子序列100%互補。於某些具體實施例中，標靶序列由與miRNA種子序列100%互補的序列所組成。於某些具體實施例中，標靶序列含有與種子序列100%互補的序列之多個拷貝(例如，二或三個拷貝)。於某些具體實施例中，100%互補的區域包含標靶序列長度的至少30%。於某些具體實施例中，標靶序列的其餘部分與miRNA具有至少約80%至約99%的互補性。於某些具體實施例中，在含有DNA正向股的表現匣中，miRNA標靶序列為miRNA的反向互補序列。Typically, the miRNA target sequence is at least 7 nucleotides to about 28 nucleotides in length, at least 8 nucleotides to about 28 nucleotides in length, and from 7 nucleotides to 28 nucleotides in length. Nucleotides, 8 nucleotides to 18 nucleotides, 12 nucleotides to 28 nucleotides, about 20 to 26 nucleotides, about 22 nucleotides, about 24 nucleotides , or about 26 nucleotides, and which contains at least one contiguous region (eg, 7 or 8 nucleotides) that is complementary to the miRNA seed sequence. In certain embodiments, the target sequence includes a sequence that has exact complementarity (100%) or partial complementarity with some mismatches to the miRNA seed sequence. In certain embodiments, the target sequence includes at least 7 to 8 nucleotides that are 100% complementary to the miRNA seed sequence. In some embodiments, the target sequence consists of a sequence that is 100% complementary to the miRNA seed sequence. In certain embodiments, the target sequence contains multiple copies (eg, two or three copies) of a sequence that is 100% complementary to the seed sequence. In certain embodiments, a 100% complementary region includes at least 30% of the length of the target sequence. In certain embodiments, the remainder of the target sequence is at least about 80% to about 99% complementary to the miRNA. In certain embodiments, in the expression cassette containing the forward strand of DNA, the miRNA target sequence is the reverse complement of the miRNA.

如此，本文提供的序列與SEQ ID NO：13的變異C9編碼序列95%至99.9%相同，被設計以避免回復成構築體中選擇的miRNA所靶向的天然人類序列。較佳地，此等序列編碼與任何病症無關的天然功能性人類C9蛋白質。例如，此蛋白質可具有SEQ ID NO：14之序列或與SEQ ID NO：14約95至約100%相同、或至少97%、至少98%、或至少99%相同的序列。As such, the sequences provided herein are 95% to 99.9% identical to the variant C9 coding sequence of SEQ ID NO: 13 and are designed to avoid reversion to the native human sequence targeted by the selected miRNA in the construct. Preferably, these sequences encode native functional human C9 proteins that are not associated with any disease disorder. For example, the protein may have the sequence of SEQ ID NO: 14 or a sequence that is about 95 to about 100% identical to SEQ ID NO: 14, or at least 97%, at least 98%, or at least 99% identical.

於某些具體實施例中，miRNA優先靶向內源性hC9基因，同時避免靶向工程化hc9基因，其中內源性C9orf72同功型2核酸序列再現於SEQ ID NO：44且經編碼的蛋白質再現於SEQ ID NO：45[參見例如，NCBI NM_018325.4(C9orf72變異體2)及Ensembl ENST00000380003.8(C9orf72-203)]。於某些具體實施例中，miRNA編碼序列包含(i) 15或16之一或多者。於某些具體實施例中，工程化hC9核酸序列為SEQ ID NO：13。於某些具體實施例中，工程化hC9核酸序列為SEQ ID NO：13，其中存有1、2、3或4個核苷酸錯誤配對。In certain embodiments, the miRNA preferentially targets the endogenous hC9 gene while avoiding targeting the engineered hc9 gene, wherein the endogenous C9orf72 isoform 2 nucleic acid sequence is reproduced in SEQ ID NO: 44 and the encoded protein Reproduced in SEQ ID NO: 45 [see, eg, NCBI NM_018325.4 (C9orf72 variant 2) and Ensembl ENST00000380003.8 (C9orf72-203)]. In certain embodiments, the miRNA coding sequence includes one or more of (i) 15 or 16. In certain embodiments, the engineered hC9 nucleic acid sequence is SEQ ID NO: 13. In certain embodiments, the engineered hC9 nucleic acid sequence is SEQ ID NO: 13, which contains 1, 2, 3 or 4 nucleotide mismatches.

於某些具體實施例中，單一核酸(例如，表現匣或含有其之載體基因體)含有工程化hC9編碼序列及至少一個miRNA編碼序列兩者，其中該miRNA特異性地靶向在工程化hC9序列中不存在的內源性人類C9序列之區域。於某些具體實施例中，人類C9編碼序列為至少一個miRNA之上游(5’)且這二個元件被間隔子或連接子序列分開。於某些具體實施例中，hC9編碼序列的終止密碼子與最5'的miRNA編碼序列的起始之間至少有75個核苷酸。於某些具體實施例中，間隔子為約75個核苷酸至約300個核苷酸、或約75個核苷酸至約250個核苷酸、或約75個核苷酸至約200個核苷酸、或約75個核苷酸至約150個核苷酸、或約75個核苷酸至約100個核苷酸、或約80個核苷酸至約300個核苷酸、或約80個核苷酸至約250個核苷酸、或約80個核苷酸至約200個核苷酸、或約80個核苷酸至約150個核苷酸、或約80個核苷酸至約100個核苷酸。可選擇地，工程化hC9編碼序列及至少一個miRNA編碼序列被約75個核苷酸分開。適合地，間隔子序列為非編碼序列，其缺少任何限制酶位。可選擇地，間隔子可包括一或多個內含子序列。於某些具體實施例中，一或多個之miRNA序列可位於該內含子中。In certain embodiments, a single nucleic acid (e.g., expression cassette or vector genome containing the same) contains both an engineered hC9 coding sequence and at least one miRNA coding sequence, wherein the miRNA specifically targets the engineered hC9 A region of the endogenous human C9 sequence that is not present in the sequence. In certain embodiments, the human C9 coding sequence is upstream (5') of at least one miRNA and the two elements are separated by a spacer or linker sequence. In certain embodiments, there are at least 75 nucleotides between the stop codon of the hC9 coding sequence and the start of the 5'most miRNA coding sequence. In certain embodiments, the spacer is about 75 nucleotides to about 300 nucleotides, or about 75 nucleotides to about 250 nucleotides, or about 75 nucleotides to about 200 nucleotides. nucleotides, or about 75 nucleotides to about 150 nucleotides, or about 75 nucleotides to about 100 nucleotides, or about 80 nucleotides to about 300 nucleotides, Or about 80 nucleotides to about 250 nucleotides, or about 80 nucleotides to about 200 nucleotides, or about 80 nucleotides to about 150 nucleotides, or about 80 nuclei nucleotides to about 100 nucleotides. Alternatively, the engineered hC9 coding sequence and at least one miRNA coding sequence are separated by approximately 75 nucleotides. Suitably, the spacer sequence is a non-coding sequence which lacks any restriction enzyme sites. Alternatively, the spacer may include one or more intronic sequences. In certain embodiments, one or more miRNA sequences may be located within the intron.

於某些具體實施例中，工程化hC9編碼序列及miRNA編碼序列係經由不同的核酸序列遞送，例如，二個以上的不同載體、包含一載體及一LNP的組合等。於某些具體實施例中，兩種不同載體為AAV載體。於某些具體實施例中，此等載體具有不同表現匣。於其他具體實施例中，此等載體具有相同衣殼。於其他具體實施例中，載體具有不同具體化。於某些具體實施例中，miRNA編碼序列經由LNP或另一非病毒遞送系統遞送。於某些具體實施例中，工程化hC9序列經由LNP或另一非病毒遞送系統遞送。於某些具體實施例中，使用二種以上的不同遞送系統(例如，病毒及非病毒、兩種不同的非病毒)之組合。於此等及其他具體實施例中，二種以上的不同載體或其他遞送系統可實質上被同時投予，或者此等系統之一或多個可於另一個之前遞送。於某些具體實施例中，工程化hC9序列為SEQ ID NO：13、或與其90%至100%相同之序列，其編碼mRNA，該mRNA不被與其共同投予的miR結合且編碼功能性人類C9orf72。In some embodiments, the engineered hC9 coding sequence and the miRNA coding sequence are delivered via different nucleic acid sequences, for example, two or more different vectors, a combination including a vector and an LNP, etc. In certain embodiments, the two different vectors are AAV vectors. In some embodiments, these carriers have different presentation boxes. In other embodiments, the vectors have the same capsid. In other embodiments, the vector has different embodiments. In certain embodiments, the miRNA coding sequence is delivered via LNP or another non-viral delivery system. In certain embodiments, engineered hC9 sequences are delivered via LNP or another non-viral delivery system. In certain embodiments, a combination of two or more different delivery systems (eg, viral and non-viral, two different non-viral) is used. In these and other embodiments, two or more different vectors or other delivery systems may be administered substantially simultaneously, or one or more of these systems may be delivered before the other. In certain embodiments, the engineered hC9 sequence is SEQ ID NO: 13, or a sequence 90% to 100% identical thereto, which encodes an mRNA that is not bound by a co-administered miR and encodes a functional human C9orf72.

如本文所使用，術語「AAV.C9orf72」或「rAAV.h9ORF72」用於指一種重組腺相關病毒，其具有AAV衣殼，於衣殼中具有載體基因體，該載體基因體包含於調節序列控制下的人類C9orf72編碼序列(例如，cDNA)。如本文所使用，術語「AAV.C9orf72.miRXXX」或「rAAV.C9orf72.miRXXX」用於指一種重組腺相關病毒，其具有AAV衣殼，於衣殼中具有載體基因體，該載體基因體包含靶向內源性人類C9ORF72編碼序列之miR。As used herein, the term "AAV.C9orf72" or "rAAV.h9ORF72" is used to refer to a recombinant adeno-associated virus having an AAV capsid with a vector genome contained in the capsid controlled by regulatory sequences The human C9orf72 coding sequence (e.g., cDNA) below. As used herein, the term "AAV.C9orf72.miRXXX" or "rAAV.C9orf72.miRXXX" is used to refer to a recombinant adeno-associated virus having an AAV capsid with a vector genome in the capsid, the vector genome containing A miR targeting the endogenous human C9ORF72 coding sequence.

可指定特定的衣殼類型，諸如例如，AAV.C9orf72或rAAV1.C9orf72，其係指具有AAV1衣殼之重組AAV；AAVhu68.C9orf72或AAVhu68.C9orf72，其係指具有AAVhu68衣殼之重組AAV。AAVrh91.C9orf72或AAVrh91.C9orf72，其係指具有AAVrh91衣殼之重組AAV。Specific capsid types can be designated, such as, for example, AAV.C9orf72 or rAAV1.C9orf72, which refers to a recombinant AAV with an AAV1 capsid; AAVhu68.C9orf72 or AAVhu68.C9orf72, which refers to a recombinant AAV with an AAVhu68 capsid. AAVrh91.C9orf72 or AAVrh91.C9orf72 refers to a recombinant AAV with AAVrh91 capsid.

「重組AAV」或「rAAV」為一種DNA酶抗性病毒顆粒，含有兩個元件，AAV衣殼及載體基因體，該載體基因體至少含有包裝在AAV衣殼內的非AAV編碼序列。除非另有說明，否則此術語可與短語「rAAV載體」互換使用。該rAAV為一種「複製缺陷型病毒」或「病毒載體」，因為其缺少任何功能性AAV rep基因或功能性AAV cap基因且不能產生子代。於某些具體實施例中，唯一的AAV序列為AAV反向末端重複序列(ITR)，通常位於載體基因體的5'及3'末端，以便使位於ITR之間的基因及調節序列包裝在AAV衣殼內。一般而言，AAV衣殼係由60個衣殼(cap)蛋白質次單元VP1、VP2、及VP3所構成，該等次單元以二十面體對稱的方式排列，比例約為1:1:10至1:1:20，取決於所選擇的AAV。可選擇各種AAV作為如上述的AAV病毒載體衣殼的來源。於一具體實施例中，AAV衣殼為AAV9衣殼或其工程化變異體。參見，SEQ ID NO：30及31。於某些具體實施例中，變異體AAV9衣殼為AAV9.PhP.eB衣殼。於某些具體實施例中，PhP.eB衣殼被選擇用於小鼠研究，且為人類中演化支F載體(例如，AAVhu68)的適合模式。於某些具體實施例中，衣殼蛋白質係藉由rAAV載體名稱中術語「AAV」後的數字、或數字及字母的組合所指定。"Recombinant AAV" or "rAAV" is a DNase-resistant viral particle that contains two elements, the AAV capsid and a vector genome that contains at least non-AAV coding sequences packaged within the AAV capsid. Unless otherwise stated, this term is used interchangeably with the phrase "rAAV vector." This rAAV is a "replication-deficient virus" or "viral vector" because it lacks any functional AAV rep gene or functional AAV cap gene and cannot produce progeny. In some embodiments, the only AAV sequences are AAV inverted terminal repeats (ITRs), which are usually located at the 5' and 3' ends of the vector genome, so that genes and regulatory sequences located between the ITRs are packaged in the AAV inside the capsid. Generally speaking, the AAV capsid is composed of 60 capsid (cap) protein subunits VP1, VP2, and VP3. These subunits are arranged in an icosahedral symmetry with a ratio of approximately 1:1:10 to 1:1:20, depending on AAV selected. Various AAVs can be selected as a source of AAV viral vector capsids as described above. In a specific embodiment, the AAV capsid is AAV9 capsid or an engineered variant thereof. See, SEQ ID NO:30 and 31. In certain embodiments, the variant AAV9 capsid is an AAV9.PhP.eB capsid. In certain embodiments, PhP.eB capsids were selected for mouse studies and are a suitable model for clade F vectors (eg, AAVhu68) in humans. In certain embodiments, the capsid protein is designated by a number following the term "AAV" in the name of the rAAV vector, or a combination of numbers and letters.

除非另有指明，本文所述的AAV衣殼、ITR、及其他選擇的AAV組件可容易地選自任何AAV，包括但不限於定義如下的AAV：AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAVrh10、AAVhu37、AAVrh32.33、AAV8bp、AAV7M8及AAVAnc80、AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9.47、AAV9(hu14)、AAV10、AAV11、AAV12、AAVrh8、AAVrh74、AAV-DJ8、AAV-DJ、AAVhu68、AAV9變異體(例如，PCT/US21/61312，2021年12月1日申請；及US臨時申請案第63/119,863號，2020年12月1日申請；及US臨時申請案第63/178,881號，2021年4月23日申請)、AAVhu95及AAVhu96 (參見，US臨時申請案第63/251,599號，2021年10月2日申請)，並未限制。參見例如，WO 2019/168961及WO 2019/169004，兩者關於具有減少的衣殼脫醯胺的新穎AAV載體及其用途；US公開專利案第2007-0036760-A1號；US公開專利案第2009-0197338-A1號；EP 1310571。亦參見WO 2003/042397 (AAV7及其他猴AAV)、US專利7790449及US專利7282199 (AAV8)、WO 2005/033321及US 7,906,111 (AAV9)、及WO 2006/110689、及WO 2003/042397 (rh.10)、WO 2005/033321、WO 2018/160582 (AAVhu68)，其在此藉由引用而併入。亦參見WO 2019/168961及WO 2019/169004，描述此等及其他AAV衣殼的脫醯胺概貌。於某些具體實施例中，與另一個演化支F衣殼AAV9相比，衣殼具有兩個編碼胺基酸差異，差異位於位置67及157，基於SEQ ID NO：34所示之VP1蛋白質的編號(參見，核苷酸序列之SEQ ID NO：32及33)。相比之下，另一個演化支F AAV (AAV9、hu31、hu31)在位置67具有Ala且在位置157具有Ala。參見例如，WO 2022/082109，提供工程化AAVhu68編碼序列，WO 2018/160582；WO 2019/169004；及WO 2019/168961，其全部藉由引用而完整併入本文。Unless otherwise specified, the AAV capsids, ITRs, and other selected AAV components described herein may be readily selected from any AAV, including, but not limited to, AAVs defined as follows: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVhu37, AAVrh32.33, AAV8bp, AAV7M8 and AAVAnc80, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12 , AAVrh8, AAVrh74, AAV-DJ8, AAV-DJ, AAVhu68, AAV9 variants (e.g., PCT/US21/61312, filed on December 1, 2021; and US Provisional Application No. 63/119,863, December 2020 1 day application; and US Provisional Application No. 63/178,881, filed on April 23, 2021), AAVhu95 and AAVhu96 (see, US Provisional Application No. 63/251,599, filed on October 2, 2021), and Unrestricted. See, for example, WO 2019/168961 and WO 2019/169004, both regarding novel AAV vectors with reduced capsid deamidation and uses thereof; US Published Patent Case No. 2007-0036760-A1; US Published Patent Case No. 2009 No. -0197338-A1; EP 1310571. See also WO 2003/042397 (AAV7 and other monkey AAVs), US Patent 7790449 and US Patent 7282199 (AAV8), WO 2005/033321 and US 7,906,111 (AAV9), and WO 2006/110689, and WO 2003/042397 (rh. 10), WO 2005/033321, WO 2018/160582 (AAVhu68), which are hereby incorporated by reference. See also WO 2019/168961 and WO 2019/169004, which describe the deamidation profile of these and other AAV capsids. In certain embodiments, compared with another clade F capsid AAV9, the capsid has two coding amino acid differences, the differences are located at positions 67 and 157, based on the VP1 protein shown in SEQ ID NO: 34 Numbering (see, SEQ ID NO: 32 and 33 for nucleotide sequences). In contrast, another clade F AAV (AAV9, hu31, hu31) has Ala at position 67 and Ala at position 157. See, for example, WO 2022/082109, which provides engineered AAVhu68 coding sequences, WO 2018/160582; WO 2019/169004; and WO 2019/168961, all of which are incorporated herein by reference in their entirety.

於某些具體實施例中，AAVhu68衣殼進一步具有下列一或多個特徵。AAVhu68衣殼蛋白質包含：由編碼SEQ ID NO：34之1至736的胺基酸序列的核酸序列的表現所產生的AAVhu68 vp1蛋白質、由SEQ ID NO：32或33所產生的vp1蛋白質、或由編碼SEQ ID NO：34之1至736預測的胺基酸序列之與SEQ ID NO：33至少70%相同的核酸序列所產生的vp1蛋白質；由編碼SEQ ID NO：34之至少約胺基酸138至736的胺基酸序列之核酸序列的表現所產生之AAVhu68 vp2蛋白質、由包含SEQ ID NO：32或33之至少核苷酸412至2211的序列所產生的vp2蛋白質、或由編碼SEQ ID NO：34之至少約胺基酸138至736之預測的胺基酸序列之與SEQ ID NO：32或33之至少核苷酸412至2211至少70%相同的核酸序列所產生的vp2蛋白質；及/或由編碼SEQ ID NO：34之至少約胺基酸203至736之預測的胺基酸序列的核酸序列的表現所產生的AAVhu68 vp3蛋白質、由包含SEQ ID NO：32或33之至少核苷酸607至2211 的序列所產生的vp3蛋白質、或由編碼SEQ ID NO：34之至少約胺基酸203至736之胺基酸序列之與SEQ ID NO：32或33之至少核苷酸607至2211至少70%相同的核酸序列所產生的vp3蛋白質。於某些具體實施例中，一種AAVhu68衣殼包含：(i) AAVhu68 vp1蛋白質、AAVhu68 vp2蛋白質及AAVhu68 vp3蛋白質的異質族群(heterogenous population)，由編碼SEQ ID NO：34的核酸序列所產生，其中AAVhu68vp1蛋白質包含在位置67的麩胺酸及在位置157的纈胺酸且AAVhu68vp2蛋白質包含在位置157的纈胺酸，編號基於SEQ ID NO：34；或(ii) AAVhu68 vp1、AAVhu68 vp2及AAVhu68 vp3蛋白質之異質族群，其中AAVhu68 vp1蛋白質為SEQ ID NO：34之胺基酸1至736(vp1)，包含在位置67的麩胺酸及在位置157的纈胺酸且進一步包含vp1蛋白質的子群體，包含基於SEQ ID NO：34中胺基酸位置之經修飾的胺基酸，其中AAVhu68 vp2蛋白質為SEQ ID NO：34之胺基酸138至736(vp2)，包含在位置157的纈胺酸且進一步包含vp2蛋白質的子群體，包含基於SEQ ID NO：34中的胺基酸位置之經修飾的胺基酸，其中AAVhu68 vp3蛋白質為SEQ ID NO：34之胺基酸203至736(vp3)，其包含vp3蛋白質的子群體，包含基於SEQ ID NO：34中胺基酸位置之經修飾的胺基酸，其中在(i)及(ii)之AAVhu68 vp1、AAVhu68 vp2及AAV hu68 vp3蛋白質包含在相對於SEQ ID NO：34中的胺基酸的位置57、329、452、512的每一者上的天冬醯胺酸-甘胺酸對中至少50%至100%脫醯胺的天冬醯胺酸(N)，其中如使用光譜測定法測定，脫醯胺的天冬醯胺酸被脫醯胺成天冬胺酸、異天冬胺酸、互變天冬胺酸/異天冬胺酸對、或其組合。於某些具體實施例中，AAVhu68衣殼包含：(a) vp1蛋白質之子群體，其中如使用質譜測定法測定，75%至100%之在vp1蛋白質位置57的N被脫醯胺；及/或(b) vp1蛋白質、vp2蛋白質、及/或vp3蛋白質之子群體，其中基於SEQ ID NO：34的編號，如使用質譜測定法測定，75%至100%之在位置329的N被脫醯胺；及/或(c)vp1蛋白質、vp2蛋白質、及/或vp3蛋白質之子群體，如使用質譜測定法測定，75%至100%之基於SEQ ID NO：34的編號在位置452的N被脫醯胺；及/或(d)vp1蛋白質、vp2蛋白質、及/或vp3蛋白質之子群體，其中如使用質譜測定法測定，75%至100%之基於SEQ ID NO：34的編號在位置512的N被脫醯胺。In certain embodiments, the AAVhu68 capsid further has one or more of the following characteristics. The AAVhu68 capsid protein includes: AAVhu68 vp1 protein produced by expression of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 34-1 to 736, vp1 protein produced by SEQ ID NO: 32 or 33, or produced by A vp1 protein produced from a nucleic acid sequence encoding the predicted amino acid sequence 1 to 736 of SEQ ID NO: 34 that is at least 70% identical to SEQ ID NO: 33; a vp1 protein produced by encoding at least about amino acid 138 of SEQ ID NO: 34 The AAVhu68 vp2 protein produced by expression of the nucleic acid sequence of the amino acid sequence to 736, the vp2 protein produced by the sequence comprising at least nucleotides 412 to 2211 of SEQ ID NO: 32 or 33, or the vp2 protein produced by encoding SEQ ID NO. : vp2 protein produced by a nucleic acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 34 that is at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO: 32 or 33; and/ or an AAVhu68 vp3 protein generated by the representation of a nucleic acid sequence encoding at least about the predicted amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 34, consisting of at least nucleotides of SEQ ID NO: 32 or 33 The vp3 protein generated from the sequence 607 to 2211, or the amino acid sequence encoding at least about amino acids 203 to 736 of SEQ ID NO: 34 and at least nucleotides 607 to 2211 of SEQ ID NO: 32 or 33 The vp3 protein produced is at least 70% identical in nucleic acid sequence. In certain embodiments, an AAVhu68 capsid includes: (i) a heterogenous population of AAVhu68 vp1 protein, AAVhu68 vp2 protein, and AAVhu68 vp3 protein, generated by the nucleic acid sequence encoding SEQ ID NO: 34, wherein The AAVhu68vp1 protein contains glutamic acid at position 67 and valine at position 157 and the AAVhu68vp2 protein contains valine at position 157, numbering based on SEQ ID NO: 34; or (ii) AAVhu68 vp1, AAVhu68 vp2 and AAVhu68 vp3 A heterogeneous population of proteins, wherein the AAVhu68 vp1 protein is amino acids 1 to 736 of SEQ ID NO: 34 (vp1), including glutamic acid at position 67 and valine at position 157 and further comprising a subpopulation of the vp1 protein , comprising modified amino acids based on the amino acid positions in SEQ ID NO: 34, wherein the AAVhu68 vp2 protein is amino acids 138 to 736 (vp2) of SEQ ID NO: 34, including valine at position 157 And further includes a subpopulation of vp2 proteins, including modified amino acids based on the amino acid positions in SEQ ID NO: 34, wherein the AAVhu68 vp3 protein is amino acids 203 to 736 (vp3) of SEQ ID NO: 34 , which includes a subpopulation of vp3 proteins, including modified amino acids based on the amino acid positions in SEQ ID NO: 34, wherein the AAVhu68 vp1, AAVhu68 vp2 and AAV hu68 vp3 proteins in (i) and (ii) include Days of at least 50% to 100% deamidation in the aspartate-glycine pair at each of positions 57, 329, 452, 512 relative to the amino acid in SEQ ID NO: 34 Aspartic acid (N), where deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, tautoaspartic acid/isoaspartic acid, as determined using spectrometric methods Amino acid pairs, or combinations thereof. In certain embodiments, AAVhu68 capsids comprise: (a) a subpopulation of the vp1 protein in which 75% to 100% of the N at position 57 of the vp1 protein is deamidated as determined using mass spectrometry; and/or (b) A subpopulation of vp1 proteins, vp2 proteins, and/or vp3 proteins in which, based on the numbering of SEQ ID NO: 34, 75% to 100% of the N at position 329 is deamidated as determined using mass spectrometry; and/or (c) a subpopulation of the vp1 protein, the vp2 protein, and/or the vp3 protein, as determined using mass spectrometry, 75% to 100% of the N at position 452 based on the number based on SEQ ID NO: 34 is deamidated ; and/or (d) a subpopulation of vp1 proteins, vp2 proteins, and/or vp3 proteins, wherein 75% to 100% of the N at position 512 based on the number based on SEQ ID NO: 34 is removed, as determined using mass spectrometry. amide.

其他適合的序列可包括例如AAVhu95[工程化VP1核酸序列SEQ ID NO：26；胺基酸序列SEQ ID NO：27及35]；AAVhu96[工程化AAVhu96 VP1核酸序列，SEQ ID NO：28；AAV hu96 VP1胺基酸序列，SEQ ID NO：29]。Other suitable sequences may include, for example, AAVhu95 [engineered VP1 nucleic acid sequence, SEQ ID NO: 26; amino acid sequences, SEQ ID NO: 27 and 35]; AAVhu96 [engineered AAVhu96 VP1 nucleic acid sequence, SEQ ID NO: 28; AAV hu96 VP1 amino acid sequence, SEQ ID NO: 29].

其他適合的AAV可包括但不限於AAVrh90 [PCT/US20/30273，2020年4月28日申請]、AAVrh91 [參見，SEQ ID NO：37及38；PCT/US20/30266，2020年4月28日申請及US臨時專利申請案第63/109,734號，2020年11月4日申請及US臨時專利申請案第63/065,616號，2020年8月14日申請]AAVrh92、AAVrh93、AAVrh91.93 [PCT/US20/30281，2020年4月28日申請]，其在此藉由引用而併入。其他適合的AAV包括AAV3B變異體，其被描述於PCT/US20/56511，2020年10月20日申請，描述AAV3B.AR2.01、AAV3B.AR2.02、AAV3B.AR2.03、AAV3B.AR2.04、AAV3B.AR2.05、AAV3B.AR2.06、AAV3B.AR2.07、AAV3B.AR2.08、AAV3B.AR2.10、AAV3B.AR2.11、AAV3B.AR2.12、AAV3B.AR2.13、AAV3B.AR2.14、AAV3B.AR2.15、AAV3B.AR2.16、或AAV3B.AR2.17，其在此藉由引用而併入。此等文件亦描述其他AAV衣殼，其可被選擇用來生產rAAV且藉由引用而併入。在從人類或非人類靈長類動物(NHP)中分離或工程化並得到充分表徵的AAV中，人類AAV2係第一個被開發為基因轉移載體的AAV；它已被廣泛用於不同標靶組織及動物模型的高效基因轉移實驗。Other suitable AAVs may include, but are not limited to, AAVrh90 [PCT/US20/30273, filed April 28, 2020], AAVrh91 [see, SEQ ID NOs: 37 and 38; PCT/US20/30266, filed April 28, 2020 Application and US Provisional Patent Application No. 63/109,734, filed on November 4, 2020 and US Provisional Patent Application No. 63/065,616, filed on August 14, 2020] AAVrh92, AAVrh93, AAVrh91.93 [PCT/ US20/30281, filed April 28, 2020], which is hereby incorporated by reference. Other suitable AAVs include AAV3B variants described in PCT/US20/56511, filed October 20, 2020, describing AAV3B.AR2.01, AAV3B.AR2.02, AAV3B.AR2.03, AAV3B.AR2. 04. AAV3B.AR2.05, AAV3B.AR2.06, AAV3B.AR2.07, AAV3B.AR2.08, AAV3B.AR2.10, AAV3B.AR2.11, AAV3B.AR2.12, AAV3B.AR2.13, AAV3B.AR2.14, AAV3B.AR2.15, AAV3B.AR2.16, or AAV3B.AR2.17, which are incorporated herein by reference. These documents also describe other AAV capsids that may be selected for the production of rAAV and are incorporated by reference. Among the well-characterized AAVs isolated or engineered from humans or non-human primates (NHP), the human AAV2 lineage was the first AAV to be developed as a gene transfer vector; it has been widely used for different targets Efficient gene transfer experiments in tissues and animal models.

如本文所使用，「載體基因體」係指包裝在形成病毒顆粒的小病毒(parvovirus)(例如，rAAV)衣殼內部的核酸序列。此種核酸序列含有AAV反向末端重複序列(ITRs)。於本文之例中，載體基因體由5’至3’最低限度含有AAV 5’ ITR、編碼序列(即，轉基因(一或多個))及AAV 3’ ITR。可選擇來自AAV2之ITR，一種異於衣殼之不同來源AAV，或可選擇除全長ITR以外者。於某些具體實施例中，ITR係來自與生產過程中提供rep功能的AAV或反式互補(transcomplementing)AAV相同的AAV來源。再者，可使用其他ITR，例如，自互補(scAAV) ITR。單股AAV及自互補(sc) AAV二者皆包含於rAAV。轉基因為一種核酸編碼序列，與載體序列異源，其編碼有興趣之多肽、蛋白質、功能性RNA分子(例如，miRNA、miRNA抑制劑)或其他基因產物。該核酸編碼序列係以在標靶組織的細胞中允許轉基因轉錄、轉譯及/或表現的方式與調節組件可操作地連接。載體基因體的適當組件係於本文中更詳細地討論。As used herein, "vector genome" refers to a nucleic acid sequence packaged inside a parvovirus (eg, rAAV) capsid that forms a viral particle. Such nucleic acid sequences contain AAV inverted terminal repeats (ITRs). In the example herein, the vector genome contains, from 5' to 3', the AAV 5' ITR, the coding sequence (i.e., the transgene(s)) and the AAV 3' ITR at a minimum. The ITR from AAV2, a different source of AAV than the capsid, or something other than the full-length ITR can be selected. In certain embodiments, the ITR is derived from the same AAV source as the AAV or transcomplementing AAV that provides the rep function during production. Furthermore, other ITRs may be used, such as self-complementary (scAAV) ITRs. Both single-stranded AAV and self-complementary (sc) AAV are included in rAAV. A transgene is a nucleic acid coding sequence, heterologous to the vector sequence, that codes for a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product of interest. The nucleic acid coding sequence is operably linked to the regulatory component in a manner that permits transcription, translation, and/or expression of the transgene in cells of the target tissue. Suitable components of vector genomes are discussed in greater detail herein.

於一例中，「載體基因體」由5’至3’，最低限度含有載體特異性序列、可操作地連接至調節控制序列之包含工程化人類C9orf72編碼序列的核酸序列與可選擇含有之靶向內源性C9orf72的miRNA序列(該調節控制序列於標靶細胞中指導其表現)，其中該載體特異性序列可為末端重複序列，該末端重複序列特異性地包裝載體基因體至病毒載體衣殼或套膜蛋白質中。例如，AAV反向末端重複用於包裝至AAV及某些其他小病毒衣殼中。In one example, a "vector genome" contains, from 5' to 3', minimally vector-specific sequences, nucleic acid sequences including engineered human C9orf72 coding sequences operably linked to regulatory control sequences, and optionally contains targeting The miRNA sequence of endogenous C9orf72 (the regulatory control sequence directs its expression in the target cell), wherein the vector-specific sequence can be a terminal repeat sequence that specifically packages the vector genome into the viral vector capsid or in mantle proteins. For example, AAV inverted terminal repeats are used for packaging into AAV and certain other small viral capsids.

於某些具體實施例中，提供一組成物，其包含適合鞘內注射的水性液體及載體儲料(stock)(例如，具有AAV衣殼的rAAV，其優先靶向中樞神經系統及/或背根神經節中的細胞(例如，CNS，包括例如神經細胞(如錐狀細胞、浦金埃氏細胞(Purkinje's cell)、顆粒細胞、梭狀細胞及聯絡神經元細胞)及神經膠細胞(如星狀細胞、寡樹突細胞、小神經膠質細胞、及室管膜細胞)，其中具有工程化hC9orf72編碼序列及/或至少一個miRNA特異性內源性hC9orf72的載體用於遞送至中樞神經系統(CNS)。於某些具體實施例中，調配包含一或多個如本文所述的載體的組成物用於枕骨下注射到腦大池內(intra-cisterna magna)。於某些具體實施例中，組成物係經由電腦斷層造影-(CT-)rAAV注射而投予。於某些具體實施例中，組成物係使用歐氏貯存器(Ommaya reservoir)而投予。於某些具體實施例中，投予患者單劑量的組成物。In certain embodiments, a composition is provided that includes an aqueous liquid suitable for intrathecal injection and a carrier stock (e.g., rAAV with an AAV capsid that preferentially targets the central nervous system and/or dorsal Cells in the root ganglion (e.g., CNS, including, for example, nerve cells (e.g., cone cells, Purkinje's cells, granule cells, spindle cells, and contact neuron cells) and glial cells (e.g., astrocytes) cells, oligodendritic cells, microglia, and ependymal cells), wherein vectors with engineered hC9orf72 coding sequences and/or at least one miRNA-specific endogenous hC9orf72 are used for delivery to the central nervous system (CNS ). In certain embodiments, compositions comprising one or more carriers as described herein are formulated for suboccipital injection into the intra-cisterna magna. In certain embodiments, compositions The agent is administered via computed tomography-(CT-)rAAV injection. In certain embodiments, the agent is administered using an Ommaya reservoir. In certain embodiments, the agent is administered Give the patient a single dose of the composition.

如本文所使用，「表現匣」係指包含生物學上有用的核酸序列(例如，編碼蛋白質、酵素或其他有用的基因產物之基因cDNA、mRNA等)及與其可操作地連接的調節序列之核酸分子，該調節序列指導或調節核酸序列及其基因產物之轉錄、轉譯及/或表現。如本文所使用，「可操作地連接的」序列包括與核酸序列連續或不連續的調節序列、及在反式(trans)或順式(cis)核酸序列中作用的調節序列兩者。此種調節序列典型上包括例如：一或多個之啟動子、強化子、內含子、科札克序列(Kozak sequence)、多腺苷酸化(polyadenylation)序列、及TATA訊息。除此之外，表現匣還可含有基因序列之上游(相對於其為5’側)的調節序列，例如，一或多個啟動子、強化子、內含子等；以及基因序列的下游(相對於其為3’側)的一或多個強化子或調節序列，例如，包含多腺苷酸化位之3’未轉譯區(3’ UTR)。於某些具體實施例中，調節序列可操作地連接至基因產物之核酸序列，其中該調節序列藉由***的核酸序列(即，5’-未轉譯區(5’UTR))而與基因產物的核酸序列分開。於某些具體實施例中，表現匣包含一或多種基因產物之核酸序列。於一些具體實施例中，表現匣可為單順反子(monocistronic)或雙順反子(bicistronic)表現匣。於其他具體實施例中，術語「轉基因」係指***標靶細胞的來自外源的一或多個DNA序列。As used herein, a "expression cassette" refers to a nucleic acid containing a biologically useful nucleic acid sequence (e.g., gene cDNA, mRNA, etc. encoding a protein, enzyme, or other useful gene product) and regulatory sequences operably linked thereto. Molecules whose regulatory sequences direct or modulate the transcription, translation and/or expression of nucleic acid sequences and their gene products. As used herein, "operably linked" sequences include both regulatory sequences that are contiguous or discontinuous with the nucleic acid sequence, and regulatory sequences that act in trans or cis nucleic acid sequences. Such regulatory sequences typically include, for example, one or more promoters, enhancers, introns, Kozak sequences, polyadenylation sequences, and TATA messages. In addition, the expression cassette may also contain regulatory sequences upstream of the gene sequence (5' side relative to it), for example, one or more promoters, enhancers, introns, etc.; and downstream of the gene sequence ( One or more enhancers or regulatory sequences (relative to its 3' side), e.g., a 3' untranslated region (3' UTR) that includes a polyadenylation site. In certain embodiments, the regulatory sequence is operably linked to the nucleic acid sequence of the gene product, wherein the regulatory sequence is coupled to the gene product via an inserted nucleic acid sequence (i.e., 5'-untranslated region (5'UTR)) of nucleic acid sequences. In certain embodiments, a cassette contains nucleic acid sequences for one or more gene products. In some embodiments, the expression cassette may be a monocistronic or bicistronic expression cassette. In other embodiments, the term "transgene" refers to one or more DNA sequences from a foreign source that are inserted into a target cell.

通常，此類可用於產生病毒載體且含有本文所述基因產物的編碼序列的表現匣，其兩側為病毒基因體的包裝訊息及其他表現控制序列(諸如本文所述者)。於某些具體實施例中，載體基因體可含有二個以上的表現匣。Typically, such expression cassettes may be used to generate viral vectors and contain the coding sequence for the gene product described herein, flanked by packaging messages for the viral genome and other expression control sequences (such as those described herein). In some embodiments, the vector genome may contain more than two expression cassettes.

於某個具體實施例中，表現匣包含C9orf72編碼序列及靶向內源性C9orf72的miRNA序列)、啟動子，且可包括其之其他調節序列，其匣可被包裝至載體(例如，rAAV、慢病毒(lentivirus)、反轉錄病毒(retrovirus)等)中。In a specific embodiment, the expression cassette includes a C9orf72 coding sequence and a miRNA sequence targeting endogenous C9orf72), a promoter, and may include other regulatory sequences thereof, and the cassette may be packaged into a vector (e.g., rAAV, lentivirus, retrovirus, etc.).

AAV 重組小病毒特別適合作為載體。如本文所述，重組小病毒可含有AAV衣殼(或波卡病毒(bocavirus)衣殼)。於某些具體實施例中，衣殼靶向背根神經節中的細胞及/或下運動神經元及/或初級感覺神經元中的細胞。於某些具體實施例中，本文提供的組成物可具有單一rAAV儲料，其包含rAAV，該rAAV包含工程化hC9orf72及特異性地靶向內源性hC9orf72的miRNA以便向下調節內源性hC9orf72水平並減少任何與hC9orf72過度表現有關的毒性。於其他具體實施例中，rAAV可包含hC9orf72且可與包含向下調節內源性hC9orf72之miRNA的不同載體共同投予。於其他具體實施例中，rAAV可包含至少一個向下調節內源性hC9orf72的miRNA及第二載體(或其他組成物)，其用來遞送hC9orf72。 AAV Recombinant small viruses are particularly suitable as vectors. As described herein, recombinant parvoviruses may contain AAV capsids (or bocavirus capsids). In certain embodiments, capsids target cells in dorsal root ganglia and/or cells in lower motor neurons and/or primary sensory neurons. In certain embodiments, compositions provided herein can have a single rAAV reservoir that includes an rAAV that includes engineered hC9orf72 and a miRNA that specifically targets endogenous hC9orf72 to downregulate endogenous hC9orf72 levels and reduce any toxicity associated with hC9orf72 overexpression. In other embodiments, rAAV may comprise hC9orf72 and may be co-administered with a different vector comprising a miRNA that downregulates endogenous hC9orf72. In other embodiments, rAAV may include at least one miRNA that down-regulates endogenous hC9orf72 and a second vector (or other composition) used to deliver hC9orf72.

例如，使用來自演化支F(例如，AAVhu68或AAV9)的AAV衣殼所產生的載體可被使用以生產載體，該載體於CNS靶向並表現hC9orf72。或者，可選擇使用來自演化支A(例如，AAV1、AAVrh91)的AAV衣殼所產生的載體。於再其他具體實施例中，其他小病毒或其他AAV病毒可為AAV衣殼的適合來源。For example, vectors generated using AAV capsids from clade F (eg, AAVhu68 or AAV9) can be used to produce vectors that target and express hC9orf72 in the CNS. Alternatively, vectors generated using AAV capsids from clade A (eg, AAV1, AAVrh91) may be chosen. In still other embodiments, other small viruses or other AAV viruses may be suitable sources of AAV capsids.

AAV1衣殼係指具有AAV vp1蛋白質、AAV vp2蛋白質及AAV vp3蛋白質的衣殼。於特定的具體實施例中，AAV1衣殼包含約1:1:10之預定比例的AAV vp1蛋白質、AAV vp2蛋白質及AAV vp3蛋白質，組裝成全部60個vp蛋白質的T1二十面體衣殼。AAV1衣殼能夠包裝基因體序列以形成AAV顆粒(例如，重組AAV，其中基因體為載體基因體)。典型地，編碼最長的vp蛋白質(即VP1)的衣殼核酸序列，於具有AAV1衣殼的rAAV生產期間係反式表現，被描述於例如US專利6,759,237、US 專利7,105,345、US專利7,186,552、US專利8,637,255、及US專利9,567,607，其在此藉由引用而併入。亦參見WO 2018/168961，其藉由引用併入。於某些具體實施例中，AAV1的特徵在於VP同功型(isoform)之異質族群的衣殼組成物，其如WO 2018/160582所定義被脫醯胺，藉由引用將其完整併入，基於如使用質譜測定法測定之衣殼中VP蛋白質總量。於某些具體實施例中，如使用質譜測定法測定，AAV衣殼在以下位置的一或多處並在以下提供的範圍內被修飾。適合的修飾包括在上述標識為脫醯胺化調節的段落中描述者，其藉由引用併入本文。於某些具體實施例中，一或多個下列位置、或N後面的甘胺酸係如本文所述地被修飾。於某些具體實施例中，構築AAV1突變體，其中位置57、383、512及/或718的N後的甘胺酸被保留(即，保持未經修飾)。於某些具體實施例中，前句中所識別的四個位置的NG與天然序列一起保留。於某些具體實施例中，將人工NG導入與WO 2018/160582中定義及識別的位置不同的位置，其藉由引用併入本文。AAV1 capsid refers to a capsid containing AAV vp1 protein, AAV vp2 protein and AAV vp3 protein. In certain embodiments, the AAV1 capsid includes a predetermined ratio of approximately 1:1:10 of AAV vp1 protein, AAV vp2 protein, and AAV vp3 protein assembled into a T1 icosahedral capsid of all 60 vp proteins. AAV1 capsids are capable of packaging genome sequences to form AAV particles (eg, recombinant AAV, where the genome is a vector genome). Typically, the capsid nucleic acid sequence encoding the longest vp protein (i.e., VP1), expressed in trans during rAAV production with AAV1 capsids, is described, for example, in US Patent 6,759,237, US Patent 7,105,345, US Patent 7,186,552, US Patent 8,637,255, and US Patent 9,567,607, which are incorporated herein by reference. See also WO 2018/168961, which is incorporated by reference. In certain embodiments, AAV1 is characterized by a capsid composition of a heterogeneous population of VP isoforms that are deamidated as defined in WO 2018/160582, which is incorporated by reference in its entirety, Based on the total amount of VP protein in the capsid as determined using mass spectrometry. In certain embodiments, the AAV capsid is modified at one or more of the following positions and within the ranges provided below, as determined using mass spectrometry. Suitable modifications include those described above in the paragraph labeled Deamidation Modulation, which is incorporated herein by reference. In certain embodiments, one or more of the following positions, or the glycine following N, is modified as described herein. In certain embodiments, AAV1 mutants are constructed in which the glycine following the N at positions 57, 383, 512, and/or 718 is retained (ie, remains unmodified). In certain embodiments, the four positions of NG identified in the preceding sentence are retained with the native sequence. In certain embodiments, artificial NG is introduced into a different location than that defined and identified in WO 2018/160582, which is incorporated herein by reference.

如本文所使用，AAVhu68衣殼係指如WO 2018/160582中所定義的衣殼，其藉由引用併入本文。如本文所述，rAAVhu68具有在生產系統中所生產的rAAVhu68衣殼，該生產系統係自AAVhu68核酸而表現衣殼。於某些具體實施例中，AAVhu68核酸序列為SEQ ID NO：32或33，編碼SEQ ID NO 34之胺基酸序列。於某些具體實施例中，AAVhu68核酸序列為SEQ ID NO：32或33，編碼SEQ ID NO：34之胺基酸序列。使用單個核酸序列vp1的生產所產生的rAAVhu68會產生vp1蛋白質、vp2蛋白質及vp3蛋白質的異質族群。此等子群體最低限度包括脫醯胺的天冬醯胺酸(N或Asn)殘基。例如，天冬醯胺酸-甘胺酸對中的天冬醯胺酸被高度脫醯胺。於某些具體實施例中，vp2及/或vp3蛋白質可被額外地或替代地從不同於vp1的核酸序列表現，例如，以改變所選擇的表現系統中vp蛋白質的比例。As used herein, AAVhu68 capsid refers to a capsid as defined in WO 2018/160582, which is incorporated herein by reference. As described herein, rAAVhu68 has a rAAVhu68 capsid produced in a production system expressing capsids from AAVhu68 nucleic acid. In certain embodiments, the AAVhu68 nucleic acid sequence is SEQ ID NO: 32 or 33, encoding the amino acid sequence of SEQ ID NO 34. In certain embodiments, the AAVhu68 nucleic acid sequence is SEQ ID NO: 32 or 33, encoding the amino acid sequence of SEQ ID NO: 34. Production of rAAVhu68 using a single nucleic acid sequence vp1 results in a heterogeneous population of vp1 proteins, vp2 proteins, and vp3 proteins. Such subpopulations include minimally deamidated aspartic acid (N or Asn) residues. For example, the aspartate in the aspartate-glycine pair is highly deamidated. In certain embodiments, vp2 and/or vp3 proteins may additionally or alternatively be expressed from nucleic acid sequences different from vp1, for example, to alter the proportion of vp proteins in the selected expression system.

包裝於AAV衣殼中並遞送到宿主細胞的基因體序列通常最低限度係由轉基因及其調節序列以及AAV反向末端重複(ITR)所組成。單股AAV及自互補(sc)AAV二者皆包含於rAAV。轉基因為與載體序列異源之核酸編碼序列，其編碼有興趣之多肽、蛋白質、功能性RNA分子(例如，miRNA、miRNA抑制劑)或其他基因產物。該核酸編碼序列係以在標靶組織的細胞中允許轉基因轉錄、轉譯及/或表現的方式與調節組件可操作地連接。The genome sequence packaged in the AAV capsid and delivered to the host cell usually consists of a minimum transgene and its regulatory sequences and the AAV inverted terminal repeat (ITR). Both single-stranded AAV and self-complementary (sc)AAV are included in rAAV. A transgene is a nucleic acid coding sequence heterologous to the vector sequence that encodes a polypeptide, protein, functional RNA molecule (eg, miRNA, miRNA inhibitor) or other gene product of interest. The nucleic acid coding sequence is operably linked to the regulatory component in a manner that permits transcription, translation, and/or expression of the transgene in cells of the target tissue.

載體的AAV序列通常包含順式作用(cis-acting) 5'及3'反向末端重複序列(參見例如B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990))。ITR序列長度為約145 bp。儘管允許對此等序列進行某種程度的輕微修飾，但較佳為實質上在分子中使用編碼ITR的整個序列。修飾此等ITR序列的能力係於本領域技術範圍內。(參見例如，如texts such as Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989)；及K. Fisher et al., J. Virol., 70:520 532 (1996)之文件)。本發明中所使用的此種分子之例為含有轉基因的「順式作用」質體，其中5’及3’ AAV ITR序列位於所選擇的轉基因序列及有關的調節元件之兩側。The AAV sequence of the vector usually contains cis-acting 5' and 3' inverted terminal repeats (see, e.g., B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The length of the ITR sequence is approximately 145 bp. Although some minor modifications to such sequences are allowed, it is preferred to use essentially the entire sequence encoding the ITR in the molecule. The ability to modify such ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, “Molecular Cloning. A Laboratory Manual,” 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70 :520 532 (1996) document). An example of such a molecule used in the present invention is a "cis-acting" plastid containing a transgene, in which the 5' and 3' AAV ITR sequences flank the selected transgene sequence and associated regulatory elements.

ITR為在載體生產過程中負責基因體的複製及包裝的遺傳元件，且為生成rAAV所需的唯一病毒順式元件。於一具體實施例中，ITR來自不同於提供衣殼的AAV。於較佳具體實施例，為了方便及加速監管批准，可使用來自AAV2之ITR序列、或其經刪除版(ΔITR)。然而，可選擇來自其他AAV來源的ITR。於ITR之來源為來自AAV2且AAV衣殼來自另一AAV來源時，生成的載體可稱為假型(pseudotyped)。一般而言，AAV載體基因體包含AAV 5’ ITR、編碼基因產物之核酸序列、及任何調節序列、以及AAV 3’ ITR。然而，此等元件之其他型態可為適合的。於一具體實施例中，提供自互補AAV。已描述5’ITR的縮短版，稱為ΔITR，其中刪除了D序列(D-sequence)及末端分割位(terminal resolution site)(trs)。於某些具體實施例中，載體基因體包括包括130個鹼基對之縮短的AAV2 ITR，其中外部「a」元件被刪除。在使用內部A元件作為模板的載體DNA擴增過程中，縮短的ITR被回復為145個鹼基對的野生型長度。於其他具體實施例中，使用全長AAV 5’及3’ITR。ITR is a genetic element responsible for genome replication and packaging during vector production, and is the only viral cis-element required to generate rAAV. In a specific embodiment, the ITR is from a different AAV than the one that provides the capsid. In a preferred embodiment, to facilitate and expedite regulatory approval, the ITR sequence from AAV2, or its deleted version (ΔITR), can be used. However, ITRs from other AAV sources can be selected. When the source of the ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be called pseudotyped. Generally, the AAV vector genome contains the AAV 5' ITR, the nucleic acid sequence encoding the gene product, and any regulatory sequences, and the AAV 3' ITR. However, other types of such components may be suitable. In a specific embodiment, self-complementary AAV is provided. A shortened version of the 5'ITR has been described, called ΔITR, in which the D-sequence and terminal resolution site (trs) are deleted. In certain embodiments, the vector genome includes a shortened AAV2 ITR including 130 base pairs, in which the external "a" element is deleted. During vector DNA amplification using the internal A element as template, the shortened ITR was restored to the wild-type length of 145 base pairs. In other embodiments, full-length AAV 5' and 3' ITRs are used.

除了載體(例如，rAAV)之上列識別的主要元件之外，載體亦包括必需的習用控制元件，其以允許轉基因在細胞中轉錄、轉譯及/或表現的方式與轉基因可操作地連接。如本文所使用，術語「表現」或「基因表現」係指來自基因的訊息被用於合成功能性基因產物的過程。基因產物可為蛋白質、肽、或核酸聚合物(如RNA、DNA或PNA)。In addition to the major elements identified above on a vector (eg, rAAV), the vector also includes the necessary conventional control elements operably linked to the transgene in a manner that allows transcription, translation, and/or expression of the transgene in the cell. As used herein, the term "expression" or "gene expression" refers to the process by which information from a gene is used to synthesize a functional gene product. Gene products can be proteins, peptides, or nucleic acid polymers (such as RNA, DNA, or PNA).

如本文所使用，術語「調節序列」或「表現控制序列」係指核酸序列，如起始序列、強化子序列、及啟動子序列，其誘導、抑制、或以其他方式控制與彼等可操作地連接的蛋白質編碼核酸序列的轉錄。調節控制元件通常含有啟動子序列作為表現控制序列之一部分，例如，位於所選擇的5’ ITR序列及編碼序列之間。於特別理想的具體實施例中，選擇用於中樞神經系統的組織特異性啟動子。例如，啟動子可為神經細胞啟動子，例如，gfaABC(1)D啟動子(Addgene #50473))、或人類Syn啟動子(序列可由Addgene獲得，Ref. #50465)。As used herein, the term "regulatory sequence" or "expression control sequence" refers to nucleic acid sequences, such as initiation sequences, enhancer sequences, and promoter sequences, that induce, inhibit, or otherwise control those operably associated with Transcription of locally linked protein-coding nucleic acid sequences. Regulatory control elements typically contain a promoter sequence as part of the expression control sequence, e.g., between the selected 5' ITR sequence and the coding sequence. In particularly desirable embodiments, tissue-specific promoters for the central nervous system are selected. For example, the promoter can be a neural cell promoter, such as the gfaABC(1)D promoter (Addgene #50473)), or the human Syn promoter (sequence available from Addgene, Ref. #50465).

其他適合的啟動子可包括例如，組成型啟動子(constitutive promoter)、可調節的啟動子[參見例如，WO 2011/126808及WO 2013/04943]、組織特異性啟動子、或對生理提示有反應的啟動子可被用於本文所述的載體。啟動子可選自不同來源，例如，人類巨細胞病毒(CMV)立即早期強化子/啟動子、SV40早期強化子/啟動子、JC多瘤病毒(JC polymovirus)啟動子、髓鞘質鹼性蛋白(MBP)或神經膠纖維酸性蛋白(glial fibrillary acidic protein)(GFAP)啟動子、單純疱疹病毒(HSV-1)潛伏相關啟動子(latency associated promoter)(LAP)、勞氏肉瘤病毒(rouse sarcoma virus)(RSV)末端長重複序列(LTR)啟動子、神經元特異性啟動子(NSE)、血小板衍生的生長因子(PDGF)啟動子、hSYN、黑色素凝集激素(melanin-concentrating hormone)(MCH)啟動子、CBA、基質金屬蛋白啟動子(matrix metalloprotein promoter)(MPP)、及雞β-肌動蛋白啟動子。除了啟動子，載體可含有一或多個其他適當的轉錄起始、終止、強化子序列、有效的RNA加工訊息諸如剪接(splicing)及多腺苷酸化(polyA)訊息；穩定細胞質的mRNA之序列，例如WPRE；增強轉譯效率之序列(即，科札克共通序列(Kozak consensus sequence))；增強蛋白質穩定性之序列；及當需要時，增強所編碼的產物之分泌的序列。適合的強化子之例為CMV強化子。其他適合的強化子包括適合所欲標靶組織適應症者。於一具體實施例中，表現匣包含表現匣包含一或多個表現強化子。於一具體實施例中，表現匣含有二或多個表現強化子。此等強化子可相同或可彼此不同。例如，強化子可包括CMV立即早期(IE)強化子。於某些具體實施例中，強化子可包括包含SEQ ID NO：3之核酸序列的CMV IE強化子(C4)。此強化子能以位置彼此相鄰的兩個拷貝的方式存在。或者，強化子的雙重拷貝可被一個或多個序列分開。於再另一具體實施例中，表現匣進一步含有內含子，例如，雞β-肌動蛋白內含子。於某些具體實施例中，表現匣包含一內含子，該內含子為包含SEQ ID NO：47的β-肌動蛋白內含子。其他適合的內含子包括本技術領域中已知者，例如，諸如WO 2011/126808所述者。適合的polyA序列之例包括例如：SV40、SV50、牛生長激素(bGH)、人類生長激素、及合成的polyA。於某些具體實施例中，polyA為SV40 poly A。於某些具體實施例中，polyA為兔球蛋白poly A (RBG)。於某些具體實施例中，polyA為包含SEQ ID NO：10之RBG polyA。可選擇地，可選擇一或多個序列以穩定mRNA。此種序列之例為經修飾的WPRE序列，其可在polyA序列的上游及編碼序列的下游被工程化[參見例如MA Zanta-Boussif, et al, Gene Therapy (2009) 16：605-619。Other suitable promoters may include, for example, constitutive promoters, regulatable promoters [see, e.g., WO 2011/126808 and WO 2013/04943], tissue-specific promoters, or responsive to physiological cues. The promoter of can be used in the vectors described herein. The promoter can be selected from different sources, for example, human cytomegalovirus (CMV) immediate early enhancer/promoter, SV40 early enhancer/promoter, JC polyomavirus promoter, myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latency associated promoter (LAP), rouse sarcoma virus ) (RSV) long terminal repeat (LTR) promoter, neuron-specific promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, melanin-concentrating hormone (MCH) promoter promoter, CBA, matrix metalloprotein promoter (MPP), and chicken β-actin promoter. In addition to the promoter, the vector may contain one or more other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing messages such as splicing and polyadenylation (polyA) messages; sequences that stabilize the cytoplasmic mRNA , such as WPRE; a sequence that enhances translation efficiency (i.e., Kozak consensus sequence); a sequence that enhances protein stability; and a sequence that enhances secretion of the encoded product, when needed. An example of a suitable enhancer is the CMV enhancer. Other suitable enhancers include those suitable for the desired target tissue indication. In one embodiment, the performance box includes one or more performance enhancers. In a specific embodiment, the expression box contains two or more expression enhancers. These enhancers may be the same or may be different from each other. For example, enhancers may include CMV immediate early (IE) enhancers. In certain embodiments, the enhancer may include the CMV IE enhancer (C4) comprising the nucleic acid sequence of SEQ ID NO: 3. This enhancer can exist as two copies located adjacent to each other. Alternatively, the double copies of the enhancer can be separated by one or more sequences. In yet another embodiment, the expression cassette further contains an intron, for example, a chicken β-actin intron. In certain embodiments, the expression cassette includes an intron that is a β-actin intron including SEQ ID NO: 47. Other suitable introns include those known in the art, for example such as those described in WO 2011/126808. Examples of suitable polyA sequences include, for example, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. In certain embodiments, polyA is SV40 polyA. In certain embodiments, polyA is rabbit globulin poly A (RBG). In certain embodiments, polyA is RBG polyA comprising SEQ ID NO: 10. Alternatively, one or more sequences can be selected to stabilize the mRNA. An example of such a sequence is a modified WPRE sequence, which can be engineered upstream of the polyA sequence and downstream of the coding sequence [see, eg, MA Zanta-Boussif, et al, Gene Therapy (2009) 16:605-619.

於某些具體實施例中，載體基因體包含組織特異性啟動子。於一些具體實施例中，組織特異性啟動子為人類突觸蛋白(synapsin)啟動子。於某些具體實施例中，人類突觸蛋白啟動子包含SEQ ID NO：6之核酸序列。於某些具體實施例中，載體基因體包含組成型啟動子，其中該啟動子為CB7啟動子或其變異體，例如，CAG啟動子。於某些具體實施例中，CB7或其變異體為雜合(hybrid)啟動子(啟動子元件)，最低限度包含人類巨細胞病毒(CMV)立即早期(IE)強化子及雞β-肌動蛋白(CB或CBA)啟動子。於某些具體實施例中，CB7啟動子或變異體係指人類巨細胞病毒(CMV)立即早期(IE)強化子(C4)、雞β-肌動蛋白(CB)啟動子、可選擇的內含子、連接雜合啟動子的元件之可選擇的間隔子序列。參見例如，具有巨細胞病毒強化子的雞β-肌動蛋白啟動子。於某些具體實施例中，CB7啟動子或啟動子元件係指人類巨細胞病毒(CMV)立即早期(IE)強化子(C4)、雞β-肌動蛋白(CB)啟動子、內含子、及連接雜合啟動子的元件之可選擇的間隔子序列。於某些具體實施例中，CB7啟動子或啟動子元件係指人類巨細胞病毒(CMV)立即早期(IE)強化子(C4)、雞β-肌動蛋白(CB)啟動子、包含具有兔β球蛋白剪接供體的雞β-肌動蛋白內含子的內含子、及連接雜合啟動子的元件之可選擇的間隔子序列。於某些具體實施例中，CB7啟動子或啟動子元件係指人類巨細胞病毒(CMV)立即早期(IE)強化子(C4)(SEQ ID NO：3)、雞β-肌動蛋白(CB)啟動子(SEQ ID NO：46)、可選擇的內含子(SEQ ID NO：47)、及連接雜合啟動子的元件之可選擇的間隔子序列。於某些具體實施例中，CB7啟動子或啟動子元件係指人類巨細胞病毒(CMV)立即早期(IE)強化子(SEQ ID NO：51)、雞β-肌動蛋白(CB)啟動子(SEQ ID NO：52)、可選擇的內含子(SEQ ID NO：53)、及連接雜合啟動子的元件之可選擇的間隔子序列。於某些具體實施例中，CB7啟動子或啟動子元件包含SEQ ID NO：4之核酸序列。於某些具體實施例中，CB7啟動子或啟動子元件包含SEQ ID NO：43之核酸序列。於某些具體實施例中，CB7啟動子或啟動子元件包含SEQ ID NO：48之核酸序列。於某些具體實施例中，CB7啟動子或啟動子元件包含SEQ ID NO：49之核酸序列。於某些具體實施例中，CB7啟動子或啟動子元件包含SEQ ID NO：50之核酸序列。較佳地，間隔子序列為非編碼的且於某些具體實施例中，可為不同長度。In certain embodiments, the vector genome includes a tissue-specific promoter. In some embodiments, the tissue-specific promoter is the human synapsin promoter. In certain embodiments, the human synapsin promoter comprises the nucleic acid sequence of SEQ ID NO: 6. In certain embodiments, the vector genome includes a constitutive promoter, wherein the promoter is a CB7 promoter or a variant thereof, for example, a CAG promoter. In certain embodiments, CB7 or a variant thereof is a hybrid promoter (promoter element), which at a minimum includes human cytomegalovirus (CMV) immediate early (IE) enhancer and chicken β-actin Protein (CB or CBA) promoter. In certain embodiments, the CB7 promoter or variant system refers to human cytomegalovirus (CMV) immediate early (IE) enhancer (C4), chicken β-actin (CB) promoter, optional intron promoter, and an optional spacer sequence connecting elements of the hybrid promoter. See, for example, chicken β-actin promoter with cytomegalovirus enhancer. In certain embodiments, the CB7 promoter or promoter element refers to human cytomegalovirus (CMV) immediate early (IE) enhancer (C4), chicken beta-actin (CB) promoter, intron , and an optional spacer sequence connecting elements of the hybrid promoter. In certain embodiments, the CB7 promoter or promoter element refers to human cytomegalovirus (CMV) immediate early (IE) enhancer (C4), chicken β-actin (CB) promoter, including rabbit The intron of the chicken β-actin intron of the β-globin splice donor, and an optional spacer sequence connecting elements of the hybrid promoter. In certain embodiments, the CB7 promoter or promoter element refers to human cytomegalovirus (CMV) immediate early (IE) enhancer (C4) (SEQ ID NO: 3), chicken beta-actin (CB ) promoter (SEQ ID NO: 46), an optional intron (SEQ ID NO: 47), and an optional spacer sequence connecting elements of the hybrid promoter. In certain embodiments, the CB7 promoter or promoter element refers to human cytomegalovirus (CMV) immediate early (IE) enhancer (SEQ ID NO: 51), chicken β-actin (CB) promoter (SEQ ID NO: 52), an optional intron (SEQ ID NO: 53), and an optional spacer sequence connecting elements of the hybrid promoter. In certain embodiments, the CB7 promoter or promoter element includes the nucleic acid sequence of SEQ ID NO: 4. In certain embodiments, the CB7 promoter or promoter element includes the nucleic acid sequence of SEQ ID NO: 43. In certain embodiments, the CB7 promoter or promoter element includes the nucleic acid sequence of SEQ ID NO: 48. In certain embodiments, the CB7 promoter or promoter element includes the nucleic acid sequence of SEQ ID NO: 49. In certain embodiments, the CB7 promoter or promoter element includes the nucleic acid sequence of SEQ ID NO: 50. Preferably, the spacer sequence is non-coding and, in certain embodiments, may be of different lengths.

於一具體實施例中，載體基因體包含：AAV 5’ ITR、啟動子、可選擇的強化子、可選擇的內含子、人類C9orf72的編碼序列(hC9orf72或huC9orf72)、poly A、及AAV 3’ ITR。於某個具體實施例中，載體基因體為AAV2 5’ ITR、CB7啟動子或其變異體、工程化C9orf72、連接子、靶向內源性C9orf72序列的miR、兔β球蛋白 poly A、及AAV2 3’ ITR。於某個具體實施例中，載體基因體為AAV2 5’ ITR、CB7啟動子或其變異體、內含子、C9orf72、兔β球蛋白poly A、及AAV2 3’ ITR。於某個具體實施例中，載體基因體為AAV2 5’ ITR、CB7啟動子或其變異體、工程化huC9orf72、連接子、miR487序列、兔β球蛋白 poly A、及AAV2 3’ ITR。huC9orf72編碼序列選自本發明說明書中彼等定義者。參見例如，SEQ ID NO：13或與其至少95%至99.9%相同的序列、如本文定義的其片段。於某些具體實施例中，其他C9orf72編碼序列可與本文提供的miR487組合。對於本發明的某些具體實施例，可對載體基因體選擇載體基因體的其他元件或此等序列上的變異。In a specific embodiment, the vector genome includes: AAV 5' ITR, promoter, selectable enhancer, selectable intron, human C9orf72 coding sequence (hC9orf72 or huC9orf72), poly A, and AAV 3 'ITR. In a specific embodiment, the vector genome is AAV2 5' ITR, CB7 promoter or variant thereof, engineered C9orf72, linker, miR targeting endogenous C9orf72 sequence, rabbit β-globin poly A, and AAV2 3'ITR. In a specific embodiment, the vector genome is AAV2 5' ITR, CB7 promoter or its variant, intron, C9orf72, rabbit β-globin poly A, and AAV2 3' ITR. In a specific embodiment, the vector genome is AAV2 5' ITR, CB7 promoter or variant thereof, engineered huC9orf72, linker, miR487 sequence, rabbit β-globin poly A, and AAV2 3' ITR. The huC9orf72 coding sequence is selected from those defined in the specification of the invention. See, for example, SEQ ID NO: 13 or a sequence at least 95% to 99.9% identical thereto, fragments thereof as defined herein. In certain embodiments, other C9orf72 coding sequences can be combined with the miR487 provided herein. For certain embodiments of the invention, other elements of the vector genome or variations in such sequences may be selected for the vector genome.

載體生產於生產AAV病毒載體(例如，重組(r)AAV)的使用，表現匣可被攜帶於任何適合的載體上，例如質體，其被遞送至包裝宿主細胞(packaging host cell)。有用於本發明之質體可經工程化而適合於原核細胞、昆蟲細胞、哺乳動物細胞等中之活體外複製及包裝。適合的轉染技術及包裝宿主細胞為本技術領域中具有通常知識者已知及/或可輕易設計。 vector production For use in the production of AAV viral vectors (eg, recombinant (r)AAV), the expression cassette can be carried on any suitable vector, such as a plasmid, which is delivered to a packaging host cell. Plastids useful in the present invention can be engineered to be suitable for in vitro replication and packaging in prokaryotic cells, insect cells, mammalian cells, and the like. Suitable transfection techniques and packaging host cells are known and/or can be readily designed by those of ordinary skill in the art.

於某些具體實施例中，生產質體包含用以包裝於衣殼內的載體基因體，其包含：(a)編碼人類C9orf72的工程化核酸序列；(b)位於(a)與(c)之間的間隔子序列；(c)至少一種對患者中的內源性人類C9orf72具有特異性的miRNA序列，其位於(a)及(b)之序列的3’；其中(a)之工程化核酸序列缺乏至少一個miRNA之靶位點，從而防止miRNA靶向工程化人類C9orf72編碼序列；(c)可操作連接至(a)和(c)之調節序列。於某些具體實施例中，生產質體包含載體基因體，該載體基因體包含SEQ ID NO：1之核酸序列、或5’ ITR–SEQ ID NO：4之表現匣–3’ ITR。In certain embodiments, the production plasmid includes a vector genome for packaging in a capsid, which includes: (a) an engineered nucleic acid sequence encoding human C9orf72; (b) located in (a) and (c) the spacer sequence between; (c) at least one miRNA sequence specific for endogenous human C9orf72 in the patient, which is located 3' of the sequence of (a) and (b); wherein the engineering of (a) The nucleic acid sequence lacks at least one miRNA target site, thereby preventing the miRNA from targeting the engineered human C9orf72 coding sequence; and (c) is operably linked to the regulatory sequences of (a) and (c). In certain embodiments, the production plasmid comprises a vector genome comprising the nucleic acid sequence of SEQ ID NO: 1, or the 5' ITR - the expression cassette of SEQ ID NO: 4 - the 3' ITR.

生產及單離適合作為載體使用之AAV之方法為本技術領域已知。一般參見例如例如，Grieger & Samulski, 2005, “Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications,” Adv. Biochem. Engin/Biotechnol. 99：119-145；Buning et al., 2008, “Recent developments in adeno-associated virus vector technology,” J. Gene Med. 10:717-733；及下列引述的參考文獻，其每一者藉由引用將其完整併入。為了將轉基因包裝到病毒粒子(virion)中，ITR是唯一需要在與包含表現匣的核酸分子相同的構築體順式的AAV組分。cap及rep基因可反式提供。Methods for producing and isolating AAV suitable for use as vectors are known in the art. See generally, for example, Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin/Biotechnol. 99: 119-145; Buning et al., 2008, “Recent developments in adeno-associated virus vector technology,” J. Gene Med. 10:717-733; and the references cited below, each of which is incorporated by reference in its entirety. In order to package a transgene into a virion, the ITR is the only AAV component that needs to be in cis in the same construct as the nucleic acid molecule containing the expression cassette. The cap and rep genes can be provided in trans.

於一具體實施例中，本文所述之表現匣被工程化至遺傳元件(例如，穿梭質體(shuttle plasmid)))，該遺傳元件將攜帶在其上的免疫球蛋白構築體序列轉移至包裝宿主細胞中以生產病毒載體。於一具體實施例中，選擇的基因元件可藉由任何適合的方法而被遞送至AAV包裝細胞(AAV packaging cell)，包括轉染、電穿孔、微脂體遞送、膜融合技術、高速DNA被覆顆粒(high velocity DNA-coated pellet)、病毒感染及原生質體(protoplast)融合。亦可製作穩定的AAV包裝細胞。或者，表現匣可用於產生AAV以外的病毒載體，或者用於活體外抗體混合物之製造。用於製作此種構築體之方法為核酸操作領域中具有通常知識者所知悉，且包括基因工程、重組工程、及合成技術。參見例如，Molecular Cloning：A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012)。In one embodiment, expression cassettes described herein are engineered to a genetic element (e.g., a shuttle plasmid) that transfers the immunoglobulin construct sequence carried thereon to the packaging host cells to produce viral vectors. In a specific embodiment, the selected genetic elements can be delivered to AAV packaging cells by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion technology, and high-speed DNA coating. Particles (high velocity DNA-coated pellet), virus infection and protoplast (protoplast) fusion. Stable AAV packaging cells can also be produced. Alternatively, the expression cassette can be used to generate viral vectors other than AAV, or for the production of antibody cocktails in vitro. Methods for making such constructs are known to those of ordinary skill in the field of nucleic acid manipulation, and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

術語「AAV中間體」或「AAV載體中間體」係指缺少包裝在其中的所欲基因體序列的組裝的rAAV衣殼。此等亦被稱為「空的(empty)」衣殼。此種衣殼可不含有表現匣的可檢測的基因體序列，或者僅含有不足以達成基因產物表現的部分包裝的基因體序列。此等空的衣殼不會將感興趣的基因轉移至宿主細胞的功能。The term "AAV intermediate" or "AAV vector intermediate" refers to an assembled rAAV capsid lacking the desired genome sequence packaged therein. These are also called "empty" capsids. Such capsids may contain no detectable genome sequences for the expression cassette, or may contain only partially packaged genome sequences that are insufficient to achieve expression of the gene product. Such empty capsids have no function in transferring the gene of interest to the host cell.

本文所述重組腺相關病毒(AAV)可使用已知技術生產。參見例如，WO 2003/042397；WO 2005/033321、WO 2006/110689；US 7588772 B2。此種方法涉及培養宿主細胞，該宿主細胞含有編碼AAV衣殼蛋白質的核酸序列；功能性rep基因；如本文所述表現匣，兩側為AAV反向末端重複(ITR)及轉基因；及足夠的輔助功能，以允許將表現匣包裝至AAV衣殼蛋白質中。本文亦提供宿主細胞，其含有編碼AAV衣殼的核酸序列；功能性rep基因；如本文所述的載體基因體；及足夠的輔助功能，以允許將載體基因體包裝至AAV衣殼蛋白質中。於一具體實施例中，宿主細胞為HEK 293細胞。此等方法在WO2017160360 A2中有更詳細的描述，其藉由引用併入本文。生產衣殼之方法、其編碼序列、及生產rAAV病毒載體之方法已被描述。參見例如，Gao, et al, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003)及US 2013/0045186A1。Recombinant adeno-associated viruses (AAV) described herein can be produced using known techniques. See, for example, WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2. Such methods involve culturing a host cell containing a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; an expression cassette as described herein, flanked by an AAV inverted terminal repeat (ITR) and a transgene; and sufficient Helper function to allow packaging of the expression cassette into the AAV capsid protein. Also provided herein are host cells containing nucleic acid sequences encoding AAV capsids; a functional rep gene; a vector genome as described herein; and sufficient accessory functions to permit packaging of the vector genome into AAV capsid proteins. In a specific embodiment, the host cell is HEK 293 cells. Such methods are described in more detail in WO2017160360 A2, which is incorporated herein by reference. Methods for producing capsids, their coding sequences, and methods for producing rAAV viral vectors have been described. See, for example, Gao, et al, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2013/0045186A1.

於一具體實施例中，提供有用於生產重組AAV之生產細胞培養物。此類細胞培養物含有在宿主細胞中表現AAV衣殼蛋白質的核酸；適於包裝至AAV衣殼中的核酸分子，例如，含有AAV ITR及編碼基因產物的非AAV核酸序列的載體基因體，該非AAV核酸序列可操作地連接至指導產物在宿主細胞中表現的序列；以及足夠的AAV rep功能及腺病毒輔助者功能，以允許將核酸分子包裝至重組AAV衣殼中。於一具體實施例中，細胞培養物係由哺乳動物細胞(例如人類胚腎293細胞等)或昆蟲細胞(例如桿狀病毒(baculovirus))組成。In a specific embodiment, a producer cell culture for producing recombinant AAV is provided. Such cell cultures contain nucleic acids that express AAV capsid proteins in host cells; nucleic acid molecules suitable for packaging into AAV capsids, e.g., vector genomes containing AAV ITRs and non-AAV nucleic acid sequences encoding gene products that are not The AAV nucleic acid sequence is operably linked to sequences that direct expression of the product in the host cell; and sufficient AAV rep function and adenovirus helper function to allow packaging of the nucleic acid molecule into a recombinant AAV capsid. In a specific embodiment, the cell culture is composed of mammalian cells (such as human embryonic kidney 293 cells, etc.) or insect cells (such as baculovirus).

典型地，rep功能來自與提供位於載體基因體兩側的ITR之AAV相同的AAV來源。於本文之例中，選擇AAV2 ITR並使用AAV2 rep。可選擇地，可選擇其他rep序列或另一rep來源(及可選擇另一ITR來源)。例如，rep可為但未限於：AAV1 rep蛋白質、AAV2 rep蛋白質；或rep 78、rep 68、rep 52、rep 40、rep68/78及rep40/52；或其片段；或其他來源。可選擇地，rep及cap序列位於細胞培養物中的相同遺傳元件上。在rep序列及cap基因之間可能有一個間隔子。此等AAV或突變體AAV衣殼序列之任一者可於指導其在宿主細胞中表現的外源調節控制序列的控制下。Typically, the rep function is derived from the same AAV source that provides the ITRs flanking the vector genome. In this example, AAV2 ITR is selected and AAV2 rep is used. Alternatively, other rep sequences or another rep source may be selected (and another ITR source may be selected). For example, rep can be, but is not limited to: AAV1 rep protein, AAV2 rep protein; or rep 78, rep 68, rep 52, rep 40, rep68/78 and rep40/52; or fragments thereof; or other sources. Alternatively, the rep and cap sequences are located on the same genetic element in cell culture. There may be a spacer between the rep sequence and the cap gene. Any of these AAV or mutant AAV capsid sequences may be under the control of exogenous regulatory control sequences that direct its expression in the host cell.

於一具體實施例中，細胞在合適的細胞培養(例如，HEK 293)細胞中製造。本文所述的基因療法載體的製備方法包括本領域眾所周知的方法，諸如用於生產基因療法載體的質體DNA的生產、載體的生產、及載體的純化。於一些具體實施例中，基因療法載體為AAV載體，且所產生的質體為編碼AAV基因體及感興趣之基因的AAV順式質體、含有AAV rep及cap基因的AAV反式質體、及腺病毒輔助質體。載體產生製程可包括諸如細胞培養之起始、細胞繼代、細胞接種、以質體DNA轉染細胞、轉染後培養基交換為無血清培養基、及含有載體的細胞及培養基的收取之方法步驟。In a specific embodiment, cells are produced in suitable cell culture (eg, HEK 293) cells. Methods for preparing gene therapy vectors described herein include methods well known in the art, such as production of plasmid DNA for producing gene therapy vectors, production of vectors, and purification of vectors. In some specific embodiments, the gene therapy vector is an AAV vector, and the generated plasmids are AAV cis-plastids encoding AAV genomes and genes of interest, AAV trans-plastids containing AAV rep and cap genes, and adenoviral helper plasmids. The vector production process may include method steps such as initiation of cell culture, cell passage, cell seeding, transfection of cells with plastid DNA, post-transfection medium exchange for serum-free medium, and collection of vector-containing cells and culture medium.

於某些具體實施例中，rAAV.C9orf72.miR之製造過程涉及以質體DNA短暫轉染HEK293細胞。藉由在PALL iCELLis生物反應器中的PEI介導之三重轉染HEK293細胞而生產單批或多批次。依序藉由澄清、TFF、親和性層析及陰離子交換層析，在可能的一次性、封閉式生物處理系統中純化所收取的AAV材料。In certain embodiments, the manufacturing process of rAAV.C9orf72.miR involves transient transfection of HEK293 cells with plasmid DNA. Single or multiple batches are produced by PEI-mediated triple transfection of HEK293 cells in PALL iCELLis bioreactors. The collected AAV material is purified sequentially by clarification, TFF, affinity chromatography and anion exchange chromatography in a possible single-use, closed bioprocessing system.

所收取的含有載體的細胞及培養基在本文中稱為粗細胞收取物。於另一系統中，藉由以基於桿狀病毒的載體感染而將基因療法載體導入昆蟲細胞中。此等生產系統的綜述，一般參見例如，Zhang et al., 2009, “Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production,” Human Gene Therapy 20:922-929，其各自之內容藉由引用將其完整併入。製造及使用此等之方法及其他AAV生產系統亦描述於下列U.S.專利，其各自之內容藉由引用而將其完整併入：5,139,941；5,741,683；6,057,152；6,204,059；6,268,213；6,491,907；6,660,514；6,951,753；7,094,604；7,172,893；7,201,898；7,229,823；及7,439,065，其在此藉由引用而併入。亦參見US臨時專利申請案第63/371,597號，2022年8月16日申請，標題為「Scalable Methods for Producing rAAV with Packaged Vector Genomes」、及US臨時專利申請案第63/371,592號，2022年8月16日申請，標題為「Scalable Methods for Downstream Purification of Recombinant Adeno-associated Virus」，兩者藉由引用而完整併入本文。粗細胞收取物之後可經歷另外的方法步驟，諸如載體收取物的濃縮、載體收取物的透析過濾、載體收取物的微流體化、載體收取物的核酸酶消化、微流體化中間體的過濾、藉由層析法的粗純化、藉由超高速離心的粗純化、藉由切向流過濾的緩衝液交換、及/或調配及過濾以製備大量載體。The recovered cells and culture medium containing the vector are referred to herein as crude cell harvest. In another system, gene therapy vectors are introduced into insect cells by infection with baculovirus-based vectors. For a general review of such production systems, see, for example, Zhang et al., 2009, “Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production,” Human Gene Therapy 20:922-929, their respective The contents are incorporated by reference in their entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which are incorporated by reference in their entireties: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 604 ; 7,172,893; 7,201,898; 7,229,823; and 7,439,065, which are hereby incorporated by reference. See also US Provisional Patent Application No. 63/371,597, filed on August 16, 2022, entitled "Scalable Methods for Producing rAAV with Packaged Vector Genomes", and US Provisional Patent Application No. 63/371,592, filed on August 2022 The application was filed on November 16, titled "Scalable Methods for Downstream Purification of Recombinant Adeno-associated Virus", both of which are fully incorporated by reference. The crude cell harvest may then undergo additional method steps, such as concentration of the carrier harvest, diafiltration of the carrier harvest, microfluidization of the carrier harvest, nuclease digestion of the carrier harvest, filtration of microfluidization intermediates, Large quantities of vectors are prepared by crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and/or formulation and filtration.

使用高鹽濃度下的兩步驟親和性層析純化，接著使用陰離子交換樹脂層析，以純化載體藥物產物並移除空的衣殼。在2016年12月9日申請的國際專利申請案第PCT/US2016/065970號中更詳細地描述此等方法，其藉由引用併入本文。AAV8之純化方法，2016年12月9日申請的國際專利申請案第PCT/US2016/065976號；及rh10之純化方法，2016年12月9日申請的國際專利申請案第PCT/US16/66013號，標題為「Scalable Purification Method for AAVrh10」，亦於2015年12月11日申請；及AAV1之純化方法，2016年12月9日申請的國際專利申請案第PCT/US2016/065974號，標題為「Scalable Purification Method for AAV1」，2015年12月11日申請，此等所有皆藉由引用併入本文。Purification using two-step affinity chromatography at high salt concentrations followed by anion exchange resin chromatography was used to purify the carrier drug product and remove empty capsids. Such methods are described in more detail in International Patent Application No. PCT/US2016/065970, filed on December 9, 2016, which is incorporated herein by reference. AAV8 purification method, International Patent Application No. PCT/US2016/065976 filed on December 9, 2016; and rh10 purification method, International Patent Application No. PCT/US16/66013 filed on December 9, 2016 , titled "Scalable Purification Method for AAVrh10", also filed on December 11, 2015; and the purification method for AAV1, International Patent Application No. PCT/US2016/065974, filed on December 9, 2016, titled " Scalable Purification Method for AAV1", filed on December 11, 2015, all of which are incorporated herein by reference.

為了計算空的(empty)及完整的(full)顆粒含量，將所選擇的樣品(例如，在本文的實施例中之碘克沙醇(iodixanol)梯度純化的製劑，其中GC的#=顆粒的#)的VP3帶(band)體積對裝載的GC顆粒作圖。所生成的線性方程式(y=mx+c)係用於計算測試物品峰的帶體積中的顆粒數量。然後將每20µL裝載的顆粒數(pt)乘以50，得到顆粒(pt)/mL。Pt/mL除以GC/mL，得到顆粒對基因體拷貝的比例(pt/GC)。Pt/mL–GC/mL得到空的pt/mL。空的pt/mL除以pt/mL並×100，得到空的顆粒的百分比。To calculate empty and full particle content, selected samples (e.g., iodixanol gradient-purified formulations in the examples herein, where GC # = particle VP3 band volume of #) plotted against loaded GC particles. The resulting linear equation (y=mx+c) is used to calculate the number of particles in the band volume of the test article peak. Then multiply the number of particles loaded per 20µL (pt) by 50 to get particles (pt)/mL. Pt/mL was divided by GC/mL to obtain the ratio of particles to genome copies (pt/GC). Pt/mL–GC/mL gives empty pt/mL. Empty pt/mL divided by pt/mL and ×100 gives the percentage of empty particles.

一般而言，用於分析空衣殼及具有包裝的基因體之AAV載體顆粒的方法為技術領域中已知，參見例如，Grimm et al., Gene Therapy (1999) 6:1322-1330；Sommer et al., Molec. Ther. (2003) 7:122-128。為了測試變性的衣殼，該方法包括對處理過的AAV儲料進行SDS-聚丙烯醯胺凝膠電泳，該電泳由能夠分離三種衣殼蛋白質的任何凝膠所組成，例如，在緩衝液中含有3-8%的Tris-乙酸鹽的梯度凝膠，然後運行凝膠直到分離樣品材料，然後將凝膠印漬到尼龍或硝化纖維素膜上，較佳為尼龍。然後將抗AAV衣殼抗體使用作為結合至變性的衣殼蛋白質的一級抗體，較佳為抗AAV衣殼單株抗體，最佳為B1抗AAV-2單株抗體(Wobus et al., J. Virol. (2000) 74:9281-9293)。然後使用二級抗體，該二級抗體與一級抗體結合且含有用於檢測與一級抗體的結合的手段，更佳為含有與其共價鍵結的檢測分子的抗IgG抗體，最佳為與辣根過氧化酶(horseradish peroxidase)共價鍵結的綿羊抗小鼠IgG抗體。使用檢測結合的方法，以半定量地確定一級抗體與二級抗體之間的結合，較佳為能夠檢測放射性同位素發射、電磁輻射或比色變化的檢測方法，最佳為化學發光檢測套組。例如，對於SDS-PAGE，可從管柱流份中取樣品，並於含有還原劑(例如DTT)的SDS-PAGE裝載緩衝液(loading buffer)中加熱，將衣殼蛋白質於預鑄的梯度聚丙烯醯胺凝膠(例如Novex)進行解析。可根據製造商的說明使用SilverXpress (Invitrogen，CA)進行銀染色或其他適合的染色方法(即SYPRO ruby或考馬斯(coomassie)染色)。於一具體實施例中，可藉由定量即時PCR(Q-PCR)測量管柱流份中的AAV載體基因體(vg)的濃度。稀釋樣品並以DNase I(或其他合適的核酸酶)消化以移除外源的DNA。核酸酶失活後，使用引子及對引子之間的DNA序列具有特異性的TaqMan™螢光探針進行進一步稀釋及擴增樣品。在Applied Biosystems Prism 7700序列檢測系統上測量每個樣品達到定義的螢光水準所需的循環數(閾值循環，Ct)。使用含有與AAV載體中含的序列相同序列的質體DNA，以於Q-PCR反應中生成標準曲線。從樣品獲得的循環閾值(Ct)數值係用於藉由將其標準化為質體標準曲線的Ct值來確定載體基因體效價(titer)。亦可使用基於數位PCR的終點分析。In general, methods for the analysis of empty capsids and AAV vector particles with packaged genomes are known in the art, see, for example, Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al. al., Molec. Ther. (2003) 7:122-128. To test denatured capsids, the method involves subjecting treated AAV stocks to SDS-polyacrylamide gel electrophoresis consisting of any gel capable of separating the three capsid proteins, e.g., in buffer A gradient gel containing 3-8% Tris-acetate is then run through the gel until the sample material is separated, and then the gel is blotted onto a nylon or nitrocellulose membrane, preferably nylon. An anti-AAV capsid antibody is then used as a primary antibody that binds to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, and most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used that binds to the primary antibody and contains a means for detecting binding to the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bonded thereto, most preferably with horseradish Sheep anti-mouse IgG antibody covalently bound to horseradish peroxidase. Use a binding detection method to semi-quantitatively determine the binding between the primary antibody and the secondary antibody, preferably a detection method capable of detecting radioisotope emission, electromagnetic radiation or colorimetric changes, preferably a chemiluminescence detection kit. For example, for SDS-PAGE, a sample can be taken from the column fraction and heated in an SDS-PAGE loading buffer containing a reducing agent (such as DTT), and the capsid protein can be concentrated in a gradient polymerization process. Analyze on an acrylamide gel (e.g. Novex). Silver staining or other suitable staining methods (ie, SYPRO Ruby or Coomassie staining) can be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions. In one embodiment, the concentration of AAV vector genome (vg) in the column fraction can be measured by quantitative real-time PCR (Q-PCR). Samples are diluted and digested with DNase I (or other suitable nuclease) to remove foreign DNA. After the nuclease is inactivated, the sample is further diluted and amplified using primers and TaqMan™ fluorescent probes specific for the DNA sequence between the primers. The number of cycles required for each sample to reach a defined fluorescence level (threshold cycle, Ct) was measured on the Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample is used to determine the vector titer by normalizing it to the Ct value of the plastid standard curve. Digital PCR-based endpoint analysis can also be used.

於一態樣，使用最適化的q-PCR方法，其利用廣效絲胺酸蛋白酶，例如蛋白酶K(如可由Qiagen購得)。更具體而言，最適化的qPCR基因體效價分析與標準分析相似，除了於DNase I消化後，將樣品以蛋白酶K緩衝液稀釋並以蛋白酶K處理，然後進行熱失活之外。適合地，以與樣品量相等的量的蛋白酶K緩衝液稀釋樣品。蛋白酶K緩衝液可濃縮至2倍以上。通常，蛋白酶K處理為約0.2mg/mL，但可於0.1mg/mL至約1mg/mL之間變化。該處理步驟通常於約55℃下進行約15分鐘，但可於較低溫度(例如約37℃至約50℃)下進行較長時間(例如約20分鐘至約30分鐘)；或者於較高的溫度(例如，高至約60℃)下進行較短的時間(例如，約5至10分鐘)。相似地，熱失活通常係於約95℃下約15分鐘，但溫度可降低(例如約70至約90℃)且時間延長(例如約20分鐘至約30分鐘)。然後將樣品稀釋(例如1000倍)，並如標準分析中所述進行TaqMan分析。In one aspect, an optimized q-PCR method is used that utilizes a broad-spectrum serine protease, such as proteinase K (eg, commercially available from Qiagen). More specifically, the optimized qPCR genome titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted in Proteinase K buffer and treated with Proteinase K, followed by heat inactivation. Suitably, the sample is diluted with an amount of proteinase K buffer equal to the amount of sample. Proteinase K buffer can be concentrated to more than 2 times. Typically, proteinase K treatment is about 0.2 mg/mL, but can vary from 0.1 mg/mL to about 1 mg/mL. The treatment step is usually carried out at about 55°C for about 15 minutes, but can be carried out at a lower temperature (for example, about 37°C to about 50°C) for a longer time (for example, about 20 minutes to about 30 minutes); or at a higher temperature. temperature (eg, up to about 60°C) for a short time (eg, about 5 to 10 minutes). Similarly, thermal inactivation is typically at about 95°C for about 15 minutes, but the temperature can be lowered (eg, from about 70 to about 90°C) and the time extended (eg, from about 20 minutes to about 30 minutes). Samples are then diluted (e.g. 1000-fold) and TaqMan assays are performed as described in Standard Assays.

另外或替代地，可使用液滴式數位PCR (droplet digital PCR)(ddPCR)。例如，已描述一種藉由ddPCR確定單股及自互補的AAV載體基因體效價的方法。參見例如，M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr；25(2):115-25. doi：10.1089/hgtb.2013.131. Epub 2014 Feb 14。Additionally or alternatively, droplet digital PCR (ddPCR) can be used. For example, a method for determining the genome titer of single-stranded and self-complementary AAV vectors by ddPCR has been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi:10.1089/hgtb.2013.131. Epub 2014 Feb 14.

簡而言之，用於從基因體缺陷的AAV中間體中分離具有包裝的基因體序列的rAAV顆粒的方法，涉及對包含重組AAV病毒顆粒及AAV衣殼中間體的懸浮液進行快速高效液相層析，其中AAV病毒顆粒及AAV中間體結合至一種經平衡於高pH的強陰離子交換樹脂，並經過鹽梯度，同時以約260及約280的紫外線吸光度來監測洗提物。可取決於所選擇的AAV而調整pH值。參見例如，WO2017/160360 (AAV9)、WO2017/100704 (AAVrh10)、WO 2017/100676 (例如，AAV8)、及WO 2017/100674 (AAV1)，其在此藉由引用而併入。於此方法中，從A260/A280之比達到反曲點時洗提的流份中收集AAV完整的衣殼。於一例中，對於親和性層析步驟，可將經透析過濾的產物應用於有效捕捉AAV2血清型的Capture Select ^TMPoros- AAV2/9親和性樹脂(Life Technologies)。於此等離子條件下，顯著百分比之殘留的細胞DNA及蛋白質流過管柱，而AAV顆粒被有效捕獲。 Briefly, a method for isolating rAAV particles with packaged genome sequences from genome-deficient AAV intermediates involves rapid high-performance liquid phase analysis of a suspension containing recombinant AAV virions and AAV capsid intermediates. Chromatography in which AAV virions and AAV intermediates are bound to a strong anion exchange resin equilibrated to high pH and passed through a salt gradient while monitoring the eluate with UV absorbance of about 260 and about 280. The pH can be adjusted depending on the AAV selected. See, for example, WO2017/160360 (AAV9), WO2017/100704 (AAVrh10), WO 2017/100676 (eg, AAV8), and WO 2017/100674 (AAV1), which are incorporated herein by reference. In this method, AAV intact capsids are collected from fractions eluted when the A260/A280 ratio reaches the inflection point. In one example, for the affinity chromatography step, the diafiltered product can be applied to Capture Select ^™ Poros-AAV2/9 affinity resin (Life Technologies) that effectively captures AAV2 serotypes. Under these plasma conditions, a significant percentage of residual cellular DNA and proteins flow through the column, and AAV particles are effectively captured.

非AAV及非病毒載體如本文使用的「載體」為一包含核酸序列之生物學或化學部分，其可被導入適當之複製或表現該核酸序列用的標靶細胞中。載體之例包括但未限於重組病毒、質體、微脂體複合物(Lipoplexes)、聚合物囊泡(Polymersome)、聚合複合物(Polyplexes)、樹枝狀聚合物(dendrimer)、細胞穿透肽(cell penetrating peptide) (CPP)結合物、磁性顆粒、或奈米顆粒。於一具體實施例中，載體為一種核酸分子，其中可***外源的或異源的或經工程化的hC9orf72編碼序列(及/或至少一miRNA)，然後可將其導入適當的標靶細胞。此種載體較佳具有一或多個複製起點、及可被***重組DNA的一或多個部位。載體通常具有可自未帶有載體的細胞中選擇帶有載體的細胞的手段，例如，彼等編碼抗藥性基因。通常的載體包括質體、病毒基因體、及「人工染色體」。本領域中具有通常知識者可使用載體之生產、製造、特性分析或定量之習用方法。 Non-AAV and non-viral vectors As used herein, a "vector" is a biological or chemical moiety containing a nucleic acid sequence that can be introduced into an appropriate target cell for the replication or expression of the nucleic acid sequence. Examples of vectors include, but are not limited to, recombinant viruses, plastids, Lipoplexes, Polymersomes, Polyplexes, dendrimers, cell-penetrating peptides ( cell penetrating peptide) (CPP) conjugates, magnetic particles, or nanoparticles. In a specific embodiment, the vector is a nucleic acid molecule into which an exogenous, heterologous or engineered hC9orf72 coding sequence (and/or at least one miRNA) can be inserted, and then introduced into an appropriate target cell. . Such vectors preferably have one or more origins of replication and one or more sites into which recombinant DNA can be inserted. Vectors often have a means to select vector-bearing cells from cells that do not bear the vector, for example, those encoding drug resistance genes. Common vectors include plasmids, viral genomes, and "artificial chromosomes." Conventional methods for production, manufacture, characterization or quantification of vectors can be used by those of ordinary skill in the art.

於一具體實施例中，載體為非病毒質體，其包含其所描述的表現匣，例如，「裸露DNA」、「裸露質體DNA」、RNA、mRNA、shRNA、RNAi等。可選擇地，經由適當裝置遞送質體或其他核酸序列，例如，經由電噴灑、電穿孔。於其他具體實施例中，核酸分子與各種組成物及奈米顆粒結合，包括例如，微胞(micelle)、微脂體(liposome)、陽離子性脂質-核酸組成物、多聚醣(poly-glycan)組成物、及其他聚合物、脂質及/或膽固醇系-核酸結合物、及其他構築體，如本文所述。參見例如，WO2014/089486、US 2018/0353616A1、US2013/0037977A1、WO2015/074085A1、US9670152B2、及US 8,853,377B2、X. Su et al, Mol. Pharmaceutics, 2011, 8 (3), pp 774–787；網路公開：March 21, 2011；WO2013/182683、WO 2010/053572及WO 2012/170930，此等全部藉由引用併入本文。In a specific embodiment, the vector is a non-viral plasmid, which contains the expression cassette described therein, for example, "naked DNA", "naked plastid DNA", RNA, mRNA, shRNA, RNAi, etc. Alternatively, plasmids or other nucleic acid sequences are delivered via appropriate means, eg, via electrospray, electroporation. In other embodiments, nucleic acid molecules are combined with various compositions and nanoparticles, including, for example, micelle, liposome, cationic lipid-nucleic acid composition, poly-glycan ) compositions, and other polymers, lipid and/or cholesterol-based-nucleic acid conjugates, and other constructs, as described herein. See, for example, WO2014/089486, US 2018/0353616A1, US2013/0037977A1, WO2015/074085A1, US9670152B2, and US 8,853,377B2, X. Su et al, Mol. Pharmaceutics, 2011, 8 (3), pp 774–78 7; net Published: March 21, 2011; WO2013/182683, WO 2010/053572 and WO 2012/170930, all of which are incorporated herein by reference.

於某個具體實施例中，非病毒載體用於遞送miRNA轉錄本，該miRNA轉錄本靶向內源性hC9orf72，在不存在於共同投予的經工程化的hC9orf72序列之位點。於一些具體實施例中，miRNA以每劑量大於約0.5 mg/kg體重的miRNA的量遞送(例如，大於約1.0 mg/kg、1.5 mg/kg、2.0 mg/kg、2.5 mg/kg、3.0 mg/kg、4.0 mg/kg、5.0 mg/kg、6.0 mg/kg、7.0 mg/kg、8.0 mg/kg、9.0 mg/kg、或10.0 mg/kg)。於一些具體實施例中，miRNA以每劑量約0.1-100 mg/kg體重之範圍的miRNA的量遞送(例如，約0.1-90 mg/kg、0.1-80 mg/kg、0.1-70 mg/kg、0.1-60 mg/kg、0.1-50 mg/kg、0.1-40 mg/kg、0.1-30 mg/kg、0.1-20 mg/kg、0.1-10 mg/kg)。於一些具體實施例中，miRNA以每劑量為下列的量或大於下列的量遞送：1 mg、5 mg、10 mg、15 mg、20 mg、25 mg、30 mg、35 mg、40 mg、45 mg、50 mg、55 mg、60 mg、65 mg、70 mg、75 mg、80 mg、85 mg、90 mg、95 mg、100 mg、150 mg、200 mg、250 mg、300 mg、350 mg、400 mg、450 mg、或500 mg。In a specific embodiment, a non-viral vector is used to deliver a miRNA transcript that targets endogenous hC9orf72 at a site that is not present in the co-administered engineered hC9orf72 sequence. In some embodiments, the miRNA is delivered in an amount of greater than about 0.5 mg/kg body weight of miRNA per dose (e.g., greater than about 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg /kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 mg/kg, or 10.0 mg/kg). In some embodiments, the miRNA is delivered in an amount of miRNA in the range of about 0.1-100 mg/kg body weight per dose (e.g., about 0.1-90 mg/kg, 0.1-80 mg/kg, 0.1-70 mg/kg , 0.1-60 mg/kg, 0.1-50 mg/kg, 0.1-40 mg/kg, 0.1-30 mg/kg, 0.1-20 mg/kg, 0.1-10 mg/kg). In some embodiments, the miRNA is delivered in an amount of or greater than the following per dose: 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg.

於某些具體實施例中，miRNA轉錄本被包封於脂質奈米顆粒(LNP)中。如本文所使用，短語「脂質奈米顆粒」係指包含一種或多種脂質(例如，陽離子性脂質、非陽離子性脂質及PEG修飾的脂質)的轉移媒劑。較佳地，脂質奈米顆粒被調配以將一種或多種miRNA遞送至一種或多種標靶細胞(例如，背根神經節、下運動神經元及/或上運動神經元、或於上述CNS所述的細胞類型)。適合的脂質之例包括例如磷脂醯基(phosphatidyl)化合物(例如，磷脂醯甘油、磷脂醯膽鹼、磷脂醯絲胺酸、磷脂醯乙醇胺、神經鞘脂、腦苷脂、及神經節苷脂)。亦考慮到聚合物作為轉移媒劑之使用，無論是單獨使用或是與其他轉移媒劑組合使用。適合的聚合物可包括例如聚丙烯酸酯、聚氰基丙烯酸烷酯、聚乳酸、聚乳酸-聚甘胺酸交酯共聚物、聚己內酯、葡聚醣、白蛋白、明膠、藻酸鹽、膠原蛋白、幾丁聚醣、環糊精、樹枝狀聚合物及聚乙亞胺。於一具體實施例中，轉移媒劑之選擇係基於其促進miRNA轉染至標靶細胞的能力。用於miRNA的有用脂質奈米顆粒包含陽離子性脂質，以包封及/或增強miRNA遞送至標靶細胞中，該標靶細胞將作為蛋白質生產的貯庫。如本文所使用，短語「陽離子性脂質」係指在選定的pH、如生理pH下，帶有淨正電荷的多種脂質種類中的任一種。考慮的脂質奈米顆粒可藉由包括採用一種或多種陽離子性脂質、非陽離子性脂質及PEG修飾的脂質的不同比例的多成分脂質混合物而製備。文獻中已描述數種陽離子性脂質，其許多為市售的。參見例如，WO2014/089486、US 2018/0353616A1、及US 8,853,377B2，其藉由引用而併入。於某些具體實施例中，LNP調配係使用常規程序進行，包含膽固醇、可離子化脂質、輔助脂質、PEG-脂質及聚合物，在被包封的mRNA周圍形成脂質雙層(Kowalski et al., 2019, Mol. Ther. 27(4):710-728)。於一些具體實施例中，LNP包含陽離子性脂質(即，N-[1-(2,3-二油醯氧基)丙基]-N,N,N-三甲基氯化銨(DOTMA)、或1,2-二油醯基-3-三甲基銨-丙烷(DOTAP))與輔助脂質DOPE。於一些具體實施例中，LNP包含可離子化脂質Dlin-MC3-DMA可離子化脂質、或哌二酮(diketopiperazine)系可離子化脂質(cKK-E12)。於一些具體實施例中，聚合物包含聚乙亞胺(PEI)、或聚(β-胺基)酯(PBAEs)。參見例如，WO2014/089486、US 2018/0353616A1、US2013/0037977A1、WO2015/074085A1、US9670152B2、及US 8,853,377B2，其藉由引用而併入。In certain embodiments, miRNA transcripts are encapsulated in lipid nanoparticles (LNPs). As used herein, the phrase "lipid nanoparticle" refers to a transfer vehicle that includes one or more lipids (eg, cationic lipids, noncationic lipids, and PEG-modified lipids). Preferably, the lipid nanoparticles are formulated to deliver one or more miRNAs to one or more target cells (e.g., dorsal root ganglia, lower motor neurons and/or upper motor neurons, or as described above in the CNS cell type). Examples of suitable lipids include, for example, phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides) . The use of polymers as transfer vehicles, either alone or in combination with other transfer vehicles, is also contemplated. Suitable polymers may include, for example, polyacrylates, polyalkyl cyanoacrylates, polylactic acid, polylactic acid-polyglycinate copolymer, polycaprolactone, dextran, albumin, gelatin, alginate , collagen, chitosan, cyclodextrin, dendrimers and polyethylenimine. In a specific embodiment, the transfer vehicle is selected based on its ability to promote the transfection of the miRNA into the target cells. Useful lipid nanoparticles for miRNA include cationic lipids to encapsulate and/or enhance delivery of the miRNA into target cells that will serve as reservoirs for protein production. As used herein, the phrase "cationic lipid" refers to any of a variety of lipid species that carry a net positive charge at a selected pH, such as physiological pH. Considered lipid nanoparticles can be prepared by including multi-component lipid mixtures in varying proportions with one or more cationic lipids, non-cationic lipids and PEG-modified lipids. Several cationic lipids have been described in the literature, many of which are commercially available. See, for example, WO2014/089486, US 2018/0353616A1, and US 8,853,377B2, which are incorporated by reference. In certain embodiments, LNP formulation is performed using conventional procedures, including cholesterol, ionizable lipids, helper lipids, PEG-lipids, and polymers to form a lipid bilayer around the encapsulated mRNA (Kowalski et al. , 2019, Mol. Ther. 27(4):710-728). In some embodiments, the LNP includes a cationic lipid (i.e., N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) , or 1,2-dioleyl-3-trimethylammonium-propane (DOTAP)) and the auxiliary lipid DOPE. In some embodiments, the LNP includes the ionizable lipid Dlin-MC3-DMA ionizable lipid, or piperine. Diketopiperazine is an ionizable lipid (cKK-E12). In some embodiments, the polymer includes polyethyleneimine (PEI) or poly(β-amino)esters (PBAEs). See, for example, WO2014/089486, US 2018/0353616A1, US2013/0037977A1, WO2015/074085A1, US9670152B2, and US 8,853,377B2, which are incorporated by reference.

於某些具體實施例中，本文所述載體為「複製缺陷型病毒」或「病毒載體」，係指合成或人工病毒顆粒，其中表現匣含有編碼經工程化的C9orf72及/或至少一個miRNA的核酸序列，該miRNA靶向在經工程化的C9orf72之序列上不存在的位點之內源性C9orf72。複製缺陷型病毒無法產生子代病毒粒子，但保留感染標靶細胞的能力。於一具體實施例中，病毒載體之基因體不包括編碼複製所需酵素的基因(該基因體可被工程化為「無膽的(gutless)」-僅含有編碼E2的核酸序列，兩側為增幅及包裝人工基因體所需的訊息)，但可能在生產過程中提供此等基因。因此，由於除非存在複製所需的病毒酵素，否則子代病毒粒子的複製及感染不會發生，而被認為可安全地用於基因療法。In certain embodiments, the vectors described herein are "replication-deficient viruses" or "viral vectors," which refer to synthetic or artificial viral particles in which the expression cassette contains a gene encoding engineered C9orf72 and/or at least one miRNA. Nucleic acid sequence that targets endogenous C9orf72 at a site that is not present in the sequence of engineered C9orf72. Replication-deficient viruses are unable to produce progeny virions but retain the ability to infect target cells. In a specific embodiment, the genome of the viral vector does not include genes encoding enzymes required for replication (the genome can be engineered to be "gutless" - containing only the nucleic acid sequence encoding E2, flanked by information required to amplify and package artificial genomes), but these genes may be provided during the production process. Therefore, it is considered safe for use in gene therapy because replication and infection of progeny virions cannot occur unless the viral enzymes required for replication are present.

如本文所使用，重組病毒載體可為任何適合的複製缺陷型病毒載體，包括例如重組腺相關病毒(AAV)、腺病毒、波卡病毒、雜合AAV/波卡病毒、單純疱疹病毒或慢病毒。As used herein, a recombinant viral vector may be any suitable replication-deficient viral vector, including, for example, recombinant adeno-associated virus (AAV), adenovirus, pocavirus, hybrid AAV/pocavirus, herpes simplex virus, or lentivirus .

如本文所使用，術語「宿主細胞」可指其中產生載體(例如，重組AAV)的包裝細胞株。宿主細胞可為原核或真核細胞(例如，人類、昆蟲、或酵母菌)，其含有藉由任一手段而被導入細胞中的外源的或異源的DNA，該手段例如為電穿孔、磷酸鈣沉澱、微注射、轉形、病毒感染、轉染、微脂體遞送、膜融合技術、高速DNA被覆顆粒、病毒感染及原生質體融合。宿主細胞之例可包括但未限於經單離的細胞、細胞培養物、大腸桿菌細胞、酵母菌細胞、人類細胞、非人類細胞、哺乳動物細胞、非哺乳動物細胞、昆蟲細胞、HEK-293細胞、肝臟細胞、腎臟細胞、中樞神經系統細胞、神經元、神經膠細胞、或幹細胞。As used herein, the term "host cell" may refer to a packaging cell strain in which a vector (eg, recombinant AAV) is produced. The host cell can be a prokaryotic or eukaryotic cell (eg, human, insect, or yeast) that contains foreign or heterologous DNA introduced into the cell by any means, such as electroporation, Calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion technology, high-speed DNA-coated particles, viral infection and protoplast fusion. Examples of host cells may include, but are not limited to, isolated cells, cell cultures, E. coli cells, yeast cells, human cells, non-human cells, mammalian cells, non-mammalian cells, insect cells, HEK-293 cells , liver cells, kidney cells, central nervous system cells, neurons, glial cells, or stem cells.

如本文所使用，術語「標靶細胞」係指欲在其中表現hC9orf72及/或miRNA之任何標靶細胞。於某些具體實施例，術語「標靶細胞」意圖指待治療C9orf72相關病症諸如ALS的受試者之細胞。標靶細胞之例包括但不限於中樞神經系統中的細胞。As used herein, the term "target cell" refers to any target cell in which hC9orf72 and/or miRNA is intended to be expressed. In certain embodiments, the term "target cells" is intended to refer to cells of a subject to be treated for a C9orf72-related disorder, such as ALS. Examples of target cells include, but are not limited to, cells in the central nervous system.

組成物本文提供一組成物，其含有至少一包含C9orf72.miR的載體(例如，rAAV.C9orf72.miR儲料)及/或至少一包含miR的載體及/或至少一包含儲料的載體，且含有可選擇的載劑、賦形劑及/或防腐劑。 Composition Provided herein is a composition comprising at least one C9orf72.miR-containing vector (eg, rAAV.C9orf72.miR reservoir) and/or at least one miR-containing vector and/or at least one reservoir-containing vector, and containing Selected carriers, excipients and/or preservatives.

如本文所使用，如本文所使用，rAAV之「儲料(stock)」係指一群rAAV。儘管由於脫醯胺作用而彼等之衣殼蛋白質具有異質性，但是儲料中的rAAV被預期有5個共有相同的載體基因體。儲料可包括具有衣殼之rAAV，該衣殼具有例如所選擇的AAV衣殼蛋白質及所選擇的生產系統的異質性脫醯胺樣式特徵。該儲料可從單一生產系統生產，或從生產系統的多次運行中儲集。可選擇各種生產系統，包括但不限於本文所述者。As used herein, the "stock" of rAAV, as used herein, refers to a population of rAAV. Five rAAVs in the stock are expected to share the same vector genome, despite heterogeneity in their capsid proteins due to deamidation. Stocks may include rAAV having capsids with heterogeneous deamidation patterns characteristic of, for example, selected AAV capsid proteins and selected production systems. The stock can be produced from a single production system or accumulated from multiple runs of a production system. A variety of production systems are available, including but not limited to those described herein.

於某些具體實施例中，組成物至少包含病毒儲料，其為重組AAV (rAAV)，適合用於單獨或與其他載體儲料或組成物組合以治療C9orf72介導的ALS或FTD。於某些具體實施例中，組成物適合用於製備治療患者的藥物。於某些具體實施例中，組成物包含病毒儲料，其為適合用於治療患者的重組AAV (rAAV)，該rAAV包含：(a)腺相關病毒衣殼、及(b)包裝於AAV衣殼中的載體基因體，該載體基因體包含AAV反向末端重複、工程化C9orf72的編碼序列、間隔子序列、至少一種miRNA的編碼序列(該miRNA特異性地靶向在不存在於工程化的人類C9orf72編碼序列之位點上的內源性人類C9orf72)、及指導編碼的基因產物表現的調節序列。於某些具體實施例中，組成物包含：獨立的載體儲料，其包含rAAV，該rAAV包含(a)腺相關病毒衣殼、及(b)包裝於AAV衣殼中的載體基因體，該載體基因體包含AAV反向末端重複、工程化人類C9orf72的編碼序列、及指導編碼的基因產物表現的調節序列；及/或獨立的載體儲料，其包含(a)腺相關病毒衣殼、及(b)包裝於AAV衣殼中的載體基因體，該載體基因體包含AAV反向末端重複、至少一個miRNA的編碼序列(該miRNA特異性地靶向在不存在於工程化C9orf72編碼序列的位點之內源性人類C9orf72)、及指導編碼的基因產物表現的調節序列。於某些具體實施例中，載體基因體包含啟動子、強化子、內含子、人類C9orf72編碼序列、及多腺苷酸化訊息。於某些具體實施例中，內含子由雞β肌動蛋白剪接供體及兔β剪接受體元件組成。於某些具體實施例中，載體基因體進一步包含位於載體基因體所有元件兩側的AAV2 5’ ITR及AAV2 3’ ITR。In certain embodiments, the composition at least includes a viral reservoir that is a recombinant AAV (rAAV) suitable for use alone or in combination with other vector stocks or compositions to treat C9orf72-mediated ALS or FTD. In certain embodiments, the compositions are suitable for use in the preparation of medicaments for treating patients. In certain embodiments, the composition includes a viral stock that is a recombinant AAV (rAAV) suitable for use in treating a patient, the rAAV comprising: (a) an adeno-associated virus capsid, and (b) packaged in an AAV capsid. A vector genome in a shell that contains AAV inverted terminal repeats, the coding sequence of engineered C9orf72, a spacer sequence, and the coding sequence of at least one miRNA that specifically targets genes not present in the engineered Endogenous human C9orf72 at the site of the human C9orf72 coding sequence, and regulatory sequences that direct expression of the encoded gene product. In certain embodiments, the composition includes: a separate vector stock comprising rAAV, the rAAV comprising (a) an adeno-associated virus capsid, and (b) a vector genome packaged in the AAV capsid, the A vector genome comprising an AAV inverted terminal repeat, the coding sequence for engineered human C9orf72, and regulatory sequences that direct expression of the encoded gene product; and/or a separate vector reservoir comprising (a) an adeno-associated virus capsid, and (b) A vector genome packaged in an AAV capsid, the vector genome comprising an AAV inverted terminal repeat, the coding sequence of at least one miRNA specifically targeting a site not present in the engineered C9orf72 coding sequence; endogenous human C9orf72), and the regulatory sequences that direct the expression of the encoded gene product. In certain embodiments, the vector genome includes a promoter, enhancer, introns, human C9orf72 coding sequence, and polyadenylation message. In certain embodiments, the intron consists of chicken β-actin splice donor and rabbit β-splice acceptor elements. In certain embodiments, the vector genome further includes an AAV2 5' ITR and an AAV2 3' ITR flanking all elements of the vector genome.

rAAV.C9orf72.miR (rAAV.hC9orf72或另一載體)可被懸浮於生理學上相容的載劑以投予至人類患者。於某些具體實施例中，為了投予至人類患者，適合地將載體懸浮於水性溶液，該水性溶液含有鹽水、界面活性劑、及生理上可相容的鹽或鹽之混合物。適合地，調整此調配物至生理上可接受的pH，例如，範圍為pH 6至9、或pH 6.5至7.5、pH 7.0至7.7、或pH 7.2至7.8。由於腦脊髓液的pH為約7.28至約7.32、或pH為7.2至7.4，對於鞘內遞送，可能期望為在此範圍內的pH；而對於靜脈內遞送，可能期望為約pH 6.8至約7.2。然而，可選擇最廣範圍及此等子範圍內的其他pH用於其他遞送途徑。rAAV.C9orf72.miR (rAAV.hC9orf72 or another vector) can be suspended in a physiologically compatible carrier for administration to human patients. In certain embodiments, for administration to a human patient, the carrier is suitably suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example in the range of pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. Since the pH of cerebrospinal fluid is about 7.28 to about 7.32, or a pH of 7.2 to 7.4, for intrathecal delivery, a pH in this range may be desired; and for intravenous delivery, a pH of about pH 6.8 to about 7.2 may be desired. . However, other pHs within the broadest range and within these subranges may be selected for other delivery routes.

於某些具體實施例中，調配物可含有不包含碳酸氫鈉的緩衝食鹽水溶液。此種調配物可含有於水中包含磷酸鈉、氯化鈉、氯化鉀、氯化鈣、氯化鎂及其混合物之一或多者的緩衝食鹽水溶液，諸如Harvard緩衝液。水溶液可進一步含有Kolliphor® P188，一種由BASF販售的泊洛沙姆(poloxamer)，其之前以商標名Lutrol® F68販賣。水溶液可具有7.2之pH或7.4之pH。In certain embodiments, the formulation may contain a buffered saline solution that does not include sodium bicarbonate. Such formulations may contain a buffered saline solution, such as Harvard buffer, containing one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and mixtures thereof in water. The aqueous solution may further contain Kolliphor® P188, a poloxamer sold by BASF and formerly sold under the trade name Lutrol® F68. The aqueous solution may have a pH of 7.2 or a pH of 7.4.

於另一具體實施例中，調配物可含有緩衝食鹽水溶液，該緩衝食鹽水溶液包含1mM磷酸鈉(Na ₃PO ₄)、150 mM氯化鈉(NaCl)、3mM氯化鉀(KCl)、1.4 mM氯化鈣(CaCl ₂)、0.8 mM氯化鎂(MgCl ₂)、及0.001% Kolliphor® 188。參見例如，harvardapparatus. com/harvard-apparatus-perfusion-fluid.html。於某些具體實施例中，較佳為Harvard緩衝液。 In another embodiment, the formulation may contain a buffered saline solution comprising 1 mM sodium phosphate (Na ₃ PO ₄ ), 150 mM sodium chloride (NaCl), 3mM potassium chloride (KCl), 1.4 mM Calcium chloride (CaCl ₂ ), 0.8 mM magnesium chloride (MgCl ₂ ), and 0.001% Kolliphor® 188. See, e.g., harvardapparatus.com/harvard-apparatus-perfusion-fluid.html. In certain embodiments, Harvard buffer is preferred.

於其他具體實施例中，調配物可含有一或多個滲透增強劑。適合的滲透增強劑之例可包括例如甘露醇、甘膽酸鈉、牛磺膽酸鈉、去氧膽酸鈉、水楊酸鈉、辛酸鈉、癸酸鈉、月桂硫酸鈉、聚氧乙烯-9-月桂醚或EDTA。In other embodiments, the formulations may contain one or more penetration enhancers. Examples of suitable penetration enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene- 9-Laureth or EDTA.

於另一具體實施例中，組成物包括載劑、稀釋劑、賦形劑及/或佐劑。鑑於轉移病毒所針對的適應症，本技術領域中具有通常知識者可容易地選擇適合的載劑。例如，一適合的載劑包括鹽水，其能以許多種緩衝溶液來調配(例如，磷酸鹽緩衝食鹽水)。其他示例性載劑包括無菌的鹽水、乳糖、蔗糖、磷酸鈣、明膠、葡聚醣、瓊脂、果膠、花生油、芝麻油、及水。緩衝液/載劑應包括防止rAAV黏附到輸液管上但不干擾活體內rAAV結合活性的成分。In another embodiment, the composition includes a carrier, diluent, excipient and/or adjuvant. Given the indication for which the virus is to be transferred, one of ordinary skill in the art can readily select a suitable carrier. For example, a suitable carrier includes saline, which can be formulated in a variety of buffer solutions (eg, phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The buffer/carrier should include ingredients that prevent adhesion of rAAV to the infusion tube but do not interfere with rAAV binding activity in vivo.

可選擇地，除了載體(例如rAAV)及載劑之外，組成物可含有其他習用的醫藥成分，諸如防腐劑、或化學穩定劑。適合的示例性防腐劑包括氯丁醇、山梨酸鉀、山梨酸、二氧化硫、沒食子酸丙酯、對羥基苯甲酸酯類(parabens)、乙基香草醛、甘油、苯酚及對氯苯酚。適合的化學穩定劑包括明膠及白蛋白。Alternatively, in addition to the carrier (eg, rAAV) and carrier, the composition may contain other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

如本文所使用，「載劑」包括任何及所有的溶劑、分散介質、媒劑、塗劑、稀釋劑、抗細菌及抗真菌劑、等張及吸收延遲劑、緩衝液、載劑溶液、懸浮液、膠體等。此種用於醫藥活性物質的介質及藥劑的用途為本技術領域中所熟知。補充的活性成分亦可併入該組成物中。用語「醫藥上可接受」係指當投予至宿主時不會產生過敏或類似的不良反應的分子實體及組成物。遞送媒劑諸如微脂體、奈米膠囊、微粒、微球、脂質顆粒、囊泡等，可用於將本發明之組成物導入適當的宿主細胞中。特別是，可將遞送轉基因的rAAV載體包封於脂質顆粒、微脂體、囊泡、奈米球或奈米顆粒等之任一者中進行調配而用於遞送。As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coating agents, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, vehicle solutions, suspensions liquid, colloid, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., can be used to introduce the composition of the present invention into appropriate host cells. In particular, rAAV vectors that deliver transgenes can be encapsulated in any of lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc. and formulated for delivery.

於一具體實施例中，組成物包括適於遞送至受試者之最終調配物，例如，為緩衝至生理上可相容的pH及鹽濃度的水性液體懸浮液。可選擇地，調配物中存在一種或多種界面活性劑。於另一具體實施例中，可將組成物作為濃縮物運輸，其進行稀釋以投予至受試者。於其他具體實施例中，組成物可被冷凍乾燥並在投予時復原。In one embodiment, the composition includes a final formulation suitable for delivery to a subject, e.g., an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition can be transported as a concentrate, which is diluted for administration to a subject. In other embodiments, the composition can be freeze-dried and reconstituted upon administration.

適合的界面活性劑或界面活性劑的組合可選自無毒的非離子界面活性劑。於一具體實施例中，選擇末端為一級羥基的雙官能嵌段共聚物界面活性劑，例如，諸如Pluronic® F68 [BASF]，亦稱為泊洛沙姆188 (Poloxamer 188)，其具有中性pH，其平均分子量為8400。可選擇其他界面活性劑及其他泊洛沙姆，即由一個聚氧伸丙基(聚(環氧丙烷))之中央疏水鏈及兩側的兩個聚氧伸乙基(聚(環氧乙烷))之親水鏈所構成的非離子三嵌段共聚物、SOLUTOL HS 15(聚乙二醇-15 羥基硬脂酸酯)、LABRASOL(聚乙二醇辛酸甘油酯(Polyoxy capryllic glyceride))、聚乙二醇10油基醚(polyoxy 10 oleyl ether)、TWEEN(聚氧乙烯山梨糖醇酐脂肪酸酯)、乙醇及聚乙二醇。於一具體實施例中，調配物含有泊洛沙姆。此等共聚物通常以字母「P」(用於泊洛沙姆)跟三個數字命名：前兩個數字×100給出聚氧伸丙基核心的近似分子量，最後一個數字×10給出聚氧伸乙基含量百分比。於一具體實施例，選擇泊洛沙姆188。界面活性劑能以高至懸浮液的約0.0005%至約0.001%之量存在。A suitable surfactant or combination of surfactants may be selected from non-toxic, non-ionic surfactants. In a specific embodiment, a difunctional block copolymer surfactant with a primary hydroxyl group at the end is selected, for example, such as Pluronic® F68 [BASF], also known as Poloxamer 188 (Poloxamer 188), which has neutral pH, its average molecular weight is 8400. You can choose other surfactants and other poloxamer, which consists of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) and two polyoxyethylene (poly(ethylene oxide)) on both sides. Nonionic triblock copolymer composed of hydrophilic chains of alkane)), SOLUTOL HS 15 (Polyethylene glycol-15 hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), Polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol and polyethylene glycol. In a specific embodiment, the formulation contains poloxamer. These copolymers are usually named with the letter "P" (for poloxamer) and three numbers: the first two numbers × 100 give the approximate molecular weight of the polyoxypropylene core, and the last number × 10 gives the polyoxypropylene core. Ethyloxyethylene content percentage. In one embodiment, poloxamer 188 is selected. The surfactant can be present in an amount up to about 0.0005% to about 0.001% of the suspension.

以足夠的量投予載體以轉染細胞並提供基因轉移及表現之足夠的水準，以提供治療益處而沒有不適當的副作用，或具有醫學上可接受的生理作用，此可由醫學領域中具有通常知識者確定。可選擇地，可使用鞘內投予以外的途徑，諸如，例如直接遞送至所欲器官(例如，肝臟(可選擇地經由肝動脈)、肺臟、心臟、眼、腎臟)、口服、吸入、鼻內、氣管內、動脈內、眼內、靜脈內、肌內、皮下、皮內、及其他腸胃外投予途徑。若需要，可合併投予途徑。Administering a vector in a sufficient amount to transfect cells and provide a sufficient level of gene transfer and expression to provide a therapeutic benefit without undue side effects, or to have a medically acceptable physiological effect, can be determined by those generally skilled in the medical field. Those who know are certain. Alternatively, routes other than intrathecal administration may be used, such as, for example, direct delivery to the desired organ (eg, liver (optionally via hepatic artery), lungs, heart, eyes, kidneys), oral, inhaled, nasal Intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. If necessary, administration routes can be combined.

載體的劑量將主要取決於諸如待治療的病況、患者的年齡、體重、及健康狀況之因子，因此於患者間可能會變化。例如，病毒載體之治療上有效的人類劑量，一般為範圍約25至約1000微升至約100 mL之溶液含有濃度約1×10 ⁹至1×10 ¹⁶的基因體病毒載體(以治療平均體重70 kg的受試者)，包括該範圍內的所有整數或分數量，且對於人類患者較佳為1.0×10 ¹²GC至1.0×10 ¹⁴GC。於一具體實施例中，調配組成物以使每劑含有至少1x10 ⁹、2x10 ⁹、3x10 ⁹、4x10 ⁹、5x10 ⁹、6x10 ⁹、7x10 ⁹、8x10 ⁹、或9x10 ⁹GC，包括該範圍內的所有整數或分數量。於另一具體實施例中，調配組成物以使每劑含有至少1x10 ¹⁰、2x10 ¹⁰、3x10 ¹⁰、4x10 ¹⁰、5x10 ¹⁰、6x10 ¹⁰、7x10 ¹⁰、8x10 ¹⁰、或9x10 ¹⁰GC，包括該範圍內的所有整數或分數量。於另一具體實施例中，調配組成物以使每劑含有至少1x10 ¹¹、2x10 ¹¹、3x10 ¹¹、4x10 ¹¹、5x10 ¹¹、6x10 ¹¹、7x10 ¹¹、8x10 ¹¹、或9x10 ¹¹GC，包括該範圍內的所有整數或分數量。於另一具體實施例中，調配組成物以使每劑含有至少1x10 ¹²、2x10 ¹²、3x10 ¹²、4x10 ¹²、5x10 ¹²、6x10 ¹²、7x10 ¹²、8x10 ¹²、或9x10 ¹²GC，包括該範圍內的所有整數或分數量。於另一具體實施例中，調配組成物以使每劑含有至少1x10 ¹³、2x10 ¹³、3x10 ¹³、4x10 ¹³、5x10 ¹³、6x10 ¹³、7x10 ¹³、8x10 ¹³、或9x10 ¹³，包括該範圍內的所有整數或分數量。於另一具體實施例中，調配組成物以使每劑含有至少1x10 ¹⁴、2x10 ¹⁴、3x10 ¹⁴、4x10 ¹⁴、5x10 ¹⁴、6x10 ¹⁴、7x10 ¹⁴、8x10 ¹⁴、或9x10 ¹⁴GC，包括該範圍內的所有整數或分數量。於另一具體實施例中，調配組成物以使每劑含有至少1x10 ¹⁵、2x10 ¹⁵、3x10 ¹⁵、4x10 ¹⁵、5x10 ¹⁵、6x10 ¹⁵、7x10 ¹⁵、8x10 ¹⁵、或9x10 ¹⁵GC，包括該範圍內的所有整數或分數量。於一具體實施例中，對於人類應用，劑量範圍可為每劑1x10 ¹⁰至約1x10 ¹²GC，包括該範圍內的所有整數或分數量。 The dosage of the vehicle will depend primarily on factors such as the condition being treated, the age, weight, and health of the patient, and therefore may vary from patient to patient. For example, a therapeutically effective human dose of a viral vector will generally range from about 25 to about 1000 microliters to about 100 mL of a solution containing a genomic viral vector at a concentration of about 1×10 ⁹ to 1×10 ¹⁶ (to treat an average body weight 70 kg subject), including all integer or fractional quantities within this range, and preferably 1.0×10 ¹² GC to 1.0×10 ¹⁴ GC for human patients. In a specific embodiment, the composition is formulated such that each dose contains at least 1x10 ⁹ , 2x10 ⁹ , 3x10 ⁹ , 4x10 ⁹ , 5x10 ⁹ , 6x10 ⁹ , 7x10 ⁹ , 8x10 ⁹ , or 9x10 ⁹ GC, including within this range All whole or fractional quantities. In another embodiment, the composition is formulated such that each dose contains at least 1x10 ¹⁰ , 2x10 ¹⁰ , 3x10 ¹⁰ , 4x10 ¹⁰ , 5x10 ¹⁰ , 6x10 ¹⁰ , 7x10 ¹⁰ , 8x10 ¹⁰ , or 9x10 ¹⁰ GC, including within this range. All integers or fractions of . In another embodiment, the composition is formulated such that each dose contains at least 1x10 ¹¹ , 2x10 ¹¹ , 3x10 ¹¹ , 4x10 ¹¹ , 5x10 ¹¹ , 6x10 ¹¹ , 7x10 ¹¹ , 8x10 ¹¹ , or 9x10 ¹¹ GC, including within this range. All integers or fractions of . In another embodiment, the composition is formulated such that each dose contains at least 1x10 ¹² , 2x10 ¹² , 3x10 ¹² , 4x10 ¹² , 5x10 ¹² , 6x10 ¹² , 7x10 ¹² , 8x10 ¹² , or 9x10 ¹² GC, including within this range. All integers or fractions of . In another embodiment, the composition is formulated such that each dose contains at least 1x10 ¹³ , 2x10 ¹³ , 3x10 ¹³ , 4x10 ¹³ , 5x10 ¹³ , 6x10 ¹³ , 7x10 ¹³ , 8x10 ¹³ , or 9x10 ¹³ , including within this range. All whole or fractional quantities. In another embodiment, the composition is formulated such that each dose contains at least 1x10 ¹⁴ , 2x10 ¹⁴ , 3x10 ¹⁴ , 4x10 ¹⁴ , 5x10 ¹⁴ , 6x10 ¹⁴ , 7x10 ¹⁴ , 8x10 ¹⁴ , or 9x10 ¹⁴ GC, including within this range. All integers or fractions of . In another embodiment, the composition is formulated such that each dose contains at least 1x10 ¹⁵ , 2x10 ¹⁵ , 3x10 ¹⁵ , 4x10 ¹⁵ , 5x10 ¹⁵ , 6x10 ¹⁵ , 7x10 ¹⁵ , 8x10 ¹⁵ , or 9x10 ¹⁵ GC, including within this range. All integers or fractions of . In one embodiment, for human use, the dosage range may be from 1x10 ¹⁰ to about 1x10 ¹² GC per dose, including all integers or fractions within this range.

於某些具體實施例中，劑量為範圍約1 x 10 ⁹GC/g腦質量至約1 x 10 ¹²GC/g腦質量。於某些具體實施例中，劑量為範圍約1 x 10 ¹⁰GC/g腦質量至約3.33 x 10 ¹¹GC/g腦質量。於某些具體實施例中，劑量為範圍約3.33 x 10 ¹¹GC/g腦質量至約1.1 x 10 ¹²GC/g腦質量。於某些具體實施例中，劑量為範圍約1.1 x 10 ¹²GC/g腦質量至約3.33 x 10 ¹³GC/g腦質量。於某些具體實施例中，劑量為低於3.33 x 10 ¹¹GC/g腦質量。於某些具體實施例中，劑量為低於1.1 x 10 ¹²GC/g腦質量。於某些具體實施例中，劑量為低於3.33 x 10 ¹³GC/g腦質量。於某些具體實施例中，劑量為約1 x 10 ¹⁰GC/g腦質量。於某些具體實施例中，劑量為約2 x 10 ¹⁰GC/g腦質量。於某些具體實施例中，劑量為約2 x 10 ¹⁰GC/g腦質量。於某些具體實施例中，劑量為約3 x 10 ¹⁰GC/g腦質量。於某些具體實施例中，劑量為約4 x 10 ¹⁰GC/g腦質量。於某些具體實施例中，劑量為約5 x 10 ¹⁰GC/g腦質量。於某些具體實施例中，劑量為6 x 10 ¹⁰GC/g腦質量。於某些具體實施例中，劑量為約7 x 10 ¹⁰GC/g腦質量。於某些具體實施例中，劑量為約8 x 10 ¹⁰GC/g腦質量。於某些具體實施例中，劑量為約9 x 10 ¹⁰GC/g腦質量。於某些具體實施例中，劑量為約1 x 10 ¹¹GC/g腦質量。於某些具體實施例中，劑量為約2 x 10 ¹¹GC/g腦質量。於某些具體實施例中，劑量為約3 x 10 ¹¹GC/g腦質量。於某些具體實施例中，劑量為約4 x 10 ¹¹GC/g腦質量。於某些具體實施例中，劑量係以rAAV的約1.44×10 ¹³至4.33×10 ¹⁴GC範圍內的固定劑量(flat dose)投予至人類。於某些具體實施例中，劑量係以rAAV的約1.44×10 ¹³至2×10 ¹⁴GC範圍內的固定劑量投予至人類。於某些具體實施例中，劑量係以rAAV的約3×10 ¹³至1×10 ¹⁴GC範圍內的固定劑量投予至人類。於某些具體實施例中，劑量係以rAAV的約5×10 ¹³至1×10 ¹⁴GC範圍內的固定劑量投予至人類。於一些具體實施例中，組成物可調配成劑量單位，以含有AAV的約1×10 ¹³至8×10 ¹⁴GC範圍內之AAV的量。於一些具體實施例中，組成物可調配成劑量單位，以含有rAAV的約1.44×10 ¹³至4.33×10 ¹⁴GC範圍內之AAV的量。於一些具體實施例中，組成物可調配成劑量單位，以含有rAAV的約3×10 ¹³至1×10 ¹⁴GC範圍內之AAV的量。於一些具體實施例中，組成物可調配成劑量單位，以含有rAAV的約5×10 ¹³至1×10 ¹⁴GC範圍內之AAV的量。 In certain embodiments, the dosage ranges from about 1 x 10 ⁹ GC/g brain mass to about 1 x 10 ¹² GC/g brain mass. In certain embodiments, the dosage ranges from about 1 x 10 ¹⁰ GC/g brain mass to about 3.33 x 10 ¹¹ GC/g brain mass. In certain embodiments, the dosage ranges from about 3.33 x 10 ¹¹ GC/g brain mass to about 1.1 x 10 ¹² GC/g brain mass. In certain embodiments, the dosage ranges from about 1.1 x 10 ¹² GC/g brain mass to about 3.33 x 10 ¹³ GC/g brain mass. In certain embodiments, the dose is less than 3.33 x ¹⁰¹¹ GC/g brain mass. In certain embodiments, the dose is less than 1.1 x 10 ¹² GC/g brain mass. In certain embodiments, the dose is less than 3.33 x ¹⁰¹³ GC/g brain mass. In certain embodiments, the dose is about 1 x 10 ¹⁰ GC/g brain mass. In certain embodiments, the dose is about 2 x 10 ¹⁰ GC/g brain mass. In certain embodiments, the dose is about 2 x 10 ¹⁰ GC/g brain mass. In certain embodiments, the dose is about 3 x 10 ¹⁰ GC/g brain mass. In certain embodiments, the dose is about 4 x ¹⁰¹⁰ GC/g brain mass. In certain embodiments, the dose is about 5 x 10 ¹⁰ GC/g brain mass. In certain embodiments, the dose is 6 x 10 ¹⁰ GC/g brain mass. In certain embodiments, the dose is about 7 x 10 ¹⁰ GC/g brain mass. In certain embodiments, the dose is about 8 x 10 ¹⁰ GC/g brain mass. In certain embodiments, the dose is about 9 x 10 ¹⁰ GC/g brain mass. In certain embodiments, the dose is about 1 x ¹⁰¹¹ GC/g brain mass. In certain embodiments, the dose is about 2 x ¹⁰¹¹ GC/g brain mass. In certain embodiments, the dose is about 3 x ¹⁰¹¹ GC/g brain mass. In certain embodiments, the dose is about 4 x ¹⁰¹¹ GC/g brain mass. In certain embodiments, the dose is administered to humans as a flat dose ranging from about 1.44×10 ¹³ to 4.33×10 ¹⁴ GC of rAAV. In certain embodiments, the dose is administered to humans at a fixed dose ranging from about 1.44×10 ¹³ to 2×10 ¹⁴ GC of rAAV. In certain embodiments, the dose is administered to humans at a fixed dose ranging from about 3×10 ¹³ to 1×10 ¹⁴ GC of rAAV. In certain embodiments, the dose is administered to humans at a fixed dose ranging from about 5×10 ¹³ to 1×10 ¹⁴ GC of rAAV. In some embodiments, the compositions may be formulated into dosage units to contain an amount of AAV in the range of about 1×10 ¹³ to 8×10 ¹⁴ GC of AAV. In some embodiments, the composition may be formulated into a dosage unit to contain an amount of rAAV in the range of about 1.44×10 ¹³ to 4.33×10 ¹⁴ GC of AAV. In some embodiments, the composition may be formulated into a dosage unit to contain an amount of rAAV in the range of about 3×10 ¹³ to 1×10 ¹⁴ GC of AAV. In some embodiments, the compositions may be formulated into dosage units to contain rAAV in an amount ranging from about 5×10 ¹³ to 1×10 ¹⁴ GC of AAV.

於某些具體實施例中，載體係以單劑投予至受試者。於某些具體實施例，冀望載體可經由多次注射(例如2劑)遞送。In certain embodiments, the carrier system is administered to the subject in a single dose. In certain embodiments, it is desired that the vector be delivered via multiple injections (eg, 2 doses).

將調整劑量以平衡治療益處與任何副作用，且此種劑量可依據採用重組載體的治療應用而變化。可監測轉基因之表現水準以確定產生病毒載體的劑量頻率，較佳為含有袖珍基因(minigene)的AAV載體。可選擇地，類似於為了治療目的所描述的劑量方案可被用於使用本文提供的組成物之免疫。Dosage will be adjusted to balance the therapeutic benefit against any side effects, and such dosage may vary depending on the therapeutic application using the recombinant vector. The expression level of the transgene can be monitored to determine the dose frequency for producing viral vectors, preferably AAV vectors containing minigenes. Alternatively, dosage regimens similar to those described for therapeutic purposes may be used for immunization using the compositions provided herein.

如本文所使用，術語「鞘內遞送」或「鞘內投予」係指經由注射至脊髓管、更具體而言為注射至蜘蛛膜下腔的投予途徑，以使其到達腦脊髓液(CSF)。鞘內遞送可包括腰椎穿刺、室內(包括腦室內(ICV))、枕骨下/腦池內穿刺、及/或C1-2穿刺。例如，可藉由腰椎穿刺手段而導入物質以在整個蜘蛛膜下腔擴散。於另一例，可注射至腦大池。As used herein, the term "intrathecal delivery" or "intrathecal administration" refers to a route of administration via injection into the spinal canal, more specifically into the subarachnoid space, so that it reaches the cerebrospinal fluid ( CSF). Intrathecal delivery may include lumbar puncture, intraventricular (including intracerebroventricular (ICV)), suboccipital/intracisternal puncture, and/or C1-2 puncture. For example, a substance may be introduced by lumbar puncture to spread throughout the subarachnoid space. In another case, the injection can be into the cistern magna.

如本文所使用，術語「腦池內遞送」或「腦池內投予」係指直接進入腦大池小腦延髓之腦脊髓液中的投予途徑，更具體而言係經由枕骨下穿刺或藉由直接注射至腦大池，或者經由永久定位的管子。As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to a route of administration directly into the cerebrospinal fluid of the cistern magna, cerebellum, and medulla oblongata, more specifically via suboccipital puncture or by Inject directly into the cistern, or via a permanently positioned tube.

包含本文所述用於抑制內源性C9orf72 (例如，在ALS患者中)的miR標靶序列的組成物，通常靶向中樞神經系統中的一種或多種不同細胞類型，包括但不限於神經元(包括例如下運動神經元及/或初級感覺神經元。此等可能包括例如錐狀細胞、浦金埃氏細胞、顆粒細胞、梭狀細胞及聯絡神經元細胞)。Compositions comprising a miR target sequence described herein for inhibiting endogenous C9orf72 (e.g., in ALS patients) typically target one or more different cell types in the central nervous system, including but not limited to neurons ( These include, for example, motor neurons and/or primary sensory neurons. These may include, for example, pyramidal cells, Purkin-Ehrlich cells, granule cells, spindle cells, and contact neuron cells).

用途本文提供的載體及組成物有用於治療具有下列的患者：C9orf72相關的病症(例如，ALS或FTD)、神經病變或與其有關的各種症狀。提供治療具有ALS或FTD的患者之組合方案或共同療法。於某些具體實施例中，此方案或共同療法包含共同投予：(a)可操作連接至調節序列的編碼工程化人類C9orf72編碼序列之重組核酸序列，該調節序列指導其在人類標靶細胞中的表現，其中人類C9orf72編碼序列具有SEQ ID NO：13的序列或與其至少95%相同的序列且由於在(b)之miRNA標靶序列中具有錯誤配對而不同於患者中的內源性人類C9orf72，及(b)至少一個miRNA的編碼序列，該miRNA對人類ALS受試者中的內源性人類C9orf72序列具有特異性，其中該mRNA可操作連接至指導其在受試者中表現的調節序列。於某些具體實施例中，miR標靶序列為miR487，具有至少SEQ ID NO：16之序列、或至少SEQ ID NO：15與5’側翼區(例如，SEQ ID NO：5)、連接子、及3’側翼區(例如，SEQ ID NO：7)之組合。於某些具體實施例中，miR標靶序列為miR.NT序列，具有至少SEQ ID NO：6之序列與5’側翼區、連接子、及一連接子和3’側翼區。參見例如，SEQ ID NO：8；或SEQ ID NO：6與SEQ ID NO：5及/或SEQ ID NO：7之組合。 use The vectors and compositions provided herein are useful for treating patients with C9orf72-related disorders (eg, ALS or FTD), neuropathy, or various symptoms related thereto. Provide combination regimens or co-therapies for the treatment of patients with ALS or FTD. In certain embodiments, the regimen or co-therapy includes co-administering: (a) a recombinant nucleic acid sequence encoding an engineered human C9orf72 coding sequence operably linked to regulatory sequences that direct its expression in human target cells Performance in wherein the human C9orf72 coding sequence has the sequence of SEQ ID NO: 13 or is at least 95% identical thereto and differs from endogenous human in the patient due to mispairing in the miRNA target sequence of (b) C9orf72, and (b) the coding sequence of at least one miRNA specific for an endogenous human C9orf72 sequence in a human ALS subject, wherein the mRNA is operably linked to a regulator that directs its expression in the subject sequence. In certain embodiments, the miR target sequence is miR487, having at least the sequence of SEQ ID NO: 16, or at least SEQ ID NO: 15 and a 5' flanking region (eg, SEQ ID NO: 5), a linker, and a combination of 3' flanking regions (eg, SEQ ID NO: 7). In certain embodiments, the miR target sequence is a miR.NT sequence, having at least the sequence of SEQ ID NO: 6 and a 5' flanking region, a linker, and a linker and a 3' flanking region. See, for example, SEQ ID NO:8; or SEQ ID NO:6 in combination with SEQ ID NO:5 and/or SEQ ID NO:7.

於某些具體實施例中，此治療具有C9orf72的患者之方案或共同療法包含共同投予：(a)可操作連接至調節序列的編碼工程化人類C9orf72編碼序列之重組核酸序列，該調節序列指導其在人類標靶細胞中的表現，其中人類C9orf72編碼序列經工程化成為由於在(b)之miRNA標靶序列中具有錯誤配對而不同於患者中的內源性人類C9orf72，及(b) a至少一個miRNA的編碼序列，該miRNA對人類受試者中的內源性人類C9orf72序列具有特異性，其中該mRNA編碼序列可操作連接至指導其在受試者中表現的調節序列，其中至少一個miRNA編碼序列具有一或多個以下序列：包含SEQ ID NO：16的miRNA編碼序列(具側翼區的miR487)。於某些具體實施例中，核酸分子進一步包含miR標靶序列為miR.NT序列，具有至少SEQ ID NO：6的序列、與5’側翼區、連接子、及一連接子和3’側翼區。參見例如，SEQ ID NO：8；或SEQ ID NO：6與SEQ ID NO：5及/或SEQ ID NO：7的組合。於某些具體實施例中，第一載體包含核酸(a)及一第二之不同的載體，包含至少一個miRNA(b)。於某些具體實施例中，第一載體為病毒載體及/或第二載體為病毒載體，且第一及第二病毒載體可為相同病毒來源或可為不同。於某些具體實施例中，第一載體為非病毒載體，第二載體為非病毒載體且第一及第二載體可為相同組成或可為不同。In certain embodiments, the regimen or co-therapy for treating a patient with C9orf72 includes co-administering: (a) a recombinant nucleic acid sequence encoding an engineered human C9orf72 coding sequence operably linked to a regulatory sequence that directs Its performance in human target cells in which the human C9orf72 coding sequence is engineered to differ from endogenous human C9orf72 in the patient due to mispairing in the miRNA target sequence of (b), and (b) a A coding sequence for at least one miRNA specific for an endogenous human C9orf72 sequence in a human subject, wherein the mRNA coding sequence is operably linked to a regulatory sequence that directs its expression in the subject, wherein at least one The miRNA coding sequence has one or more of the following sequences: the miRNA coding sequence comprising SEQ ID NO: 16 (miR487 with flanking regions). In certain embodiments, the nucleic acid molecule further comprises a miR target sequence that is a miR.NT sequence, having at least the sequence of SEQ ID NO: 6, and a 5' flanking region, a linker, and a linker and a 3' flanking region. . See, for example, SEQ ID NO:8; or SEQ ID NO:6 in combination with SEQ ID NO:5 and/or SEQ ID NO:7. In certain embodiments, a first vector includes nucleic acid (a) and a second, different vector includes at least one miRNA (b). In certain embodiments, the first vector is a viral vector and/or the second vector is a viral vector, and the first and second viral vectors may be of the same viral origin or may be of different origins. In some embodiments, the first vector is a non-viral vector, the second vector is a non-viral vector and the first and second vectors may be of the same composition or may be different.

可選擇地，本文提供的載體及組成物可用於與一或多種選自下列的協同療法組合：可用於降低某些患者發病率的ALS管理的可取得的批准治療包括利魯唑(riluzole)及依達拉奉(edaravone)。利魯唑為一種口服麩胺酸鹽抑制劑，已被證明可延緩某些ALS患者的呼吸器依賴或氣管切開術的開始。依達拉奉為一種IV投予的神經保護劑，在減緩ALS患者的身體功能喪失方面取得了適度的成功。具ALS的患者亦可受益於多專科照護，包括輔助通訊裝置的實施、營養支持、呼吸器輔助、控制疾病症狀的藥物治療、心理支持以及身體、職業和言語治療。其他適合的協同療法可包括乙醯胺酚(acetaminophen)、及/或非類固醇消炎藥(NSAIDs)。於某些具體實施例中，載體可與涉及一種或多種類固醇(例如強體松(prednisone))的免疫調節方案組合遞送。Alternatively, the vectors and compositions provided herein may be used in combination with one or more synergistic therapies selected from: Available approved treatments for the management of ALS that may reduce morbidity in certain patients include riluzole; Edaravone. Riluzole, an oral glutamate inhibitor, has been shown to delay ventilator dependence or the initiation of tracheostomy in some ALS patients. Edaravone is an IV-administered neuroprotective agent that has had modest success in slowing the loss of body function in ALS patients. People with ALS can also benefit from multi-specialty care, including implementation of assistive communication devices, nutritional support, ventilator assistance, medications to control disease symptoms, psychological support, and physical, occupational and speech therapy. Other suitable synergistic therapies may include acetaminophen, and/or non-steroidal anti-inflammatory drugs (NSAIDs). In certain embodiments, the vector may be delivered in combination with an immunomodulatory regimen involving one or more steroids (eg, prednisone).

如本文所使用，術語電腦斷層造影(CT)係指放射線攝影術，其中藉由電腦自沿軸線製成的一系列平面截面影像而建構身體結構的三維影像。As used herein, the term computed tomography (CT) refers to radiography in which a three-dimensional image of body structures is constructed by computer from a series of planar cross-sectional images taken along an axis.

當指核酸或其片段時，術語「實質上同源」或「實質上相似」係指在與另一核酸(或其互補股)的適當的核苷酸***或刪除進行最佳比對時，於至少約95%至99%的比對序列中有核苷酸序列同一性。較佳地，該同源為全長序列、或其開讀框、或長度至少為15個核苷酸的其他適合的片段。本文描述適合的片段之例。When referring to a nucleic acid or a fragment thereof, the terms "substantially homologous" or "substantially similar" mean when optimally aligned with an appropriate nucleotide insertion or deletion of another nucleic acid (or its complementary strand). There is nucleotide sequence identity in at least about 95% to 99% of the aligned sequences. Preferably, the homology is the full-length sequence, or its open reading frame, or other suitable fragment of at least 15 nucleotides in length. This article describes examples of suitable fragments.

於核酸序列之上下文中，術語「序列同一性」、「序列同一性百分比」、或「百分比相同」係指兩個序列中當比對以獲得最大對應性時為相同的殘基。序列同一性比較之長度理想可為整個基因體之全長、基因編碼序列之全長、或至少約500至5000個核苷酸之片段。然而，較小片段中的同一性亦為理想，例如至少約9個核苷酸、通常至少約20至24個核苷酸、至少約28至32個核苷酸、至少約36個以上之核苷酸。相似地，可容易地確定整個蛋白質之全長、或其片段的胺基酸序列的「序列同一性百分比」。適合地，片段為至少約8個胺基酸長且可多至約700個胺基酸。本文描述適合的片段之例。In the context of nucleic acid sequences, the terms "sequence identity," "percent sequence identity," or "percent identical" refer to the residues in two sequences that are identical when aligned for maximum correspondence. The length of the sequence identity comparison can ideally be the full length of the entire genome, the full length of the gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides. However, identity in smaller fragments is also desirable, such as at least about 9 nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides. glycosides. Similarly, the "percent sequence identity" of the amino acid sequence of an entire protein, or a fragment thereof, can be readily determined. Suitably, the fragment is at least about 8 amino acids long and can be up to about 700 amino acids long. This article describes examples of suitable fragments.

術語「高度保留」意指至少80%同一性，較佳為至少90%同一性，更佳為超過97%同一性。藉由使用本技術領域中具有通常知識者已知的演算法及電腦程式，本技術領域中具有通常知識者可容易地確定同一性。The term "highly conserved" means at least 80% identity, preferably at least 90% identity, and more preferably more than 97% identity. Identity can be readily determined by one of ordinary skill in the art by using algorithms and computer programs known to those of ordinary skill in the art.

除非上限範圍另有說明，否則應理解，同一性百分比為最低同一性水準，涵括所有更高水準的同一性，多至與參考序列具有100%同一性。除非另有說明，否則應理解，同一性百分比為最低同一性水準，涵括所有更高水準的同一性，多至與參考序列具有100%同一性。例如，「95%同一性」及「至少95%同一性」可交換地使用且包括與參考序列具有95、96、97、98、99、多至100%及其間的所有分數之同一性。Unless the upper range indicates otherwise, it is understood that percent identity is the lowest level of identity and encompasses all higher levels of identity, up to 100% identity with the reference sequence. Unless otherwise stated, it is understood that percent identity is the lowest level of identity and encompasses all higher levels of identity, up to 100% identity with the reference sequence. For example, "95% identity" and "at least 95% identity" are used interchangeably and include identities to a reference sequence of 95, 96, 97, 98, 99, up to 100%, and all fractions therebetween.

除非另有指明，數值將被理解為遵從常規的數學四捨五入規則。Unless otherwise indicated, numerical values will be understood as subject to ordinary mathematical rounding rules.

一般而言，當提及兩個不同腺相關病毒之間的「同一性」、「同源性」、或「相似性」時，參照「比對」序列來確定「同一性」、「同源性」、或「相似性」。「比對」序列或「比對」係指多個核酸序列或蛋白質(胺基酸)序列，與參考序列相比，通常含有缺失或增加的鹼基或胺基酸的校正。在示例中，使用公開的AAV9序列作為參考點進行AAV比對。使用多種公開或市售的多序列比對程式中的任何一種進行比對。此種程式之例包括：「Clustal Omega」、「Clustal W」、「CAP Sequence Assembly」、「MAP」、及「MEME」，此等可通過網際網路上的網站伺服器進行。此種程式之其他來源為本技術領域中具有通常知識者所知悉。或者，亦可使用Vector NTI應用程式。本領域中亦有許多可用於測量核苷酸序列同一性的已知演算法，包括彼等含於上述程式中者。作為另一例，可使用GCG版本6.1的程式Fasta™，而比較多核苷酸序列。Fasta™提供查詢序列及檢索序列之間最佳重疊區域的比對及序列同一性百分比。例如，核酸序列之間的序列同一性百分比可使用Fasta™及其如GCG版本6.1中所提供的內定參數(字長為6，得分矩陣的NOPAM因子)而確定，其藉由引用併入本文。多序列比對程式亦可用於胺基酸序列，例如，「Clustal Omega」、「Clustal X」、「MAP」、「PIMA」、「MSA」、「BLOCKMAKER」、「MEME」、及「Match-Box」程式。一般而言，儘管本技術領域中具有通常知識者可依需要改變此等設定，但此等程式之任一者皆可於內定設定下使用。或者，本技術領域中具有通常知識者可利用另一種演算法或電腦程式，該演算法或電腦程式至少提供如所引用的演算法及程式所提供的同一性或比對水準。參見例如，J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999)。Generally speaking, when referring to "identity", "homology" or "similarity" between two different adeno-associated viruses, reference is made to "aligning" sequences to determine "identity", "homology" "Sex" or "similarity". An "aligned" sequence or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences that typically contain corrections of deleted or added bases or amino acids compared to a reference sequence. In the example, AAV alignment is performed using the published AAV9 sequence as a reference point. Alignments were performed using any of a variety of published or commercially available multiple sequence alignment programs. Examples of such programs include: "Clustal Omega", "Clustal W", "CAP Sequence Assembly", "MAP", and "MEME", which are available through website servers on the Internet. Other sources of such programs are known to those of ordinary skill in the art. Alternatively, use the Vector NTI app. There are also many algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the above program. As another example, polynucleotide sequences can be compared using the program Fasta™ of GCG version 6.1. Fasta™ provides alignment and sequence identity percentage of the best overlap region between the query sequence and the search sequence. For example, the percent sequence identity between nucleic acid sequences can be determined using Fasta™ and its default parameters (word length 6, NOPAM factor of the scoring matrix) as provided in GCG version 6.1, which is incorporated herein by reference. Multiple sequence alignment programs can also be used for amino acid sequences, such as "Clustal Omega", "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" ” program. In general, any of these programs can be used with default settings, although one of ordinary skill in the art can change these settings as necessary. Alternatively, one of ordinary skill in the art may utilize another algorithm or computer program that provides at least the same level of identity or comparison as provided by the cited algorithms and programs. See, for example, J. D. Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

應注意的是，術語「一(a或an)」係指一個(種)或多個(種)。如此，術語「一」(a或an)、「一個(種)或以上」及「至少一個(種)」於本文中可互換使用。It should be noted that the term "a" or "an" refers to one or more. As such, the terms "a" (a or an), "one or more" and "at least one" are used interchangeably herein.

詞語「包含」(comprise、comprises、及comprising)被包括性地而不是排他性地解釋。詞語「由…組成」(consist、consisting)及其變體被排他性地而不是包括性地解釋。儘管說明書中的多個具體實施例使用「包含」語句來呈現，但在其他情況下，相關具體實施例亦意圖使用「由…組成」或「實質上由…組成」語句來解釋及描述。The words "comprise," "comprises," and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist, consisting" and their variations are to be interpreted exclusively and not inclusively. Although many specific embodiments in the specification are presented using "comprises" statements, in other cases, relevant specific embodiments are also intended to be explained and described using "composed of" or "consisting essentially of" statements.

如本文所使用，除非另有指明，術語「約」意指與給定參考值間有10% (±10%，例如，±1、±2、±3、±4、±5、±6、±7、±8、±9、±10、或其兩者之間的值)的變動。As used herein, unless otherwise specified, the term "about" means 10% (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or values in between).

如本文所使用，「疾病」、「病症」及「病況」可互換使用，以表示受試者的不正常狀態。As used herein, "disease," "disease," and "condition" are used interchangeably to refer to an abnormal state of a subject.

如本文所使用，術語「C9orf72相關症狀」或「症狀」係指具有ALS症狀的患者中的症狀，包括例如，持續性無力，此可能有不同的表現，一些患者有一個或多個肢體的孤立無力，而其他人最初表現出延髓無力(bulbar weakness)，其會影響控制言語、吞嚥和咀嚼的肌肉。其他表現包括肌張力和肌腱反射異常、進行性肌肉無力的跡象、尤其是在軀幹及四肢的肌肉萎縮、與無法控制運動相關的痙攣。臨床症狀從肌束震顫、肌肉痙攣、步態障礙、無法步行、手臂和手部功能喪失，到言語和吞嚥困難以及呼吸困難。吸入性肺炎和呼吸功能不全為此等患者的常見死因。大約29%的C9orf72重複擴增攜帶者沒有出現ALS 症狀。反而，他們被診斷出患有額顳葉癡呆症(FTD)，其係一種進行性腦部疾病，會影響人格、行為、語言和認知。有些人甚至會出現這兩種病況的特徵，並被診斷為患有ALS-FTD變體。As used herein, the term "C9orf72-related symptoms" or "symptoms" refers to symptoms in patients with symptoms of ALS, including, for example, persistent weakness, which may manifest differently, and some patients have isolation of one or more limbs. Weakness, while others initially present with bulbar weakness, which affects the muscles that control speech, swallowing, and chewing. Other findings include abnormal muscle tone and tendon reflexes, signs of progressive muscle weakness, muscle atrophy especially in the trunk and extremities, and spasticity associated with an inability to control movement. Clinical symptoms range from fasciculations, muscle spasms, gait disturbance, inability to walk, loss of arm and hand function, to speech and swallowing difficulties, and breathing difficulties. Aspiration pneumonia and respiratory insufficiency are common causes of death in these patients. Approximately 29% of carriers of the C9orf72 repeat expansion do not develop ALS symptoms. Instead, they were diagnosed with frontotemporal dementia (FTD), a progressive brain disease that affects personality, behavior, language and cognition. Some people even develop features of both conditions and are diagnosed with the ALS-FTD variant.

如本文所使用，「患者」或「受試者」意指用於臨床研究的雄性或雌性人類、及動物模型(包括例如狗、非人類齡長類、囓齒類或其他適合的模型)。於一具體實施例中，此等方法及組成物之受試者為診斷具有C9orf72相關病症的人類。此種病症可包括於C9orf72基因具有缺陷的患者，例如，如與肌肉萎縮性脊髓側索硬化症(ALS)或額顳葉癡呆症(FTD)、或兩者(C9FTD/ALS)相關者。C9orf72重複擴增亦被鑑定為其他神經退行性疾病的罕見原因，包括帕金森氏症(Parkinson's disease)、進行性核上性麻痺(progressive supranuclear palsy)、共濟失調、皮質基底節症候群(corticobasal syndrome)、類亨丁頓氏病症候群(Huntington disease-like syndrome)、庫賈氏病(Creutzfeldt-Jakob disease)及阿茲海默症。於某些具體實施例中，此等方法及組成物之人類受試者為出生前、新生兒、嬰兒、學步兒、學齡前、小學生、青少年、年輕成人或成人。於另一具體實施例中，此等方法及組成物之受試者為兒科患者。As used herein, "patient" or "subject" means male or female humans, and animal models (including, for example, dogs, non-human species, rodents, or other suitable models) used in clinical studies. In a specific embodiment, the subjects of these methods and compositions are humans diagnosed with C9orf72-related disorders. Such disorders may include patients with defects in the C9orf72 gene, such as those associated with amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD), or both (C9FTD/ALS). C9orf72 repeat expansion has also been identified as a rare cause of other neurodegenerative diseases, including Parkinson's disease, progressive supranuclear palsy, ataxia, and corticobasal syndrome. ), Huntington disease-like syndrome, Creutzfeldt-Jakob disease and Alzheimer's disease. In certain embodiments, the human subject of these methods and compositions is prenatal, newborn, infant, toddler, preschooler, elementary school student, adolescent, young adult, or adult. In another embodiment, the subjects of these methods and compositions are pediatric patients.

如本文所使用，術語「治療水平」意指C9orf72活性為健康對照之至少約5%、約8%、約10%、約15%、約20%、約25%、約30%、約35%、約40%、約45%、約50%、約55%、約60%、約65%、約70%、約75%、約80%、約85%、約90%、約95%、約100%、大於100%、約2倍、約3倍、或約5倍。用於測量hC9orf72活性的適合分析為本領域已知。於一些具體實施例中，一種或多種次單元蛋白質的此種治療水平可造成C9orf72相關的ALS或FTD症狀的緩解；逆轉某些C9orf72相關的症狀及/或防止ALS或FTD相關的某些症狀進行；或其任何組合。於某些具體實施例中，藉由無氣管切開述的存活、肺功能改善措施來衡量治療效果，例如，如藉由用力肺活量(FVC)或緩慢肺活量(SVC)來衡量。可使用ALS功能評定量表(ALSFRS-R)確定治療效果的其他合適測量值，其評量粗大動作任務(gross motor tasks)(在床上翻身、走路和爬樓梯)、精細動作任務(fine motor tasks)(切食物、寫字和穿衣/衛生狀況)、延髓功能(說話、吞嚥和流涎)及呼吸功能(呼吸困難、端坐呼吸和需要換氣支持))(Cedarbaum et al., (1999). "The ALSFRS-R：a revised ALS functional rating scale that incorporates assessments of respiratory function. BDNF ALS Study Group (Phase III)." J Neurol Sci. 169(1-2):13-21)。精確的肢體等長強度測試(ATLIS)、肌肉萎縮性脊髓側索硬化症-特異性生活質量簡表(ALSSQOL-SF)。另外或可選擇地，可測量適合的生物標誌物以評估功效。適合的生物標誌物包括例如神經絲重鏈(NFH)和神經絲輕鏈(NFL)、二肽重複蛋白、tau蛋白及/或神經影像學。As used herein, the term "therapeutic level" means C9orf72 activity that is at least about 5%, about 8%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35% of healthy controls. , about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, greater than 100%, about 2 times, about 3 times, or about 5 times. Suitable assays for measuring hC9orf72 activity are known in the art. In some embodiments, such therapeutic levels of one or more subunit proteins may result in amelioration of C9orf72-related ALS or FTD symptoms; reverse certain C9orf72-related symptoms and/or prevent progression of certain ALS or FTD-related symptoms. ; or any combination thereof. In some embodiments, the therapeutic effect is measured by measures of improvement in survival without tracheostomy and lung function, for example, as measured by forced vital capacity (FVC) or slow vital capacity (SVC). Other appropriate measures of treatment effectiveness may be determined using the ALS Functional Rating Scale (ALSFRS-R), which assesses gross motor tasks (turning over in bed, walking, and climbing stairs), fine motor tasks ) (cutting food, writing, and dressing/hygiene), bulbar function (speaking, swallowing, and salivation), and respiratory function (dyspnea, orthopnea, and need for ventilatory support)) (Cedarbaum et al., (1999) . "The ALSFRS-R: a revised ALS functional rating scale that incorporates assessments of respiratory function. BDNF ALS Study Group (Phase III)." J Neurol Sci. 169(1-2):13-21). Accurate Limb Isometric Strength Test (ATLIS), Amyotrophic Lateral Sclerosis-Specific Quality of Life Short Form (ALSSQOL-SF). Additionally or alternatively, suitable biomarkers can be measured to assess efficacy. Suitable biomarkers include, for example, neurofilament heavy chain (NFH) and neurofilament light chain (NFL), dipeptide repeat protein, tau protein, and/or neuroimaging.

於某些具體實施例中，藉由本文提供的組成物及方案遞送的人類C9orf72具有功能性內源性野生型蛋白質的胺基酸序列。於某些具體實施例中，該序列為SEQ ID NO：14或45之胺基酸序列或與功能性人類C9orf72蛋白質為約95至100%同一性之功能性蛋白質。In certain embodiments, human C9orf72 delivered by the compositions and protocols provided herein has the amino acid sequence of a functional endogenous wild-type protein. In certain embodiments, the sequence is the amino acid sequence of SEQ ID NO: 14 or 45 or a functional protein that is about 95 to 100% identical to a functional human C9orf72 protein.

在本文中的術語「表現」以其最廣泛的含義使用，且包含RNA的產生或RNA及蛋白質的產生。關於RNA，術語「表現」或「轉譯」特別涉及肽或蛋白質的產生。表現可為短暫的或可為穩定的。The term "expression" is used herein in its broadest sense and includes the production of RNA or the production of RNA and protein. With respect to RNA, the term "expression" or "translation" relates specifically to the production of peptides or proteins. Manifestations may be transient or may be stable.

另外可選擇地，表現匣(及載體基因體)於UTR中可包含一或多個背根神經節(drg)-miRNA靶向序列，例如，以降低drg-毒性及/或軸突病變(axonopathy)。參見例如，2019年12月20日申請之PCT/US2019/67872，現已公開為WO 2020/132455；PCT/US2021/032002、現已公開為WO2021/231579、2020年5月12日申請之US臨時專利申請案第63/023593號、及2020年6月12日申請之US臨時專利申請案第63/038488號、及US臨時專利申請案第63/279,561號，所有標題皆為「Compositions for Drg-Specific Reduction of Transgene Expression」，此等完整併入本文。於一些具體實施例中，表現匣可經由遺傳元件(例如，質體)遞送至包裝宿主細胞並包裝到病毒載體的衣殼中(例如，病毒顆粒)。Alternatively, the expression cassette (and vector genome) may include one or more dorsal root ganglion (drg)-miRNA targeting sequences in the UTR, for example, to reduce drg-toxicity and/or axonopathy. ). See, for example, PCT/US2019/67872, filed on December 20, 2019, now published as WO 2020/132455; PCT/US2021/032002, now published as WO2021/231579, and US Provisional filed on May 12, 2020 Patent Application No. 63/023593, US Provisional Patent Application No. 63/038488 filed on June 12, 2020, and US Provisional Patent Application No. 63/279,561, all titled "Compositions for Drg- Specific Reduction of Transgene Expression", which is incorporated herein in its entirety. In some embodiments, expression cassettes can be delivered to packaging host cells via genetic elements (eg, plastids) and packaged into the capsids of viral vectors (eg, viral particles).

如本文所使用，術語「可操作地連接的」係指與有興趣的基因鄰接的表現控制序列、及以反式或以一距離地作用而控制有興趣的基因之表現控制序列兩者。As used herein, the term "operably linked" refers to both expression control sequences that are contiguous with a gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

當使用於提及之蛋白質或核酸時，術語「異源的」表示該蛋白質或核酸包含在自然界中彼此之間未發現有相同關係的兩個以上的序列或子序列。例如，通常重組產生的核酸，具有二個以上來自無關基因的序列，其排列以產生新的功能性核酸。例如，於一具體實施例中，核酸具有來自一個基因的啟動子，其被安排以指導來自不同基因的編碼序列的表現。如此，參照編碼序列，該啟動子為異源的。When used in reference to a protein or nucleic acid, the term "heterologous" means that the protein or nucleic acid contains more than two sequences or subsequences that are not found to be identically related to each other in nature. For example, nucleic acids typically produced recombinantly have two or more sequences from unrelated genes arranged to produce a new functional nucleic acid. For example, in one embodiment, a nucleic acid has a promoter from one gene arranged to direct the expression of coding sequences from a different gene. As such, the promoter is heterologous with respect to the coding sequence.

如本文所述，調節元件包含但未限於：啟動子；強化子；轉錄因子；轉錄終止子；有效的RNA加工訊息，如剪接及多腺苷酸化訊息(polyA)；穩定細胞質mRNA的序列，例如土撥鼠肝炎病毒(WHP)轉錄後調節元件(WPRE)；提高轉譯效率的序列(即，科札克共通序列)。As described herein, regulatory elements include, but are not limited to: promoters; enhancers; transcription factors; transcription terminators; efficient RNA processing messages, such as splicing and polyadenylation messages (polyA); sequences that stabilize cytoplasmic mRNA, such as Woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE); sequence that increases translation efficiency (i.e., Kozak consensus sequence).

本發明上下文中的術語「轉譯」係關於一個在核糖體的過程，其中mRNA股控制胺基酸序列的組裝以產生蛋白質或肽。The term "translation" in the context of the present invention refers to a process in the ribosome in which the mRNA strand controls the assembly of amino acid sequences to produce proteins or peptides.

於一具體實施例中，本文提供用於包裝質體的載體基因體包含SEQ ID NO：17，包括縮短的AAV2-5’ ITR；包含C4強化子、CB7啟動子、工程化C9orf72編碼序列及C9miR487標靶序列的表現匣；WPRE元件；及polyA訊息(例如，SEQ ID NO：18或與其至少97%相同的序列之表現匣)、及縮短的AAV2-3’ AAV。於某些具體實施例中，例如，經包裝的rAAV載體，載體基因體包含全長5’ ITR及全長3’ ITR。於某些具體實施例中，載體基因體包含上述載體元件，不具WPRE元件。於某些具體實施例中，載體基因體包含scAAV。In a specific embodiment, the vector genome provided herein for packaging plasmids includes SEQ ID NO: 17, including shortened AAV2-5' ITR; including C4 enhancer, CB7 promoter, engineered C9orf72 coding sequence and C9miR487 A representation box of the target sequence; a WPRE element; and a polyA message (e.g., a representation box of SEQ ID NO: 18 or a sequence at least 97% identical thereto), and shortened AAV2-3' AAV. In certain embodiments, such as packaged rAAV vectors, the vector genome includes a full-length 5' ITR and a full-length 3' ITR. In some embodiments, the vector genome includes the above-mentioned vector elements without the WPRE element. In certain embodiments, the vector genome includes scAAV.

於某些具體實施例中，rAAV或另一載體可含有表現匣，該表現匣在單獨載體中含有miR487靶向序列及C9orf72編碼序列[參見例如，SEQ ID NO：19]或單獨表現匣。於某些具體實施例中，WPRE元件可從表現匣去除及/或以另一基因體元件替代。In certain embodiments, rAAV or another vector may contain an expression cassette containing the miR487 targeting sequence and the C9orf72 coding sequence in a separate vector [see, eg, SEQ ID NO: 19] or a separate expression cassette. In certain embodiments, the WPRE element can be removed from the expression cassette and/or replaced with another genomic element.

除非於本說明書中另有定義，否則本文所用的技術及科學術語具有與本領域中具有通常知識者及參照公開文本所通常理解的相同含義，公開文本為本領域中具有通常知識者提供了本申請案中所使用之許多術語的一般指引。Unless otherwise defined in this specification, technical and scientific terms used herein have the same meanings as commonly understood by a person of ordinary skill in the art with reference to the disclosure, which provides the information to a person of ordinary skill in the art. General guidance on many terms used in applications.

下列實施例僅為說明性的且未意圖限制本發明。The following examples are illustrative only and are not intended to limit the invention.

實施例下列實施例為說明性且未意圖限制本發明。 Example The following examples are illustrative and not intended to limit the invention.

實施例1：C9orf72載體策略我們生成rAAV，該rAAV包含：(1)包含靶向內源性C9orf72之miR序列的表現匣，(2)包含此等miR序列及工程化C9orf72 cDNA的組合的表現匣，該工程化C9orf72 cDNA在表達匣中被miR靶向的C9orf72區域中具有修飾，因此不會被miR靶向。為了減弱(knock down)內源性C9orf72，檢查具有表現各種miRNA序列的載體基因體的rAAV：miR.NT(陰性對照)、miR32-101(陽性對照)、及miR487。 Example 1: C9orf72 vector strategy We generate rAAV that contains: (1) a expression cassette containing miR sequences targeting endogenous C9orf72, (2) a expression cassette containing a combination of these miR sequences and an engineered C9orf72 cDNA in The C9orf72 region in the expression cassette that is targeted by miRs has modifications in it and is therefore not targeted by miRs. To knock down endogenous C9orf72, rAAV with vector genomes expressing various miRNA sequences was examined: miR.NT (negative control), miR32-101 (positive control), and miR487.

在293 HEK細胞株中以順式質體使用習用三重轉染法構築載體，該順式質體包含要包裝的載體基因體，由5’ ITR、間隔子序列、表現匣、間隔子序列、及3’ ITR所構成。縮短的(130 bp) 5’-及3’ ITRs位於此順式質體中；在複製及包裝期間，此等恢復到全長145 bp 5’及3’ ITRs。此順式質體與包含轉染和包裝所需的Ad輔助基因的反式質體及包含編碼AAV衣殼的VP1基因的反式質體共轉染。於某些小鼠研究中，使用稱為AAV9-eB的AAV9突變體。In the 293 HEK cell line, a conventional triple transfection method was used to construct the vector using a cis-plastid. The cis-plastid contains the vector gene to be packaged, consisting of 5' ITR, spacer sequence, expression cassette, spacer sequence, and Composed of 3' ITR. Shortened (130 bp) 5'- and 3' ITRs are located in this cis-acting plasmid; during replication and packaging, these revert to the full-length 145 bp 5' and 3' ITRs. This cis-plastid was co-transfected with a trans-plastid containing the Ad accessory genes required for transfection and packaging and a trans-plastid containing the VP1 gene encoding the AAV capsid. In some mouse studies, a mutant AAV9 called AAV9-eB is used.

實施例2：Tg(C9orf72_3)系112小鼠模型的概念驗證研究在Tg(C9orf72_3)系112小鼠模型中評估本文所述的rAAV構築體減弱突變體C9orf72 RNA及DPR的能力。經由單次腦室內(ICV)注射，將rAAV投予至成年Tg(C9orf72_3)系112小鼠。選擇劑量範圍以評估從最大可行劑量開始的半對數增量。媒劑處理的轉基因和非轉基因小鼠作為對照。注射後30天，犧牲小鼠，收集腦及脊髓用於分析。選擇30天的時間點以允許有足夠的時間達到C9orf72 RNA和DPR蛋白質的穩態水平。藉由定量rtPCR使用外顯子特異性引子測量總C9orf72 mRNA，並對GAPDH表現進行標準化。使用對C9orf72的第一內含子具有特異性的引子，藉由rtPCR對含有異常重複的轉錄本進行量化。使用Mesoscale Discovery平台，藉由免疫測定法測量DPRs (poly-GP)。 Example 2: Proof-of-concept study of Tg(C9orf72_3) line 112 mouse model The ability of the rAAV constructs described herein to attenuate mutant C9orf72 RNA and DPR was evaluated in the Tg(C9orf72_3) line 112 mouse model. rAAV was administered to adult Tg(C9orf72_3) line 112 mice via a single intracerebroventricular (ICV) injection. The dose range was chosen to evaluate semi-logarithmic increments starting from the maximum feasible dose. Vehicle-treated transgenic and non-transgenic mice served as controls. Thirty days after injection, mice were sacrificed, and brains and spinal cords were collected for analysis. The 30-day time point was chosen to allow sufficient time to reach steady-state levels of C9orf72 RNA and DPR protein. Total C9orf72 mRNA was measured by quantitative rtPCR using exon-specific primers and normalized to GAPDH performance. Transcripts containing aberrant repeats were quantified by rtPCR using primers specific for the first intron of C9orf72. DPRs (poly-GP) were measured by immunoassay using the Mesoscale Discovery platform.

實施例3：Tg(C9orf72_3)系112小鼠模型中最小有效劑量(MED)的鑑定在Tg(C9orf72_3)系112小鼠模型中評估多劑量的選定的rAAV載體。劑量包括最大可行劑量和超過30倍劑量範圍的半對數增量。rAAV由受過訓練的人員經由單次腦室內(ICV)注射投予至成年Tg(C9orf72_3)系112小鼠。媒劑處理的轉基因和非轉基因小鼠作為對照。每天進行兩次臨床觀察，每週測量體重。對於所有非計劃的死亡，將對完整的組織列表進行全面的大體病理學和組織病理學分析，並酌情進行其他分析以確定可能的死因。注射後90天，犧牲小鼠。選擇90天的時間點來評估突變轉錄本減弱的持久性。收集腦、脊髓、心臟、肺、肝臟、脾臟、腎臟、食道、胃、大腸和小腸、腸系膜和頸部淋巴結、腎上腺、及性腺，檢查大體病理學，並進行組織病理學處理。在組織病理學發現的情況下，對免疫細胞浸潤進行適用的免疫組織化學染色。收集血液用於血清化學檢測和全血細胞計數。如上述，在腦和脊髓中測量含有內含子的C9orf72 RNA及DPR。顯著降低突變體C9orf72 mRNA及DPR表現水平的最低劑量被認為是MED。顯著性將藉由與媒劑對照組的適當統計比較而確定。腦和脊髓的部分以及收集的所有其他組織被固定並包埋在石蠟中用於組織病理學分析。 Example 3: Identification of minimum effective dose (MED) in Tg(C9orf72_3) line 112 mouse model Multiple doses of selected rAAV vectors were evaluated in the Tg(C9orf72_3) line 112 mouse model. Doses include the maximum feasible dose and semi-log increments over a 30-fold dose range. rAAV was administered to adult Tg(C9orf72_3) line 112 mice via a single intracerebroventricular (ICV) injection by trained personnel. Vehicle-treated transgenic and non-transgenic mice served as controls. Clinical observations were made twice daily and body weight was measured weekly. For all unplanned deaths, a comprehensive gross pathology and histopathology analysis of the complete tissue list will be performed, with additional analyzes performed as appropriate to determine the likely cause of death. Ninety days after injection, mice were sacrificed. The 90-day time point was chosen to assess the attenuated persistence of mutant transcripts. The brain, spinal cord, heart, lungs, liver, spleen, kidneys, esophagus, stomach, large and small intestines, mesenteric and cervical lymph nodes, adrenal glands, and gonads were collected, examined for gross pathology, and processed for histopathology. In the case of histopathological findings, appropriate immunohistochemical staining was performed for immune cell infiltration. Blood was collected for serum chemistry testing and complete blood count. Intron-containing C9orf72 RNA and DPR were measured in brain and spinal cord as described above. The lowest dose that significantly reduced mutant C9orf72 mRNA and DPR expression levels was considered the MED. Significance will be determined by appropriate statistical comparisons with vehicle controls. Sections of the brain and spinal cord, as well as all other tissues collected, were fixed and embedded in paraffin for histopathological analysis.

圖1A至1D提供來自11-14週齡小鼠(C9 L112 Het)脊髓的qPCR結果，該小鼠注射(靜脈內-尾靜脈)3x10 ¹¹GC/100 µl之rAAV-PHP.eb-CB7.CI.C9miR. WPRE.rBG，miR為NT或PBS、miR487、miR32、或miR32-101。圖1A提供C9內含子剪接的引子在脊髓的結果。圖1B提供C9內含子保留的引子在脊髓的結果。圖1C及1D提供來自腦的C9內含子剪接的引子(圖1C)或C9內含子保留的引子(圖1D)的qPCR結果。 Figures 1A to 1D provide qPCR results from spinal cords from 11-14 week old mice (C9 L112 Het) injected (i.v. - tail vein) with 3x10 ¹¹ GC/100 µl of rAAV-PHP.eb-CB7.CI .C9miR. WPRE.rBG, the miR is NT or PBS, miR487, miR32, or miR32-101. Figure 1A provides the results of C9 intron splicing primers in the spinal cord. Figure 1B provides the results of the C9 intron-retaining primer in the spinal cord. Figures 1C and ID provide qPCR results for C9 intron spliced primers (Figure 1C) or C9 intron retained primers (Figure 1D) from brain.

圖2A-2D提供在poly(GP) Meso Scale Discovery (MSD)-免疫分析中可溶部分DPR蛋白質病理學評估的結果。C57BL/6J-Tg(C9orf72_i3)112Lutzy/J (JR：023099)小鼠顯示在1及3個月齡時在腦溶胞產物及在12個月齡時在脊髓溶胞產物中poly(GP)可溶部分顯著增加，與作為對照之NCAR相比。隨著小鼠年齡的增長，在(G ₄C2)149小鼠中觀察到小鼠腦中可溶性部分的DPR減少。數據表示為平均值±SD。以rAAV和媒劑或包含miRNA的rAAV處理C9缺陷小鼠中的poly (GP)反應。圖2A顯示在6、9 & 12個月齡的腦溶胞產物中(G ₄C ₂) ₁₄₉小鼠顯示顯著增加poly(GP)可溶部分，與(G ₄C ₂) ₁₄₉對照比較。圖2B及2C顯示隨著小鼠年齡的增長，預計(G ₄C ₂) ₁₄₉小鼠中可溶部分中的DPR會減少(圖2B)，因為它們在不溶部分中積累(圖2C)。 Figures 2A-2D provide the results of pathology assessment of the soluble fraction of DPR proteins in a poly(GP) Meso Scale Discovery (MSD)-immunoassay. C57BL/6J-Tg(C9orf72_i3)112Lutzy/J (JR: 023099) mice show poly(GP) activity in brain lysates at 1 and 3 months of age and in spinal cord lysates at 12 months of age. The soluble fraction was significantly increased compared to NCAR as a control. As mice age, a decrease in DPR in the soluble fraction of the mouse brain is observed in (G ₄ C2)149 mice. Data are expressed as mean ± SD. Poly (GP) response in C9-deficient mice treated with rAAV and vehicle or miRNA-containing rAAV. Figure 2A shows that (G ₄ C ₂ ) ₁₄₉ mice displayed a significant increase in poly(GP) soluble fraction in brain lysates at 6, 9 & 12 months of age compared to (G ₄ C ₂ ) ₁₄₉ controls. Figures 2B and 2C show that as mice age, DPRs in the soluble fraction in (G ₄ C ₂ ) ₁₄₉ mice are expected to decrease (Figure 2B) because they accumulate in the insoluble fraction (Figure 2C).

圖3提供存活曲線，其中對不同組的野生型對照(WT/NCAR)雌性或雄性小鼠或僅接受PBS(VEH)或接受3x10 ¹¹之兩種不同的rAAV其中之一的半合子/TG小鼠在數週至14週齡內繪製存活百分比：AAV-1係具有載體基因體為CB7.CI.C9miR487.WPRE.rBG之AAV PHP.eB衣殼，以及AAV-2係具有載體基因體為CB7.CI.C9miR487.WPRE.rBG之AAV PHP.eB衣殼，在4週齡時經由尾靜脈注射。 Figure 3 provides survival curves for different groups of wild-type control (WT/NCAR) female or male mice that received either PBS alone (VEH) or 3x10 ^hemizygous /TG mice with one of two different rAAVs. Percent survival of mice plotted from weeks to 14 weeks of age: AAV-1 line with AAV PHP.eB capsid with vector genotype CB7.CI.C9miR487.WPRE.rBG, and AAV-2 line with vector genotype CB7. The AAV PHP.eB capsid of CI.C9miR487.WPRE.rBG was injected via the tail vein at 4 weeks of age.

圖4提供來自圖3描述的動物研究的按組別(雄性和雌性一起)的體重，如從開始(4週齡)到終止的研究。Figure 4 provides body weights by group (males and females together) from the animal study described in Figure 3, as from start (4 weeks of age) to termination of the study.

圖5提供來自圖3描述的動物研究的按組別的雌性體重，如從開始(4週齡)到終止的研究。Figure 5 provides female body weights by group from the animal study described in Figure 3, as from the start (4 weeks of age) to the end of the study.

圖6提供來自圖3描述的動物研究的按組別的雄性體重，如從開始(4週齡)到終止的研究。Figure 6 provides male body weights by group from the animal study described in Figure 3, from the start (4 weeks of age) to the end of the study.

圖7A及7B提供野生型小鼠(WT/NCAR媒劑)(第1組)、僅接受PBS(媒劑)的半合子/TG小鼠及接受3x10 ¹¹兩種不同rAAV之一者的兩個治療組的腦中poly(GP)反應：AAV-1係具有載體基因體為CB7.CI. C9miR487.WPRE.rBG之AAV PHP.eB衣殼，及AAV-2係具有載體基因體為CB7.CI.C9miR487.WPRE.rBG 之AAV PHP.eB衣殼；在4週齡時經由尾靜脈注射。圖7A已對背景進行校正且圖7B未對背景進行校正。 Figures 7A and 7B provide wild-type mice (WT/NCAR vehicle) (Group 1), hemizygous/TG mice that received PBS (vehicle) only, and two that received 3x10 ¹¹ of one of two different rAAVs Poly(GP) response in the brain of the treatment group: AAV-1 line has the AAV PHP.eB capsid with the vector gene body CB7.CI. C9miR487.WPRE.rBG, and AAV-2 line has the vector gene body CB7.CI .C9miR487.WPRE.rBG AAV PHP.eB capsid; injected via tail vein at 4 weeks of age. Figure 7A has the background corrected and Figure 7B has not.

實施例4：人類iPSC衍生的運動神經元樣的細胞之脫靶分析此研究評估rAAV轉導人類運動神經元樣細胞後的脫靶基因減弱。運動神經元樣細胞將根據標準規程從誘導的多能幹細胞(iPSC)中分化(Bianchi et al, 2018, Rapid and efficient differentiation of functional motor neurons from human iPSC for neural injury modelling. Stem Cell Res. 2018 Oct;32:126-134. doi：10.1016/j.scr.2018.09.006. Epub 2018 Sep 26. PMID：30278374.)。藉由形態學和膽鹼乙醯轉移酶染色確認表型。以rAAV轉導細胞。對照細胞以rAAV載體處理，該rAAV載體具有與rAAV測試載體相同的衣殼，其中rAAV測試載體不攜帶miRNA或對照細胞將不接受任何處理。收集細胞用於RNA分離和RNA-seq分析。鑑定了被rAAV測試載體下調的轉錄本。對於每個下調的轉錄本，藉由序列同源性鑑定潛在的miRNA標靶序列，並將評估恒河猴中相應標靶序列之間的同源性程度，以便預測與脫靶基因減弱相關的毒性藉由NHP毒理學研究可被預測的可能性。 Example 4: Off-target analysis of human iPSC-derived motor neuron-like cells This study evaluates off-target gene attenuation after rAAV transduction of human motor neuron-like cells. Motor neuron-like cells will be differentiated from induced pluripotent stem cells (iPSCs) according to standard protocols (Bianchi et al, 2018, Rapid and efficient differentiation of functional motor neurons from human iPSCs for neural injury modeling. Stem Cell Res. 2018 Oct; 32:126-134. doi: 10.1016/j.scr.2018.09.006. Epub 2018 Sep 26. PMID: 30278374.). Phenotype was confirmed by morphology and choline acetyltransferase staining. Cells were transduced with rAAV. Control cells are treated with a rAAV vector that has the same capsid as the rAAV test vector, where the rAAV test vector does not carry the miRNA or the control cells will not receive any treatment. Cells were collected for RNA isolation and RNA-seq analysis. Transcripts downregulated by the rAAV test vector were identified. For each downregulated transcript, potential miRNA target sequences are identified through sequence homology, and the degree of homology between corresponding target sequences in rhesus monkeys will be assessed to predict toxicity associated with off-target gene attenuation. Possibilities that can be predicted through NHP toxicology studies.

實施例5：非人類靈長類的毒理學研究在成年恒河猴(大約3-10歲)中進行一項為期90天之符合GLP的安全性研究，以研究ICM投予後rAAV測試載體的毒理學。選擇90天評估期是因為此允許轉基因表現有足夠的時間達到穩定高原。選擇動物的年齡以代表意圖的成年患者群體。下表中概述研究設計。恒河猴接受三種劑量水平的rAAV.C9orf72.MiR (3.00 x 10 ¹²GC總量、1.00 x 10 ¹³GC總量、或3.00 x 10 ¹³GC總量；每劑N=3)或媒劑(ITFFB；N=2)。選擇的劑量水平與計劃的MED中評估的劑量水平相當，當按腦質量縮放時(假設成年小鼠大腦為0.4克，成年恒河猴腦為90克)，此等劑量涵蓋建議的臨床劑量水平範圍。使用相同於用於臨床試驗的載體遞送裝置對NHP進行投劑。在毒理學研究開始之前優化載體遞送裝置和投予程序，以確保可重複和準確的載體遞送。實際投予的載體劑量水平和任何與設備相關的載體損失將在此研究報告中提供。將進行基線神經系統檢查、完整的身體檢查、體重和日常觀察，包括食慾評估、臨床病理學(具有差異的細胞計數、臨床化學和凝血組)、CSF化學和CSF細胞學。在rAAV測試載體或媒劑投予後，每天監測動物的痛苦跡象和異常行為。血液和CSF臨床病理學評估和神經學檢查在rAAV測試載體或媒劑投予後30天每週進行一次，之後每30天進行一次。在基線和此後的每個30天時間點，抗AAV NAb和細胞毒性T淋巴細胞(CTL)對rAAV的反應藉由干擾素γ(IFN-γ)酶聯免疫斑點(ELISpot)試驗進行評估。 Example 5: Toxicology Study in Non-Human Primates A 90-day GLP-compliant safety study was conducted in adult rhesus monkeys (approximately 3-10 years old) to study the rAAV test vector following ICM administration. Toxicology. The 90-day evaluation period was chosen because this allows sufficient time for transgene performance to plateau. The age of the animals was chosen to represent the intended adult patient population. The study design is outlined in the table below. Rhesus monkeys received three dose levels of rAAV.C9orf72.MiR (3.00 x 10 ¹² total GC, 1.00 x 10 ¹³ total GC, or 3.00 x 10 ¹³ total GC; N=3 per dose) or vehicle (ITFFB ;N=2). The dose levels selected are comparable to those evaluated in the planned MED and, when scaled by brain mass (assuming 0.4 g for an adult mouse brain and 90 g for an adult rhesus monkey brain), cover recommended clinical dose levels Scope. NHPs were administered using the same vector delivery device used in clinical trials. Optimize the vector delivery device and administration procedure prior to the start of toxicology studies to ensure reproducible and accurate vector delivery. Actual vector dose levels administered and any device-related vector losses will be provided in this study report. A baseline neurological examination, complete physical examination, weight, and daily observations including appetite assessment, clinical pathology (with differential cell count, clinical chemistry, and coagulation panel), CSF chemistry, and CSF cytology will be performed. Following rAAV test vector or vehicle administration, animals were monitored daily for signs of distress and abnormal behavior. Blood and CSF clinicopathological assessments and neurological examinations were performed weekly for 30 days after rAAV test vector or vehicle administration and every 30 days thereafter. At baseline and at each 30-day time point thereafter, anti-AAV NAb and cytotoxic T lymphocyte (CTL) responses to rAAV were assessed by interferon gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay.

rAAV或媒劑投予後90天，將動物安樂死。收穫了一份完整的組織清單(腦、脊髓、DRG、周圍神經、心臟、肺臟、肝臟、脾臟、腎臟、食道、胃、大腸和小腸、腸系膜和頸部淋巴結、腎上腺、及性腺），適當稱重，並進行組織病理學分析。此外，從肝臟、脾臟和骨髓中採集淋巴細胞，以評估屍檢時此等器官中是否存在對載體衣殼有反應的T細胞。藉由組織樣本中的qPCR評估載體生物分布。載體基因體亦可在血清和CSF樣本中進行量化。藉由分析在尿液和糞便中檢測到的載體基因體來評估載體分泌。Animals were euthanized 90 days after rAAV or vehicle administration. A complete tissue inventory was harvested (brain, spinal cord, DRG, peripheral nerves, heart, lungs, liver, spleen, kidneys, esophagus, stomach, large and small intestines, mesenteric and cervical lymph nodes, adrenal glands, and gonads), appropriately weighed weight and perform histopathological analysis. In addition, lymphocytes were collected from the liver, spleen, and bone marrow to assess the presence of T cells in these organs that responded to the vector capsid at autopsy. Vector biodistribution was assessed by qPCR in tissue samples. Vector genomes can also be quantified in serum and CSF samples. Vector secretion was assessed by analyzing vector genomes detected in urine and feces.

表.符合恒河猴GLP的毒理學研究組名 1 2 3 4 N 2 3 3 3 性別/年齡 M+F M+F M+F M+F 年齡 3–10歲 3–10歲 3–10歲 3–10歲體重 3–12 kg 3–12 kg 3–12 kg 3–12 kg 測試物媒劑 rAAV.C9orf72.MiR rAAV.C9orf72.MiR rAAV.C9orf72.MiR ROA ICM ICM ICM ICM 載體劑量 (總劑量) N/A 3.00 x 10 ¹²GC 1.00 x 10 ¹³GC 3.00 x 10 ¹³GC 屍檢日 90 90 90 90 媒劑將由ITFFB (具0.001% Pluronic F-68的人工CSF)所組成。縮寫：CSF，腦脊髓液； F，女性； GLP，良好實驗室規範； GC，基因體拷貝；ICM，腦大池內；ITFFB，鞘內最終製劑緩衝液；M，男性；N，動物數量；N/A，不適用；ROA，投予途徑。 Table. Rhesus monkey GLP compliant toxicology studies Group name 1 2 3 4 N 2 3 3 3 gender/age M+F M+F M+F M+F age 3–10 years old 3–10 years old 3–10 years old 3–10 years old weight 3–12kg 3–12kg 3–12kg 3–12kg test object medium rAAV.C9orf72.MiR rAAV.C9orf72.MiR rAAV.C9orf72.MiR ROA ICM ICM ICM ICM Vehicle dose (total dose) N/A 3.00 x 10 ¹² GC 1.00 x 10 ^13GC 3.00 x 10 ^13GC autopsy day 90 90 90 90 The vehicle will consist of ITFFB (artificial CSF with 0.001% Pluronic F-68). Abbreviations : CSF, cerebrospinal fluid; F, female; GLP, good laboratory practice; GC, genome copy; ICM, intracisternal; ITFFB, intrathecal final formulation buffer; M, male; N, number of animals; N /A, not applicable; ROA, route of investment.

實施例6：首次人體臨床試驗計畫書概要第1/2期開放標籤、多中心劑量遞增研究以評估遞送至具有肌肉萎縮性脊髓側索硬化症(ALS)的成年受試者的腦大池(ICM)中的單劑量rAAV.C9orf72.miR之安全性及耐受性，該肌肉萎縮性脊髓側索硬化症(ALS)係由 C9orf72基因的第一內含子中致病性六核苷酸重複擴增所引起。受試者數量：多至12個可評估之受試者目的： 主要的安全性：● 通過以下評量，評估rAAV.C9orf72.miR在投予單次ICM劑量後12個月內的安全性及耐受性： ○ 不良事件(AEs)及嚴重不良事件(SAEs) ○ 生命體徵和身體檢查 ○ 神經學檢查 ○ 心電圖(ECG) ○ 感覺神經傳導研究(用於評估DRG毒性) ○ 實驗室評估(血清化學、血液學、凝血研究、肝功能測試[LFTs]、尿液分析及CSF化學和細胞學) ○ 載體(衣殼)之免疫原性 主要功效：● 評估rAAV.C9orf72.miR對治療後12個月內無氣管切開存活率的影響 次要：● 基於以下終點，評估在單次ICM劑量投予後rAAV.C9orf72.MiR在12個月內的藥效學及生物學活性： ○ 在CSF中之DPR (polyGP蛋白質)水平 ○ 在CSF及血清中之神經絲輕鏈(NFL)水平 ● 評估rAAV.C9orf72.MiR在投予單次ICM劑量後12個月的功效，如以下方式測量： ○ 永久換氣時間 ○ ALS功能評定量表(ALSFRS-R) ○ 肺功能，肺計量(SVC) 探測：● 進一步評估rAAV.C9orf72.MiR在投予單次ICM劑量後12個月內的功效，如下方式測量： ○ 肢體等長強度精確測試(ATLIS)試驗 ○ 肌肉萎縮性脊髓側索硬化症-特異性生活質量簡表(ALSSQOL-SF) ● 進一步評估在單次ICM劑量後12個月內rAAV.C9orf72.MiR的藥效學作用，如下方式測量： ○ CSF中的神經絲重鏈(NFH) ○ CSF中的p-Tau：t-Tau比率 ○ 腦MRI上運動皮質區和皮質下結構的腦室體積變化及萎縮 ○ 皮質脊髓束的擴散張量磁共振影像(Diffusion-tensor magnetic resonance imaging (DT-MRI)) 研究設計：此為在具有ALS的成年受試者中藉由單次ICM注射投予的rAAV.C9orf72.MiR之第1/2期、FIH、多中心、開放標籤、單臂、劑量遞增研究，ALS係由 C9orf72基因的第一內含子中致病性六核苷酸重複擴增所引起。將在12個月內評估安全性及耐受性、藥效學、及臨床功效，並在rAAV.C9orf72.MiR投予後5年內對所有受試者進行追蹤，以長期評估安全性及耐受性、藥效學、疾病進展、及臨床結果。此試驗將由一個篩選階段所組成，以確定從大約第–35天到第–1天，群組1和群組2中每個潛在受試者的資格。確認合格後，受試者將接受基線評估，包括腦部MRI、CSF收集的LP、抽血、尿液收集、生命體徵(vitals)、EGG、身體檢查及臨床評估。基線評估將在第–1天和第0天進行，資格將在投予rAAV.C9Oorf72.MiR之前的基線處重新確認。加入群組3的受試者將從大約第–120天到第–90天經歷上述相同的篩選評估，然後將進入90天的觀察期，在此期間將評量疾病表現。在預治療階段結束時，將評估疾病進展，進展迅速的受試者將接受rAAV.C9orf72.MiR。疾病進展緩慢的受試者將不會繼續參加試驗。在治療期期間，受試者將在第0天早上入院。受試者將在第0天接受單次ICM劑量的rAAV.C9orf72.MiR，並在投劑後至少留院24小時進行觀察。隨後的研究訪視將在治療後第7天、第14天、第30天、3個月、6個月、9個月和12個月進行，以及LTFU訪視將在投劑後18個月、24個月和每年至5年進行。此試驗將由以下三個作為單次ICM注射投予的rAAV.C9orf72.MiR之群組所組成： ● 群組1 (低劑量)：三名符合條件的受試者(受試者#1-3)將依次編入群組中並投予低劑量的rAAV.C9orf72.MiR，每個受試者間有4週的安全觀察期。若未觀察到SRT，則在群組1中的第三名受試者投予rAAV.C9orf72.MiR後4週，獨立的安全委員會將評估所有可用的安全數據。 ● 群組2 (高劑量)：若決定繼續進行，將依次編入三名符合條件的受試者(受試者#4–6)並給予高劑量的rAAV.C9orf72.MiR，每個受試者間有4週的安全觀察期。若未觀察到SRT，安全委員會將在受試者#6投劑後4週評估所有可用的安全性數據，包括來自群組1受試者的安全性數據。 ● 群組3 (MTD)：在安全委員會提出積極建議之前，將編入大約6名被定義為快速進展者(受試者#7–12)的符合條件的受試者，並以MTD投予單次ICM劑量的rAAV.C9orf72.MiR。此群組中受試者的投劑不會與每個受試者之間的4週安全觀察期錯開，且在沒有SRTs的情況下，於此群組不需要安全委員會審查。基於已發布的存活及生物標記物數據的建模以及預期的效果大小，可在最終方案中進一步調整建議的樣本大小。累計地，我們預期在高劑量或低劑量群組任一者有9名受試者入組，而總共有12名受試者(跨所有劑量)。納入標準群組1及群組2： ● ≥25歲且≤75歲的男性及女性 ● 基於世界神經病學聯合會修訂標準的明確、很可能或臨床可能的ALS診斷 ● C9orf72GGGGCC致病的重複擴增＞36次重複 ● ECAS總分數≥105 ● 在篩選訪視後18個月內出現ALS疾病症狀，包括四肢無力 ● 用力肺活量(FVC)測量值≥性別、身高和年齡預測值的70% ● 未使用過利魯唑，或者，若有使用利魯唑，在篩選訪視前至少30天使用穩定劑量的利魯唑，並願意在試驗期間維持利魯唑劑量。群組3： ● 必須符合上述所有納入標準才有資格編入預處理階段。 ● 在90天治療前導入期(run-in phase)結束時，必須確認上述資格標準，並且受試者必須在90天導入期期間經歷ALSFRS-R總分下降3分或以上。排除標準 ● 永久性氣管切開術或換氣 ● 被診斷患有ALS-FTD或ALS並伴有認知障礙的患者 ● 任何肢體的Ashworth改良評分≥4 ● 無法提供完全同意或缺乏充分接觸之可以提供同意的具合法授權的看護者 ● MRI、ICM遞送或LP的禁忌症(例如，局部感染、血小板減少症、凝血病、顱內壓升高(由於佔位性病灶(space occupying lesion)、螢光影像的禁忌症的[ICP]) ● 免疫功能不全的患者 ● 具有人類免疫缺陷病毒(HIV)或者C型肝炎的陽性檢測結果的患者 ● 非由於 C9orf72重複擴增所引起的惡性腫瘤或慢性中樞神經系統疾病 ● 研究者的意見認為可能對患者構成風險的藥物，諸如免疫抑制藥物或全身性皮質類固醇。非類固醇類抗炎藥(NSAID)若在篩選前30天以穩定劑量使用並且同意在試驗期間保持相同劑量，則可接受 ● 研究者的意見認為可能導致與C9orf72重複擴增無關的認知障礙的任何併發疾病，包括神經梅毒、腦積水、中風、小血管缺血性疾病、不受控制的甲狀腺功能減退症或維生素缺乏症 ● 於有生育能力的女性，在篩選訪視時通過血清妊娠試驗證實為陽性尿液，在投予研究產品前第1天通過血清結果證實為陽性尿液，或不願在試驗期間進行額外的妊娠試驗。 ● 於有生育潛力的男性和女性，不願使用醫學上可接受的雙重屏障避孕方法(諸如與殺***劑一起使用的避孕套/隔膜)或從篩選之日到載體投予後12個月期間禁慾 ● 在篩選前30天內或在該臨床試驗中使用的研究產品的5個半衰期內，以較長者為準，以研究產品編入任何其他臨床試驗 ● 先前的基因治療 ● 編入30天內需要住院的任何急性疾病 ● 任何當前或先前的病況、身體檢查或實驗室試驗發現，研究者認為，此等發現會使受試者處於不適當的風險或會干擾研究產品的評估或受試者安全性或研究結果的解釋研究產品 rAAV.C9orf72.MiR 投予途徑及程序 rAAV.C9orf72.MiR將在第0天通過實時CT引導下枕下注射到腦大池，以作為單劑投予給住院受試者。在第0天，與研究相關的研究藥房將準備含有適當力價的5.6 mL rAAV.C9orf72.MiR的注射器，並將其運送到手術室。在研究藥物投予之前，受試者將被麻醉、插管，準備注射部位並使用無菌技術覆蓋。將進行LP以移除預定體積的CSF，之後將IT注射碘造影劑以幫助可視化腦大池的相關解剖結構。IV造影劑可在針***之前或期間作為IT造影劑的替代方法進行投予。使用IV或IT造影劑的決定由執行該程序的介入醫生自行決定。在CT螢光鏡引導下，將一根脊椎穿刺針(22–25 G)送入腦大池。可使用更大的穿刺針來協助針的放置。確認穿刺針放置後，將擴充組連接到脊髓穿刺針上並注入CSF。根據介入醫生的判斷，可以將含有造影劑的注射器連接到延長裝置並注射少量以確認穿刺針放置在腦大池中。確認穿刺針放置後，將含有rAAV.C9orf72.MiR的注射器連接到擴充組。注射器內容物將在1-2分鐘內緩慢注射，遞送5.0 mL的體積。安全性評估在研究計劃中指定的時間進行安全性評估，包括AEs及SAEs的收集、身體和神經學檢查、生命體徵、臨床實驗室測試（血清化學、血液學、凝血、LFT、尿液分析）、ECG、神經傳導研究、以及CSF細胞學和化學(細胞計數、蛋白質、葡萄糖)。 Example 6: Summary of First-in-Human Clinical Trial Proposal Phase 1/2 open-label, multicenter dose-escalation study to evaluate a single dose of rAAV.C9orf72.miR delivered to the cistern magna (ICM) of adult subjects with amyotrophic lateral sclerosis (ALS) Safety and Tolerability Amyotrophic lateral sclerosis (ALS) is caused by a pathogenic hexanucleotide repeat expansion in the first intron of the C9orf72 gene. Number of subjects: Up to 12 evaluable subjects Purpose: Main safety: ● Evaluate the safety and tolerability of rAAV.C9orf72.miR within 12 months after a single ICM dose through the following assessments: ○ Adverse events (AEs) and serious adverse events (SAEs) ○ Vital signs and physical examination ○ Neurologic examination ○ Electrocardiogram (ECG) ○ Sensory nerve conduction studies (for assessment of DRG toxicity) ○ Laboratory evaluation (serum chemistry, hematology, coagulation studies, liver function tests [LFTs], urine Analysis and CSF Chemistry and Cytology) ○ Immunogenicity of the vector (capsid) Primary Efficacy: ● Evaluate the impact of rAAV.C9orf72.miR on tracheostomy-free survival 12 months after treatment Secondary: ● Based on the following endpoints, To evaluate the pharmacodynamics and biological activity of rAAV.C9orf72.MiR over 12 months after a single ICM dose administration: ○ DPR (polyGP protein) levels in CSF ○ Neurofilament light chain ( NFL) levels● To assess the efficacy of rAAV.C9orf72.MiR 12 months after a single ICM dose, as measured by: ○ Permanent ventilation time ○ ALS Functional Rating Scale (ALSFRS-R) ○ Lung function, lung Measurement (SVC) Detection: ● To further evaluate the efficacy of rAAV.C9orf72.MiR over 12 months after a single ICM dose, as measured by: ○ Precision Test of Limb Isometric Strength (ATLIS) test ○ Muscular Atrophic Spinal Cord Thoracic Sclerosis-Specific Quality of Life Short Form (ALSSQOL-SF) ● To further evaluate the pharmacodynamic effects of rAAV.C9orf72.MiR 12 months after a single ICM dose, as measured by: ○ Neurofilament weight in CSF chain (NFH) ○ p-Tau:t-Tau ratio in CSF ○ Ventricular volume changes and atrophy of motor cortex areas and subcortical structures on brain MRI ○ Diffusion-tensor magnetic resonance imaging of corticospinal tracts imaging (DT-MRI)) Research design: This is a Phase 1/2, FIH, multicenter, open-label, single-arm, dose-escalation study of rAAV.C9orf72.MiR administered via a single ICM injection in adult subjects with ALS administered by It is caused by a pathogenic hexanucleotide repeat expansion in the first intron of the C9orf72 gene. Safety and tolerability, pharmacodynamics, and clinical efficacy will be assessed within 12 months, and all subjects will be followed for 5 years after rAAV.C9orf72.MiR administration to assess long-term safety and tolerability properties, pharmacodynamics, disease progression, and clinical outcomes. The trial will consist of a screening phase to determine eligibility for each potential subject in Cohorts 1 and 2 from approximately Day –35 to Day –1. After confirmation of eligibility, subjects will undergo a baseline assessment, including brain MRI, LP of CSF collection, blood draw, urine collection, vital signs, EGG, physical examination and clinical evaluation. Baseline assessments will be conducted on Days –1 and 0, and eligibility will be reaffirmed at baseline prior to administration of rAAV.C9Oorf72.MiR. Subjects enrolled in Cohort 3 will undergo the same screening assessments described above from approximately Day –120 to –90 and will then enter a 90-day observation period during which disease manifestations will be assessed. At the end of the pre-treatment phase, disease progression will be assessed and subjects with rapid progression will receive rAAV.C9orf72.MiR. Subjects whose disease progresses slowly will not continue in the trial. During the treatment period, subjects will be admitted to the hospital on the morning of Day 0. Subjects will receive a single ICM dose of rAAV.C9orf72.MiR on Day 0 and remain in the hospital for observation for at least 24 hours after dosing. Subsequent study visits will be at 7, 14, 30, 3, 6, 9, and 12 months post-treatment, and the LTFU visit will be at 18 months post-dose , 24 months and every year to 5 years. This trial will consist of the following three cohorts of rAAV.C9orf72.MiR administered as a single ICM injection: ● Cohort 1 (low dose): three eligible subjects (Subjects #1-3 ) will be sequentially organized into groups and administered low doses of rAAV.C9orf72.MiR, with a 4-week safety observation period between each subject. If no SRT is observed, an independent safety committee will evaluate all available safety data 4 weeks after administration of rAAV.C9orf72.MiR to the third subject in Cohort 1. ● Cohort 2 (High Dose): If the decision is made to proceed, three eligible subjects (Subjects #4–6) will be enrolled in sequence and given high doses of rAAV.C9orf72.MiR, each subject There is a 4-week safety observation period. If no SRT is observed, the Safety Committee will evaluate all available safety data, including safety data from Cohort 1 subjects, 4 weeks after subject #6 dosing. ● Cohort 3 (MTD): Approximately 6 eligible subjects, defined as rapid progressors (Subjects #7–12), will be enrolled and administered a single MTD dose prior to a positive safety committee recommendation. Sub-ICM doses of rAAV.C9orf72.MiR. Dosing of subjects in this cohort will not be staggered by the 4-week safety observation period between each subject, and in the absence of SRTs, no safety committee review is required for this cohort. The recommended sample size can be further adjusted in the final protocol based on modeling of published survival and biomarker data and expected effect sizes. Cumulatively, we expect to enroll 9 subjects in either the high-dose or low-dose cohorts, for a total of 12 subjects (across all doses). inclusion criteria Cohort 1 and Cohort 2: ● Men and women aged ≥ 25 years and ≤ 75 years ● Definite, probable or clinically possible diagnosis of ALS based on the revised criteria of the World Federation of Neurology ● C9orf72 GGGGCC pathogenic repeat expansion> 36 repeats ● Total ECAS score ≥ 105 ● ALS disease symptoms, including limb weakness, within 18 months of the screening visit ● Forced vital capacity (FVC) measurement ≥ 70% of predicted for gender, height, and age ● Not used riluzole, or, if riluzole is used, be on a stable dose of riluzole for at least 30 days before the screening visit and be willing to maintain the riluzole dose for the duration of the trial. Group 3: ● Must meet all the above inclusion criteria to be eligible for inclusion in the pre-processing stage. ● At the end of the 90-day pre-treatment run-in phase, the above eligibility criteria must be confirmed, and subjects must experience a 3-point or greater decrease in ALSFRS-R total score during the 90-day run-in phase. Exclusion criteria ● Permanent tracheostomy or ventilation ● Patients diagnosed with ALS-FTD or ALS with cognitive impairment ● Ashworth Modified Score ≥ 4 in any extremity ● Inability to provide full consent or lack of adequate contact with the means to provide consent Legally authorized caregiver ● Contraindications to MRI, ICM delivery, or LP (e.g., local infection, thrombocytopenia, coagulopathy, elevated intracranial pressure due to space occupying lesion, contraindications to fluoroscopic imaging [ICP]) ● Immunocompromised patients ● Patients with positive test results for human immunodeficiency virus (HIV) or hepatitis C ● Malignant tumors or chronic central nervous system diseases not caused by C9orf72 repeat expansion ● Drugs that, in the opinion of the investigator, may pose a risk to the patient, such as immunosuppressive drugs or systemic corticosteroids. Nonsteroidal anti-inflammatory drugs (NSAIDs) if used at a stable dose 30 days before screening and agree to maintain the same dose during the trial , is acceptable ● Any concurrent disease that, in the opinion of the investigator, may cause cognitive impairment unrelated to the C9orf72 repeat expansion, including neurosyphilis, hydrocephalus, stroke, small vessel ischemic disease, and uncontrolled hypothyroidism or vitamin deficiency ● In women of childbearing potential who have a positive urine test confirmed by a serum pregnancy test at the screening visit, a positive urine test confirmed by a serum pregnancy test on day 1 prior to administration of study product, or who are unwilling to participate in the trial An additional pregnancy test during the period. ● In men and women of childbearing potential who are unwilling to use a medically acceptable double barrier method of contraception (such as condoms/diaphragms used with spermicide) or who are carriers from the date of screening Abstinence during the 12-month period after administration ● Prior gene therapy ● During the 30 days prior to screening or the 5 half-lives of the investigational product used in that clinical trial, whichever is longer, with the investigational product enrolled in any other clinical trial ● Code any acute illness requiring hospitalization within 30 days ● Any current or previous medical condition, physical examination, or laboratory test finding that, in the opinion of the investigator, would place the subject at inappropriate risk or interfere with the evaluation of the investigational product or subject safety or interpretation of study results. research products rAAV.C9orf72.MiR Investment methods and procedures rAAV.C9orf72.MiR will be administered as a single dose to hospitalized subjects on day 0 via real-time CT-guided suboccipital injection into the cistern magna. On Day 0, syringes containing 5.6 mL of rAAV.C9orf72.MiR at the appropriate dose will be prepared by the study-associated research pharmacy and transported to the operating room. Prior to study drug administration, subjects will be anesthetized, intubated, and the injection site prepared and covered using sterile technique. LP will be performed to remove a predetermined volume of CSF, followed by IT to inject iodinated contrast agent to help visualize the relevant anatomy of the cistern magna. IV contrast media can be administered before or during needle insertion as an alternative to IT contrast media. The decision to use IV or IT contrast media is at the discretion of the interventionalist performing the procedure. Under CT fluoroscopic guidance, a spinal puncture needle (22–25 G) was advanced into the cistern. A larger needle can be used to assist with needle placement. After confirming needle placement, connect the expansion set to the spinal needle and inject CSF. At the discretion of the interventionalist, a syringe containing contrast material can be attached to the extension device and a small amount injected to confirm needle placement in the cistern. After confirming needle placement, connect the syringe containing rAAV.C9orf72.MiR to the expansion set. The contents of the syringe will be injected slowly over 1-2 minutes, delivering a volume of 5.0 mL. safety assessment Safety assessments will be conducted at the times specified in the study plan, including collection of AEs and SAEs, physical and neurological examinations, vital signs, clinical laboratory tests (serum chemistry, hematology, coagulation, LFT, urinalysis), ECG, Nerve conduction studies, and CSF cytology and chemistry (cell count, protein, glucose).

本說明書中列出的所有專利、專利公開案及其他出版物，如2022年1月10日申請之US臨時專利申請案第63/298,046號，以及其序列表，藉由引用併入本文。儘管已經參考特定較佳具體實施例描述本發明，但應當理解，可於不脫離本發明的精神的情況下進行修改。此種修改意圖落入所附申請專利權利的範疇內。All patents, patent publications and other publications listed in this specification, such as US Provisional Patent Application No. 63/298,046 filed on January 10, 2022, and its sequence listing, are incorporated herein by reference. Although the invention has been described with reference to certain preferred embodiments, it will be understood that modifications may be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended patent application.

無without

圖1A至1D提供來自11-14週齡小鼠(C9 L112 Het)脊髓的qPCR結果，該小鼠注射(靜脈內-尾靜脈) 3 x 10 ¹¹GC/100 µl之rAAV-PHP.eb-CB7.CI.C9miR.WPRE.rBG，miR為NT或PBS、miR487、miR32、或miR32-101。圖1A提供C9內含子剪接的引子在脊髓的結果。圖1B提供C9內含子保留的引子在脊髓的結果。圖1C及1D提供來自腦的C9內含子剪接的引子(圖1C)或C9內含子保留的引子(圖1D)的qPCR結果。圖2A-2D提供在poly(GP) Meso Scale Discovery (MSD)-免疫分析中可溶部分DPR蛋白質病理學評估的結果。C57BL/6J-Tg(C9orf72_i3)112Lutzy/J (JR：023099)小鼠顯示在1及3個月齡時在腦溶胞產物及在12個月齡時在脊髓溶胞產物中poly(GP)可溶部分顯著增加，與作為對照之NCAR相比。隨著小鼠年齡的增長，在(G ₄C ₂)149小鼠中觀察到小鼠腦中可溶性部分的DPR減少。數據表示為平均值±SD。以rAAV和媒劑或包含miRNA的rAAV處理C9缺陷小鼠中的poly (GP)反應。圖2A顯示在6、9 & 12個月齡的腦溶胞產物中(G ₄C ₂) ₁₄₉小鼠顯示顯著增加poly(GP)可溶部分，與(G ₄C ₂) ₁₄₉對照比較。圖2B及2C顯示隨著小鼠年齡的增長，預計(G ₄C ₂) ₁₄₉小鼠中可溶部分中的DPR會減少(圖2B)，因為它們在不溶部分中積累(圖2C)。圖3提供存活曲線，其中對不同組的野生型對照(WT/NCAR)雌性或雄性小鼠或僅接受PBS(VEH)或接受3x10 ¹¹之兩種不同的rAAV其中之一的半合子/TG小鼠在數週至14週齡內繪製存活百分比：AAV-1係具有載體基因體為CB7.CI.C9miR487.WPRE.rBG之AAV PHP.eB衣殼，以及AAV-2係具有載體基因體為CB7.CI.C9miR487.WPRE.rBG之AAV PHP.eB衣殼，在4週齡時經由尾靜脈注射。圖4提供來自圖3描述的動物研究的按組別(雄性和雌性一起)的體重，如從開始(4週齡)到終止的研究。圖5提供來自圖3描述的動物研究的按組別的雌性體重，如從開始(4週齡)到終止的研究。圖6提供來自圖3描述的動物研究的按組別的雄性體重，如從開始(4週齡)到終止的研究。圖7A及7B提供野生型小鼠(WT/NCAR媒劑)(第1組)、僅接受PBS(媒劑)的半合子/TG小鼠及接受3x10 ¹¹兩種不同rAAV之一者的兩個治療組的腦中poly(GP)反應：AAV-1係具有載體基因體為CB7.CI.C9miR487.WPRE.rBG之AAV PHP.eB衣殼，及AAV-2係具有載體基因體為CB7.CI.C9miR487.WPRE.rBG 之AAV PHP.eB衣殼；在4週齡時經由尾靜脈注射。圖7A已對背景進行校正且圖7B未對背景進行校正。 Figures 1A to 1D provide qPCR results from spinal cords from 11-14 week old mice (C9 L112 Het) injected (i.v. - tail vein) with 3 x 10 ¹¹ GC/100 µl of rAAV-PHP.eb-CB7 .CI.C9miR.WPRE.rBG, the miR is NT or PBS, miR487, miR32, or miR32-101. Figure 1A provides the results of C9 intron splicing primers in the spinal cord. Figure 1B provides the results of the C9 intron-retaining primer in the spinal cord. Figures 1C and ID provide qPCR results for C9 intron spliced primers (Figure 1C) or C9 intron retained primers (Figure 1D) from brain. Figures 2A-2D provide the results of pathology assessment of the soluble fraction of DPR proteins in a poly(GP) Meso Scale Discovery (MSD)-immunoassay. C57BL/6J-Tg(C9orf72_i3)112Lutzy/J (JR: 023099) mice show poly(GP) activity in brain lysates at 1 and 3 months of age and in spinal cord lysates at 12 months of age. The soluble fraction was significantly increased compared to NCAR as a control. As mice age, a decrease in DPR in the soluble fraction of the mouse brain is observed in (G ₄ C ₂ )149 mice. Data are expressed as mean ± SD. Poly (GP) response in C9-deficient mice treated with rAAV and vehicle or miRNA-containing rAAV. Figure 2A shows that (G ₄ C ₂ ) ₁₄₉ mice displayed a significant increase in poly(GP) soluble fraction in brain lysates at 6, 9 & 12 months of age compared to (G ₄ C ₂ ) ₁₄₉ controls. Figures 2B and 2C show that as mice age, DPRs in the soluble fraction in (G ₄ C ₂ ) ₁₄₉ mice are expected to decrease (Figure 2B) because they accumulate in the insoluble fraction (Figure 2C). Figure 3 provides survival curves for different groups of wild-type control (WT/NCAR) female or male mice that received either PBS alone (VEH) or 3x10 ^hemizygous /TG mice with one of two different rAAVs. Percent survival of mice plotted from weeks to 14 weeks of age: AAV-1 line with AAV PHP.eB capsid with vector genotype CB7.CI.C9miR487.WPRE.rBG, and AAV-2 line with vector genotype CB7. The AAV PHP.eB capsid of CI.C9miR487.WPRE.rBG was injected via the tail vein at 4 weeks of age. Figure 4 provides body weights by group (males and females together) from the animal study described in Figure 3, as from start (4 weeks of age) to termination of the study. Figure 5 provides female body weights by group from the animal study described in Figure 3, as from the start (4 weeks of age) to the end of the study. Figure 6 provides male body weights by group from the animal study described in Figure 3, from the start (4 weeks of age) to the end of the study. Figures 7A and 7B provide wild-type mice (WT/NCAR vehicle) (Group 1), hemizygous/TG mice that received PBS (vehicle) only, and two that received 3x10 ¹¹ of one of two different rAAVs Poly(GP) response in the brain of the treatment group: AAV-1 line has the AAV PHP.eB capsid with the vector gene body CB7.CI.C9miR487.WPRE.rBG, and AAV-2 line has the vector gene body CB7.CI .C9miR487.WPRE.rBG AAV PHP.eB capsid; injected via tail vein at 4 weeks of age. Figure 7A has the background corrected and Figure 7B has not.

TW202340467A_112101016_SEQL.xmlTW202340467A_112101016_SEQL.xml

無。without.

Claims

一種重組腺相關病毒(rAAV)，其包含AAV衣殼及包裝於其中的載體基因體，其中該載體基因體包含： (a)編碼功能性人類C9蛋白質之工程化人類C9orf72編碼序列； (b)位於(a)與(c)之間的間隔子序列； (c)至少一個miRNA編碼序列，該miRNA編碼序列對C9orf72患者中的內源性人類c9orf72核酸序列的靶位點具有特異性；其中(a)之該工程化核酸序列缺少(c)之至少一個miRNA的靶位點，因而防止至少一個miRNA靶向該工程化c9orf72編碼序列；及 (d)可操作連接至(a)和(c)之調節序列，該調節序列指導其在細胞中的表現。 A recombinant adeno-associated virus (rAAV), which includes an AAV capsid and a vector genome packaged therein, wherein the vector genome includes: (a) An engineered human C9orf72 coding sequence encoding a functional human C9 protein; (b) The spacer sequence located between (a) and (c); (c) at least one miRNA coding sequence specific for a target site of an endogenous human c9orf72 nucleic acid sequence in a C9orf72 patient; Wherein (a) the engineered nucleic acid sequence lacks the target site of at least one miRNA of (c), thereby preventing at least one miRNA from targeting the engineered c9orf72 coding sequence; and (d) is operably linked to a regulatory sequence of (a) and (c) that directs its expression in the cell.

如請求項1之rAAV，其中該AAV衣殼係選自AAVhu68、AAVrh91、AAV9、AAVhu95、AAVhu96、或AAV1衣殼。Such as the rAAV of claim 1, wherein the AAV capsid is selected from AAVhu68, AAVrh91, AAV9, AAVhu95, AAVhu96, or AAV1 capsid.

如請求項1或2之rAAV，其中該工程化C9ORF72編碼序列具有SEQ ID NO：13之核酸序列或與其至少80%相同的序列。The rAAV of claim 1 or 2, wherein the engineered C9ORF72 coding sequence has the nucleic acid sequence of SEQ ID NO: 13 or a sequence that is at least 80% identical to it.

如請求項1至3中任一項之rAAV，其中該工程化C9orf72編碼序列具有SEQ ID NO：13之核酸序列或與其至少約90%相同的序列。The rAAV of any one of claims 1 to 3, wherein the engineered C9orf72 coding sequence has the nucleic acid sequence of SEQ ID NO: 13 or a sequence that is at least about 90% identical to it.

如請求項1至4中任一項之rAAV，其中該至少一個miRNA包含一或多個miRNA靶向序列之序列，其包含5’側翼區、至少SEQ ID NO：15 (miR487)或與SEQ ID NO：15至少99%相同的序列、及3’側翼區，其中該至少一個miRNA不結合至(a)之該工程化C9orf72編碼序列或其編碼的傳訊RNA(mRNA)。The rAAV of any one of claims 1 to 4, wherein the at least one miRNA includes a sequence of one or more miRNA targeting sequences, which includes a 5' flanking region, at least SEQ ID NO: 15 (miR487) or is identical to SEQ ID NO: 15 (miR487). NO: 15 is at least 99% identical to the sequence, and the 3' flanking region, wherein the at least one miRNA does not bind to the engineered C9orf72 coding sequence of (a) or its encoded signaling RNA (mRNA).

如請求項5之rAAV，其中該5’側翼選自SEQ ID NO：5或SEQ ID NO：22之序列。The rAAV of claim 5, wherein the 5' flank is selected from the sequence of SEQ ID NO: 5 or SEQ ID NO: 22.

如請求項1至6中任一項之rAAV，其中該間隔子的長度為75個核苷酸至約250個核苷酸。The rAAV of any one of claims 1 to 6, wherein the spacer has a length of 75 nucleotides to about 250 nucleotides.

如請求項1至7中任一項之rAAV，其中該至少一個miRNA編碼序列係在該工程化C9orf72編碼序列的3’側。The rAAV of any one of claims 1 to 7, wherein the at least one miRNA coding sequence is on the 3' side of the engineered C9orf72 coding sequence.

如請求項1至7中任一項之rAAV，其中該至少一個miRNA編碼序列係位於內含子序列中。The rAAV of any one of claims 1 to 7, wherein the at least one miRNA coding sequence is located in an intron sequence.

如請求項1至9中任一項之rAAV，其中該至少一個miRNA編碼序列進一步包含一個或多於一個的miRNA編碼序列。The rAAV of any one of claims 1 to 9, wherein the at least one miRNA coding sequence further includes one or more than one miRNA coding sequence.

如請求項1至10中任一項之rAAV，其中在該載體基因體中的該調節序列進一步包含組成型啟動子(constitutive promoter)。The rAAV of any one of claims 1 to 10, wherein the regulatory sequence in the vector genome further includes a constitutive promoter.

如請求項11之rAAV，其中該啟動子為CB7啟動子，該CB7啟動子包含細胞巨大病毒(cytomegalovirus)立即早期強化子及雞β-肌動蛋白啟動子。Such as the rAAV of claim 11, wherein the promoter is a CB7 promoter, and the CB7 promoter includes a cytomegalovirus immediate early enhancer and a chicken β-actin promoter.

如請求項1至10中任一項之rAAV，其中該調節序列包含神經元特異性啟動子。The rAAV of any one of claims 1 to 10, wherein the regulatory sequence includes a neuron-specific promoter.

一種醫藥組成物，其包含如請求項1至13中任一項之rAAV、及醫藥上可接受的水性懸浮液、賦形劑、及/或稀釋劑。A pharmaceutical composition comprising the rAAV of any one of claims 1 to 13, and a pharmaceutically acceptable aqueous suspension, excipient, and/or diluent.

一種適合治療具有C9orf72相關病症的患者之如請求項1至13中任一項之重組AAV或如請求項14之組成物。A recombinant AAV according to any one of claims 1 to 13 or a composition according to claim 14 suitable for treating patients with C9orf72-related disorders.

一種於製造用於治療具有C9orf72相關病症的患者的藥物中使用的如請求項1至12中任一項之重組AAV。A recombinant AAV according to any one of claims 1 to 12 for use in the manufacture of a medicament for the treatment of patients with a C9orf72-related disorder.