TW201936201A - Non-viral production and delivery of genes - Google Patents

Non-viral production and delivery of genes Download PDF

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TW201936201A
TW201936201A TW107145131A TW107145131A TW201936201A TW 201936201 A TW201936201 A TW 201936201A TW 107145131 A TW107145131 A TW 107145131A TW 107145131 A TW107145131 A TW 107145131A TW 201936201 A TW201936201 A TW 201936201A
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dna
virus
sequence
cdsdna
certain embodiments
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珊希爾 拉姆
喬爾 施耐德
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美商堅固生物科技公司
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/20—Pseudochromosomes, minichrosomosomes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2820/00—Vectors comprising a special origin of replication system
    • C12N2820/60—Vectors comprising a special origin of replication system from viruses
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention described herein pertains to the production of closed-end double-stranded DNA (cdsDNA) that can be easily amplified and produced in large quantity, for use in, for example, therapeutic use, such as gene therapy.

Description

基因之非病毒生產及遞送Non-viral production and delivery of genes

用於諸如基因療法之目的的基因遞送至靶細胞為眾所周知的，特別是用於治療諸如囊腫性纖維化及某些癌症之疾病。該術語包括將任何核酸材料(諸如基因或基因的一部分)遞送或引入靶細胞以糾正一些基因缺陷，以及基因疫苗接種及在適合之宿主細胞中活體外生產商業上有用之蛋白質。Delivery of genes to target cells for purposes such as gene therapy is well known, particularly for the treatment of diseases such as cystic fibrosis and certain cancers. The term includes the delivery or introduction of any nucleic acid material (such as a gene or a portion of a gene) into a target cell to correct some genetic defects, as well as gene vaccination and in vitro production of a commercially useful protein in a suitable host cell.

細胞遞送系統通常分為三大類，亦即涉及直接注射裸DNA或RNA之細胞遞送系統、利用病毒或經基因修飾之病毒之細胞遞送系統以及利用非病毒遞送劑之細胞遞送系統。儘管作為遞送劑之病毒具有高效率及高細胞選擇性之優點，但其亦具有毒性、產生發炎反應及難以處理大DNA片段之缺點。Cell delivery systems are generally divided into three broad categories, namely cell delivery systems involving direct injection of naked DNA or RNA, cell delivery systems utilizing viral or genetically modified viruses, and cell delivery systems utilizing non-viral delivery agents. Although the virus as a delivery agent has the advantages of high efficiency and high cell selectivity, it also has the disadvantages of toxicity, inflammatory reaction, and difficulty in handling large DNA fragments.

因此，需要提供經改良之細胞遞送系統，其包括任何所關注之DNA片段的非病毒遞送。Accordingly, there is a need to provide improved cell delivery systems that include non-viral delivery of any DNA fragment of interest.

本發明之一個態樣提供一種DNA構築體，其包含：(1)骨架序列，其包含支持在真核(例如哺乳動物)或原核細胞中自我複製之序列；(2)嵌段，其包含：(a)所關注之DNA片段；(b)側接所關注之DNA片段的一對末端序列，其中該等末端序列為反向末端重複序列(ITR)、長末端重複序列(LTR)或內部重複序列或端粒序列；及(c)側接ITR或LTR對之一對原核端粒酶(protelomerase)識別序列。在某些實施例中，嵌段或所關注之DNA片段經組態以能夠在真核細胞之整個生命週期中染色體外存在。One aspect of the invention provides a DNA construct comprising: (1) a backbone sequence comprising a sequence that supports self-replication in a eukaryotic (eg, mammalian) or prokaryotic cell; (2) a block comprising: (a) a DNA fragment of interest; (b) a pair of terminal sequences flanked by the DNA fragment of interest, wherein the terminal sequences are inverted terminal repeats (ITRs), long terminal repeats (LTRs) or internal repeats a sequence or telomere sequence; and (c) a pair of ITR or LTR pairs to a protelomerase recognition sequence. In certain embodiments, the block or DNA fragment of interest is configured to be capable of extrachromosomally throughout the life cycle of a eukaryotic cell.

在某些實施例中，ITR來自雙股DNA病毒，諸如AAV。在某些實施例中，AAV為AAV1-AAV10中之任一者。In certain embodiments, the ITR is from a double-stranded DNA virus, such as AAV. In certain embodiments, the AAV is any one of AAV1-AAV10.

在某些實施例中，LTR來自DNA病毒，諸如HSV。In certain embodiments, the LTR is from a DNA virus, such as HSV.

在某些實施例中，原核端粒酶識別序列中之至少一者包含至少14 bp長度之完美反向重複DNA序列或其變體。In certain embodiments, at least one of the prokaryotic telomerase recognition sequences comprises a perfect inverted repeat DNA sequence of at least 14 bp in length or a variant thereof.

在某些實施例中，原核端粒酶識別序列中之至少一者包含嗜溫性噬菌體完美反向重複序列之22 bp共同序列。In certain embodiments, at least one of the prokaryotic telomerase recognition sequences comprises a 22 bp common sequence of a mesophilic phage perfect inverted repeat.

在某些實施例中，原核端粒酶識別序列中之至少一者來自大腸桿菌噬菌體N15 (諸如由大腸桿菌N15 TelN原核端粒酶識別之大腸桿菌噬菌體)、土壤桿菌克雷伯氏菌(Klebsiella )噬菌體Phi KO2、耶爾森氏菌(Yersinia )噬菌體PY54、鹽單胞菌(Halomonas )噬菌體phiHAP-1及弧菌(Vibrio )噬菌體VP882，或伯氏疏螺旋體(Borrelia burgdorferi )。In certain embodiments, at least one of the prokaryotic telomerase recognition sequences is from E. coli bacteriophage N15 (such as E. coli phage recognized by E. coli N15 TelN protel telomerase), Klebsiella ( Klebsiella) ) phage Phi KO2, Yersinia (Yersinia) phage PY54, Halomonas (of Halomonas) phage phiHAP-1 and Vibrio (Vibrio) phage VP882, or Borrelia burgdorferi (Borrelia burgdorferi).

在某些實施例中，原核端粒酶識別序列中之至少一者包含至少30個、至少40個、至少60個、至少80個或至少100個鹼基對長度之完美反向重複序列。In certain embodiments, at least one of the prokaryotic telomerase recognition sequences comprises a perfect inverted repeat of at least 30, at least 40, at least 60, at least 80, or at least 100 base pairs in length.

在某些實施例中，所關注之DNA片段包含在真核啟動子及/或增強子控制下/可操作地連接於真核啟動子及/或增強子之所關注之編碼序列，及視情況選用之真核轉錄終止序列。In certain embodiments, the DNA fragment of interest comprises a coding sequence of interest under the control of a eukaryotic promoter and/or enhancer/operably linked to a eukaryotic promoter and/or enhancer, and optionally The eukaryotic transcription termination sequence was selected.

在某些實施例中，所關注之編碼序列可包含編碼抗原之DNA疫苗，該抗原：(1)用於治療或預防諸如癌症、過敏症、毒性及病原體感染之病況(例如，真菌，病毒，諸如人類乳頭瘤病毒(HPV)、HIV、HSV2/HSV1、流感病毒(A、B及C型)、脊髓灰質炎病毒、RSV病毒、鼻病毒、輪狀病毒、A型肝炎病毒、諾沃克病毒組(Norwalk Virus Group)、腸病毒、星狀病毒、麻疹病毒、副流感病毒、腮腺炎病毒、水痘-帶狀疱疹病毒、細胞巨大病毒、埃-巴二氏病毒(Epstein-Barr virus)、腺病毒、風疹病毒、人類T細胞淋巴瘤I型病毒(HTLV-I)、B型肝炎病毒(HBV)、C型肝炎病毒(HCV)、D型肝炎病毒、痘病毒、馬爾堡(Marburg)及埃博拉(Ebola))；細菌(諸如結核分枝桿菌(Mycobacterium tuberculosis)、披衣菌屬(Chlamydia)、淋病奈瑟氏菌(Neisseria gonorrhoeae)、志賀桿菌屬(Shigella)、沙門氏菌屬(Salmonella)、霍亂弧菌(Vibrio cholerae)、梅毒螺旋體(Treponema pallidum)、假單胞菌屬(Pseudomonas)、百日咳博德特氏菌(Bordetella pertussis)、布氏桿菌屬(Brucella)、土拉熱弗朗西絲菌(Francisella tularensis)、幽門螺旋桿菌(Helicobacter pylori)、鉤端螺旋體(Leptospira interrogans)、嗜肺性退伍軍人桿菌(Legionella pneumophila)、鼠疫耶爾森氏菌(Yersinia pestis)、鏈球菌屬(Streptococcus)(A型及B型)、肺炎球菌屬(Pneumococcus)、腦膜炎雙球菌屬(Meningococcus)、流感嗜血桿菌(Haemophilus influenza)(b型)、剛地弓形蟲(Toxoplasma gondii)、彎曲桿菌病(Campylobacteriosis)、卡他莫拉菌(Moraxella catarrhalis)、多諾萬病(Donovanosis)及放線菌病(Actinomycosis))；真菌病原體(諸如念珠菌病(Candidiasis)及麴菌病(Aspergillosis))；寄生蟲病原體(諸如絛蟲屬(Taenia)、吸蟲、蛔蟲、阿米巴病(Amoebiasis)、梨形鞭毛蟲病(Giardiasis)、隱胞子蟲屬(Cryptosporidium)、住血吸蟲屬(Schistosoma)、肺炎肺囊蟲(Pneumocystis carinii)、滴蟲病(Trichomoniasis)及旋毛蟲病(Trichinosis)；(2)來自腺病毒科(adenoviridae)(例如人類腺病毒)、疱疹病毒科(herpesviridae)(例如HSV-1、HSV-2、EBV、CMV及VZV)、乳多空病毒科(papovaviridae)(例如HPV)、痘病毒科(poxyiridae)(例如天花及牛痘)、細小病毒科(parvoviridae)(例如小病毒B19)、呼腸孤病毒科(reoviridae)(例如輪狀病毒)、冠狀病毒科(coronaviridae)(例如SARS)、黃病毒科(flaviviridae)(例如黃熱病、西尼羅河病毒、登革熱、C型肝炎及蜱媒腦炎)、小核糖核酸病毒科(picornaviridae)(例如脊髓灰質炎、鼻病毒及A型肝炎)、披膜病毒科(togaviridae)(例如風疹病毒)、絲狀病毒科(filoviridae)(例如馬爾堡及埃博拉)、副黏病毒科(paramyxoviridae)(例如副流感病毒、呼吸道合胞病毒、腮腺炎及麻疹)、彈狀病毒科(rhabdoviridae)(例如狂犬病病毒)、布尼亞病毒科(bunyaviridae)(例如漢坦病毒(Hantaan virus))、正黏病毒科(orthomyxoviridae)(例如A型、B型及C型流感病毒)、反轉錄病毒科(retroviridae)(例如HIV及HTLV)及肝DNA病毒科(hepadnaviridae)(例如B型肝炎)之成員；(3)來自造成獸醫學疾病之病原體，諸如病毒病原體、呼腸孤病毒(例如非洲馬病或藍舌病毒)及疱疹病毒(例如馬疱疹)、***病毒、蜱媒腦炎病毒、登革熱病毒、SARS、西尼羅河病毒及漢坦病毒；(4)來自免疫缺陷病毒，諸如SIV或貓免疫缺陷病毒；(5)係新抗原(例如由癌症/腫瘤中之突變基因編碼)、腫瘤抗原，諸如睪丸抗原(例如MAGE家族之成員(MAGE 1、2、3等)、NY-ESO-1及SSX-2)、分化抗原(例如酪胺酸酶、gp100、PSA、Her-2及CEA)、突變的自身抗原及病毒性腫瘤抗原(例如來自致癌HPV類型之E6及/或E7)、MART-1、Melan-A、p97、β-HCG、GaINAc、MAGE-1、MAGE-2、MAGE-4、MAGE-12、MUC1、MUC2、MUC3、MUC4、MUC18、CEA、DDC、P1A、EpCam、黑素瘤抗原gp75、Hker 8、高分子量黑素瘤抗原、K19、Tyr1、Tyr2、pMel 17基因家族之成員、c-Met、PSM (***黏蛋白抗原)、PSMA (***特異性膜抗原)、***分泌蛋白、α-胎蛋白、CA 125、CA 19.9、TAG-72、BRCA-1及BRCA-2抗原。In certain embodiments, the coding sequence of interest may comprise a DNA vaccine encoding an antigen that: (1) is used to treat or prevent conditions such as cancer, allergy, toxicity, and pathogen infection (eg, fungi, viruses, Such as human papillomavirus (HPV), HIV, HSV2/HSV1, influenza virus (types A, B and C), poliovirus, RSV virus, rhinovirus, rotavirus, hepatitis A virus, norovirus group (Norwalk Virus Group), enterovirus, astrovirus, measles virus, parainfluenza virus, mumps virus, varicella-zoster virus, giant cell virus, Epstein-Barr virus, adenovirus , rubella virus, human T-cell lymphoma type I virus (HTLV-I), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, poxvirus, Marburg and Ebo Ebola; bacteria (such as Mycobacterium tuberculosis, Chlamydia, Neisseria gonorrhoeae, Shigella, Salmonella, cholera) Vibrio cholerae, plum Treponema pallidum, Pseudomonas, Bordetella pertussis, Brucella, Francisella tularensis, Helicobacter pylori ), Leptospira interrogans, Legionella pneumophila, Yersinia pestis, Streptococcus (types A and B), pneumococcal ( Pneumococcus), Meningococcus, Haemophilus influenza (type b), Toxoplasma gondii, Campylobacteriosis, Moraxella catarrhalis , Donovanosis and Actinomycosis; fungal pathogens (such as Candidiasis and Aspergillosis); parasitic pathogens (such as Taenia, trematode, Aphids, Amoebiasis, Giardiasis, Cryptosporidium, Schistosoma, Pneumocystis (Pneumoc) Ystis carinii), Trichomoniasis and Trichinosis; (2) from the adenoviridae (eg human adenovirus), herpesviridae (eg HSV-1, HSV-2) , EBV, CMV, and VZV), papovaviridae (eg, HPV), poxyiridae (eg, smallpox and vaccinia), parvoviridae (eg, small virus B19), regia Reviralidae (eg, rotavirus), coronaviridae (eg, SARS), flaviviridae (eg, yellow fever, West Nile virus, dengue, hepatitis C, and sputum encephalitis), Piporaviridae (eg, polio, rhinovirus, and hepatitis A), togaviridae (eg, rubella virus), filoviridae (eg, Marburg and Ebola) ), Paramyxoviridae (eg, parainfluenza virus, respiratory syncytial virus, mumps and measles), rhabdoviridae (eg rabies virus), Bunyaviridae (eg Han) Hantan virus), Orthomycosis Member of the family orthomyxoviridae (eg, influenza A, B, and C), retroviridae (eg, HIV and HTLV), and hepadnaviridae (eg, hepatitis B); 3) From pathogens causing veterinary diseases, such as viral pathogens, reoviruses (such as African horse disease or bluetongue virus) and herpes viruses (such as horse herpes), foot-and-mouth disease virus, tick-borne encephalitis virus, dengue virus, SARS , West Nile virus and Hantavirus; (4) from immunodeficiency virus, such as SIV or feline immunodeficiency virus; (5) new antigen (for example, encoded by a mutant gene in cancer/tumor), tumor antigen, such as testicular antigen (eg members of the MAGE family (MAGE 1, 2, 3, etc.), NY-ESO-1 and SSX-2), differentiation antigens (eg tyrosinase, gp100, PSA, Her-2 and CEA), mutations themselves Antigen and viral tumor antigens (eg E6 and/or E7 from oncogenic HPV types), MART-1, Melan-A, p97, β-HCG, GaINAc, MAGE-1, MAGE-2, MAGE-4, MAGE- 12, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, P1A, EpCam, melanoma antigen gp75, Hker 8, high score Melanoma antigen, K19, Tyr1, Tyr2, members of the pMel 17 gene family, c-Met, PSM (prostate mucin antigen), PSMA (prostate specific membrane antigen), prostate secretory protein, alpha-fetoprotein, CA 125, CA 19.9, TAG-72, BRCA-1 and BRCA-2 antigens.

在某些實施例中，所關注之編碼序列包含用於基因療法之治療性DNA分子，其中該治療性DNA分子：(1)在患有由功能基因之功能異常形式(例如杜興氏肌肉萎縮症(Duchenne muscular dystrophy)、囊腫性纖維化、高歇氏病(Gaucher's Disease)及腺苷脫胺酶(ADA)缺乏症、發炎疾病、自體免疫、慢性及感染性疾病、AIDS、癌症、神經疾病、心血管疾病、高膽固醇血症、各種血液病症(包括各種貧血症、地中海貧血症及血友病，及肺氣腫)及實體腫瘤之基因)引起之基因病症的個體中表現該功能基因；(2)編碼毒性肽(亦即化學治療劑，諸如蓖麻毒素、白喉毒素及眼鏡蛇毒因子)、腫瘤抑制基因(諸如p53)、編碼轉化致癌基因之反義mRNA序列的基因、諸如腫瘤壞死因子(TNF)及其他細胞介素之抗腫瘤肽、或轉化致癌基因之反式顯性陰性突變體；(3)編碼活性RNA形式(例如小干擾RNA (siRNA、miRNA、shRNA)或小活化RNA (saRNA)；或(4)編碼CRISPR/Cas組分(諸如Cas9酶或sgRNA)。In certain embodiments, the coding sequence of interest comprises a therapeutic DNA molecule for gene therapy, wherein the therapeutic DNA molecule: (1) is suffering from a functionally abnormal form of a functional gene (eg, Duchenne muscle atrophy) Duchenne muscular dystrophy, cystic fibrosis, Gaucher's Disease and adenosine deaminase (ADA) deficiency, inflammatory disease, autoimmune, chronic and infectious diseases, AIDS, cancer, nerve The functional gene is expressed in individuals with genetic disorders caused by diseases, cardiovascular diseases, hypercholesterolemia, various blood disorders (including various anemia, thalassemia and hemophilia, and emphysema) and genes of solid tumors (2) encoding toxic peptides (ie, chemotherapeutic agents such as ricin, diphtheria toxin, and cobra venom factor), tumor suppressor genes (such as p53), genes encoding antisense mRNA sequences that convert oncogenes, such as tumor necrosis; Anti-tumor peptides of factors (TNF) and other interleukins, or trans-dominant negative mutants that convert oncogenes; (3) encode active RNA forms (eg, small interfering RNAs (siRNA, miRNA, shRNA) Small activating RNA (saRNA); or (4) encode CRISPR / Cas component (such as an enzyme or Cas9 sgRNA).

本發明之另一態樣提供藉由使主題DNA構築體與識別一對原核端粒酶識別序列之原核端粒酶接觸而產生之封閉末端雙股DNA (cdsDNA)。在某些實施例中，主題cdsDNA經組態以能夠在真核細胞之整個生命週期中染色體外存在。Another aspect of the invention provides a closed terminal double stranded DNA (cdsDNA) produced by contacting a subject DNA construct with a prokaryotic telomerase that recognizes a pair of prokaryotic telomerase recognition sequences. In certain embodiments, the subject cdsDNA is configured to be capable of extrachromosomally throughout the life cycle of a eukaryotic cell.

本發明之另一態樣提供封閉末端雙股DNA (cdsDNA)，其包含：(a)所關注之DNA片段；(b)側接所關注之DNA片段的一對末端序列，其中該等末端序列為(例如雙股DNA病毒的)反向末端重複序列(ITR)、DNA病毒(諸如HSV)之長末端重複序列(LTR)或內部重複序列、或端粒序列；及(c)側接該對末端序列之一對半原核端粒酶識別序列，其中該等半原核端粒酶識別序列中之每一者形成cdsDNA之一個封閉末端。在某些實施例中，cdsDNA或所關注之DNA片段經組態以能夠在真核細胞之整個生命週期中染色體外存在。Another aspect of the invention provides a closed-end double-stranded DNA (cdsDNA) comprising: (a) a DNA fragment of interest; (b) a pair of terminal sequences flanked by a DNA fragment of interest, wherein the terminal sequences An inverted terminal repeat (ITR) of (eg, a double-stranded DNA virus), a long terminal repeat (LTR) or internal repeat of a DNA virus (such as HSV), or a telomere sequence; and (c) flanked by the pair One of the terminal sequences is a half-prokaryotic telomerase recognition sequence, wherein each of the semi-prokaryotic telomerase recognition sequences forms a closed end of the cds DNA. In certain embodiments, the cds DNA or DNA fragment of interest is configured to be capable of extrachromosomally throughout the life cycle of a eukaryotic cell.

本發明之另一態樣提供包含主題cdsDNA之醫藥組合物。Another aspect of the invention provides a pharmaceutical composition comprising the subject cdsDNA.

在某些實施例中，cdsDNA係由奈米粒子(諸如LNP或基於聚合物之NP)包圍。In certain embodiments, the cdsDNA is surrounded by nanoparticle (such as LNP or polymer based NP).

在某些實施例中，奈米粒子為SNALP (穩定核酸-脂質粒子)、AtuPLEX、DACC、DBTC、RONDEL、DPC (Dynamic PolyConjugate)、SMARTICLE、DiLA2 或EnCore。In certain embodiments, the nanoparticles are SNALP (stabilized nucleic acid-lipid particles), AtuPLEX, DACC, DBTC, RONDEL, DPC (Dynamic PolyConjugate), SMARTCLE, DiLA 2, or EnCore.

本發明之另一態樣提供產生無細胞封閉末端雙股DNA (cdsDNA)之方法，該方法包含：(1)在真核(例如哺乳動物)或原核細胞中擴增DNA構築體後，分離主題DNA構築體；(2)使用不會在該嵌段內消解之核酸內切酶使DNA構築體線性化；(3)使DNA構築體與識別一對原核端粒酶識別序列之原核端粒酶接觸以釋放cdsDNA；(4)在步驟(2)及(3)之後，用核酸外切酶移除不是cdsDNA之線性化DNA構築體或其片段；(5)富集或純化cdsDNA。Another aspect of the invention provides a method of producing cell-free blocked terminal double-stranded DNA (cdsDNA), the method comprising: (1) separating a subject after amplification of the DNA construct in a eukaryotic (eg, mammalian) or prokaryotic cell a DNA construct; (2) linearization of the DNA construct using an endonuclease that does not digest in the block; (3) a DNA construct and a prokaryotic telomerase that recognizes a pair of protelomerase recognition sequences Contact to release cdsDNA; (4) after step (2) and (3), remove the linearized DNA construct or fragment thereof that is not cdsDNA with an exonuclease; (5) enrich or purify the cds DNA.

在某些實施例中，步驟(2)及(3)以任何順序或同時在步驟(1)之後進行。In certain embodiments, steps (2) and (3) are performed in any order or simultaneously after step (1).

本發明之另一態樣提供產生封閉末端雙股DNA (cdsDNA)之方法，該方法包含：(1)在真核(例如哺乳動物)或原核細胞中擴增DNA構築體後，分離主題DNA構築體；(2)使DNA構築體與識別一對原核端粒酶識別序列之原核端粒酶接觸以釋放cdsDNA；(3)富集或純化cdsDNA。Another aspect of the invention provides a method of producing a closed-end double-stranded DNA (cdsDNA), the method comprising: (1) isolating a subject DNA construct after amplifying a DNA construct in a eukaryotic (eg, mammalian) or prokaryotic cell (2) contacting the DNA construct with prokaryotic telomerase that recognizes a pair of prokaryotic telomerase recognition sequences to release cdsDNA; (3) enriching or purifying cds DNA.

在某些實施例中，該方法進一步包含將cdsDNA囊封於奈米粒子(諸如SNALP、AtuPLEX、DACC、DBTC、RONDEL、DPC、SMARTICLE、DiLA2 或EnCore)中。In certain embodiments, the method further comprises encapsulating the cdsDNA in a nanoparticle (such as SNALP, AtuPLEX, DACC, DBTC, RONDEL, DPC, SMARTICLE, DiLA 2, or EnCore).

本發明之另一態樣提供將所關注之靶基因(GOI)遞送至靶細胞中之方法，該方法包含使靶細胞與包含主題cdsDNA之組合物接觸。Another aspect of the invention provides a method of delivering a target gene of interest (GOI) into a target cell, the method comprising contacting the target cell with a composition comprising the subject cdsDNA.

在某些實施例中，靶細胞係活體外、離體或活體內接觸。In certain embodiments, the target cell line is contacted ex vivo, ex vivo or in vivo.

在某些實施例中，靶GOI為用於治療DMD (杜興氏肌肉萎縮症)之野生型迷你-或微型肌縮蛋白基因、DMD途徑中之基因(諸如卵泡抑素或其人類IgG融合物，或IKKβ或NF-κB途徑之抑制劑)、或與DMA之基因修飾物(諸如SPP1或LTBP4)相關之構築體，且其中靶細胞為肌肉(例如骨骼肌諸如脛骨前肌細胞、心肌、平滑肌、或膈肌、肱三頭肌、比目魚肌、脛骨前肌、腓腸肌、伸趾長肌、腹直肌、股四頭肌及其組合之肌肉，或如以引用的方式併入之WO2017152090A2之表1中所述之肌肉)。在某些實施例中，DMD之至少一種症狀或特徵在強度、嚴重性或頻率方面有所降低，或延遲發作(例如，DMD之至少一種症狀或特徵選自由肌肉萎縮、肌無力、肌肉脆性、肌肉壞死、肌肉纖維化、關節攣縮、骨骼變形、心肌病、吞咽受損、腸和膀胱功能受損、肌肉缺血、認知障礙、行為功能障礙、社交障礙、脊柱側凸和呼吸功能受損組成之群)。在某些實施例中，治療導致肌肉質量相對於對照增加(例如，相對於對照增加至少10%、20%、30%、40%、50%、60%、70%、80%、90%、100%、150%、200%或500%)。在某些實施例中，治療導致肌肉再生、肌肉力量增加、柔韌性增加、運動範圍增加、耐力增加、易疲勞性減少、血流量增加、認知改善、肺功能改善、炎症抑制、肌纖維化減少及/或肌肉壞死減少。在某些實施例中，該方法進一步包含投與一或多種額外治療劑，諸如抗Fit-1抗體或其片段、艾達沙隆(edasalonexent)、龐日魯單抗(pamrevlumab)、潑尼松(prednisone)、地夫可特(deflazacort)、RNA調節治療劑、外顯子跳躍治療劑及任何其他基因療法。In certain embodiments, the target GOI is a wild type mini- or micro-muscle protein gene for use in the treatment of DMD (Duchenne Muscular Dystrophy), a gene in the DMD pathway (such as follistatin or a human IgG fusion thereof) , or an inhibitor of the IKKβ or NF-κB pathway), or a construct associated with a genetic modification of DMA (such as SPP1 or LTBP4), and wherein the target cells are muscles (eg, skeletal muscles such as tibialis anterior muscle cells, myocardium, smooth muscle) Or muscles of the diaphragm, triceps, soleus, tibialis anterior, gastrocnemius, toe long, rectus abdominis, quadriceps and combinations thereof, or Table 1 of WO2017152090A2, incorporated by reference. The muscles described in the). In certain embodiments, at least one symptom or characteristic of the DMD is reduced in intensity, severity, or frequency, or delayed onset (eg, at least one symptom or characteristic of DMD is selected from muscle atrophy, muscle weakness, muscle fragility, Muscle necrosis, muscle fibrosis, joint contracture, skeletal deformity, cardiomyopathy, swallowing damage, impaired bowel and bladder function, muscle ischemia, cognitive impairment, behavioral dysfunction, social disorder, scoliosis and impaired respiratory function Group). In certain embodiments, the treatment results in an increase in muscle mass relative to the control (eg, an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, relative to the control, 100%, 150%, 200% or 500%). In certain embodiments, treatment results in muscle regeneration, increased muscle strength, increased flexibility, increased range of motion, increased endurance, reduced fatigue, increased blood flow, improved cognitive function, improved lung function, reduced inflammation, decreased muscle fibrosis, and / or reduced muscle necrosis. In certain embodiments, the method further comprises administering one or more additional therapeutic agents, such as an anti-Fit-1 antibody or fragment thereof, edasalonexent, pamrevlumab, prednisone (prednisone), deflazacort, RNA-modulating therapeutics, exon skipping therapeutics, and any other gene therapy.

在某些實施例中，所關注之靶基因(GOI)每日、每週兩次、每週一次、每月一次、兩月一次或每2、3、4、5、6、9、12、18、24、36、60、72或更多個月一次遞送至靶細胞。In certain embodiments, the target gene of interest (GOI) is daily, twice weekly, weekly, monthly, bimonthly, or every 2, 3, 4, 5, 6, 9, 12, Delivery to target cells at 18, 24, 36, 60, 72 or more months.

本發明之一個相關態樣提供包含主題cdsDNA或主題醫藥組合物中之任一者之組合物的用途，其係用於製造將所關注之靶基因(GOI)活體內遞送至靶細胞中之藥物。A related aspect of the invention provides the use of a composition comprising any one of the subject cdsDNA or a subject pharmaceutical composition for the manufacture of a medicament for the in vivo delivery of a target gene of interest (GOI) to a target cell .

預期本文所述實施例中之任一者或多者，包括僅在實例中或僅在本發明之一個態樣下描述的彼等實施例，可與任一或多個實施例組合，除非明確否定或不當。It is contemplated that any one or more of the embodiments described herein, including those embodiments described only in the examples or only one aspect of the invention, may be combined with any one or more embodiments unless explicitly Negative or improper.

相關申請案之引用
本申請案主張2017年12月14日申請之美國臨時申請案第62/598,532號之申請日權益，該臨時申請案之全部內容以引用的方式併入本文中。 Related applications of reference <br/> This application claims the benefit of the filing date of the application of US Provisional Application No. 62 of / 598,532 December 14, 2017, the entire contents of this provisional application is incorporated herein by reference in .

1. 概述
本文所述之本發明之組合物及方法部分基於以下發現：所關注之DNA片段側接一對末端序列，諸如基於某些雙股DNA病毒之反向末端重複序列(ITR)、或某些DNA病毒之長末端重複序列(LTR)或內部重複序列的末端序列，且進一步側接一對原核端粒酶識別序列，可有效擴增以產生包含此類所關注之DNA片段的無細胞封閉末端雙股DNA (cdsDNA)分子，隨後可將cdsDNA封裝於某些奈米粒子中，諸如基於脂質之奈米粒子(LNP)或基於聚合物之奈米粒子(NP)作為醫藥組合物。1. SUMMARY The compositions and methods of the invention described herein are based in part on the discovery that a DNA fragment of interest is flanked by a pair of end sequences, such as an inverted terminal repeat (ITR) based on certain double-stranded DNA viruses, or The terminal sequence of the long terminal repeat (LTR) or internal repeat of certain DNA viruses, and further flanked by a pair of prokaryotic telomerase recognition sequences, which can be efficiently amplified to produce cell-free cells containing such DNA fragments of interest. The terminal double-stranded DNA (cdsDNA) molecule is blocked, and the cds DNA can then be encapsulated in certain nanoparticles, such as lipid-based nanoparticle (LNP) or polymer-based nanoparticle (NP) as a pharmaceutical composition.

本文所提供之本發明提供一種大量或大規模製造/生產任何所關注之無細胞DNA片段(例如治療性DNA分子)的方式，該片段可用於例如基因療法而不使用任何病毒載體。The invention provided herein provides a means of producing or producing any cell-free DNA fragment of interest (e.g., a therapeutic DNA molecule) in large or large scale, which fragment can be used, for example, in gene therapy without the use of any viral vector.

本發明之不同態樣更詳細地描述於下文單獨的部分中。Different aspects of the invention are described in more detail in separate sections below.

2. 所關注之DNA片段
所關注之DNA片段理論上可為任何DNA序列，包括非編碼序列，或編碼蛋白質、RNA (諸如非轉譯RNA，其可用於RNAi、反義抑制，小活化RNA或用於CRISPR/Cas之sgRNA，僅舉幾例)等之序列。2. The DNA fragment of interest for the DNA fragment of interest can theoretically be any DNA sequence, including non-coding sequences, or encoding proteins, RNA (such as non-translated RNA, which can be used for RNAi, antisense suppression, small activating RNA or The sequence of CRISPR/Cas sgRNA, to name a few).

在某些實施例中，所關注之DNA片段的長度為約100 bp、200 bp、500 bp、1 kb、2 kb、3 kb、4 kb、5 kb、10 kb、15 kb、20 kb、25 kb、30 kb、50 kb、100 kb、150 kb、300 kb、500 kb、750 kb、1000 kb、1500 kb、2000 kb、5000 kb或更長。In certain embodiments, the DNA fragment of interest is about 100 bp, 200 bp, 500 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 15 kb, 20 kb, 25 Kb, 30 kb, 50 kb, 100 kb, 150 kb, 300 kb, 500 kb, 750 kb, 1000 kb, 1500 kb, 2000 kb, 5000 kb or longer.

在某些實施例中，所關注之DNA片段編碼功能蛋白、或其功能結構域或部分。舉例而言，所編碼之功能蛋白可為與缺乏功能蛋白相關/由缺乏功能蛋白引起之疾病中缺少的蛋白質。所編碼之功能蛋白可為DMD小基因或全長DMD基因。In certain embodiments, a DNA fragment of interest encodes a functional protein, or a functional domain or portion thereof. For example, the encoded functional protein can be a protein that is deficient in a disease associated with the absence of a functional protein or caused by a lack of a functional protein. The functional protein encoded can be a DMD minigene or a full length DMD gene.

在某些實施例中，所關注之DNA片段編碼設計用於拮抗靶基因之轉錄及/或轉譯的反義寡核苷酸。In certain embodiments, the DNA fragment of interest encodes an antisense oligonucleotide designed to antagonize transcription and/or translation of a target gene.

在某些實施例中，所關注之DNA片段編碼設計用於拮抗靶基因表現之RNAi構築體。RNAi構築體可產生siRNA、shRNA (短髮夾RNA)或miRNA (微RNA)。In certain embodiments, the DNA fragment of interest encodes an RNAi construct designed to antagonize the expression of a target gene. RNAi constructs can produce siRNA, shRNA (short hairpin RNA) or miRNA (microRNA).

在某些實施例中，所關注之DNA片段不編碼設計用於拮抗靶基因之轉錄及/或轉譯的反義寡核苷酸。In certain embodiments, the DNA fragment of interest does not encode an antisense oligonucleotide designed to antagonize transcription and/or translation of a target gene.

在某些實施例中，所關注之DNA片段不編碼設計用於拮抗靶基因表現之RNAi構築體。RNAi構築體可產生siRNA、shRNA (短髮夾RNA)或miRNA (微RNA)。In certain embodiments, the DNA fragment of interest does not encode an RNAi construct designed to antagonize the expression of a target gene. RNAi constructs can produce siRNA, shRNA (short hairpin RNA) or miRNA (microRNA).

在某些實施例中，所關注之DNA片段編碼設計用於拮抗靶基因表現之小活化RNA (saRNA)。In certain embodiments, the DNA fragment of interest encodes a small activating RNA (saRNA) designed to antagonize the expression of a target gene.

小活化RNA (saRNA)為靶向基因啟動子以在稱為RNAa之過程中誘導轉錄基因活化的小雙股RNA (dsRNA)。已知小dsRNA，諸如siRNA及微小RNA (miRNA)，係進化保守RNAi之觸發物，其經由抑制轉錄、降解互補mRNA或阻斷蛋白質轉譯而始終導致基因沉默。dsRNA亦可藉由靶向基因啟動子中之選定序列而充當saRNA。Small activating RNA (saRNA) is a small double-stranded RNA (dsRNA) that targets a gene promoter to induce transcriptional gene activation in a process called RNAa. Small dsRNAs, such as siRNA and microRNAs (miRNAs), are known to be triggers of evolutionarily conserved RNAi, which ultimately result in gene silencing by inhibiting transcription, degrading complementary mRNA, or blocking protein translation. The dsRNA can also act as a saRNA by targeting a selected sequence in a gene promoter.

在某些實施例中，saRNA為21個核苷酸長，在各股之3'端具有2個核苷酸的突出端。藉由遵循一組已知規則且視情況在培養細胞中測試，可在1至2 kb啟動子區內設計數種saRNA。In certain embodiments, the saRNA is 21 nucleotides in length with a 2 nucleotide overhang at the 3' end of each strand. Several saRNAs can be designed in the 1 to 2 kb promoter region by following a known set of rules and optionally testing in cultured cells.

在某些實施例中，saRNA經設計以靶向與蛋白質編碼基因之啟動子序列重疊的非編碼轉錄物。In certain embodiments, the saRNA is designed to target a non-coding transcript that overlaps with a promoter sequence of a protein-encoding gene.

化學合成之saRNA及表現為shRNA之saRNA已用於活體外及活體內實驗，包括用於治療癌症、肝病、局部缺血及***功能障礙之動物模型及人類臨床試驗。Chemically synthesized saRNAs and saRNAs that display shRNA have been used in in vitro and in vivo experiments, including animal models for the treatment of cancer, liver disease, ischemia and erectile dysfunction, and human clinical trials.

在某些實施例中，所關注之DNA片段為DNA疫苗。DNA疫苗通常編碼感染性生物體DNA之修飾形式。向個體投與DNA疫苗，隨後其表現所選擇之感染性生物體的蛋白質，從而引發針對該蛋白質之免疫反應，該蛋白質通常為保護性的。DNA疫苗亦可在癌症免疫療法中編碼腫瘤抗原或新抗原。In certain embodiments, the DNA fragment of interest is a DNA vaccine. DNA vaccines typically encode a modified form of DNA of an infectious organism. The DNA vaccine is administered to the individual, which then expresses the protein of the selected infectious organism, thereby eliciting an immune response against the protein, which is typically protective. DNA vaccines can also encode tumor antigens or new antigens in cancer immunotherapy.

DNA疫苗可包含編碼抗原之核酸序列，用於治療或預防許多病況，包括但不限於癌症、過敏症、毒性及病原體感染，該病原體諸如但不限於真菌；病毒，包括人類乳頭瘤病毒(HPV)、HIV、HSV2/HSV1、流感病毒(A、B及C型)、脊髓灰質炎病毒、RSV病毒、鼻病毒、輪狀病毒、A型肝炎病毒、諾沃克病毒組、腸病毒、星狀病毒、麻疹病毒、副流感病毒、腮腺炎病毒、水痘-帶狀疱疹病毒、細胞巨大病毒、埃-巴二氏病毒、腺病毒、風疹病毒、人類T細胞淋巴瘤I型病毒(HTLV-I)、B型肝炎病毒(HBV)、C型肝炎病毒(HCV)、D型肝炎病毒、痘病毒、馬爾堡及埃博拉；細菌，包括結核分枝桿菌、披衣菌屬、淋病奈瑟氏菌、志賀桿菌屬、沙門氏菌屬、霍亂弧菌、梅毒螺旋體、假單胞菌屬、百日咳博德特氏菌、布氏桿菌屬、土拉熱弗朗西絲菌、幽門螺旋桿菌、鉤端螺旋體、嗜肺性退伍軍人桿菌、鼠疫耶爾森氏菌、鏈球菌屬(A型及B型)、肺炎球菌屬、腦膜炎雙球菌屬、流感嗜血桿菌(b型)、剛地弓形蟲、彎曲桿菌病、卡他莫拉菌、多諾萬病及放線菌病；真菌病原體，包括念珠菌病及麴菌病；寄生蟲病原體，包括絛蟲屬、吸蟲、蛔蟲、阿米巴病、梨形鞭毛蟲病、隱胞子蟲屬、住血吸蟲屬、肺炎肺囊蟲、滴蟲病及旋毛蟲病。DNA疫苗可包含編碼來自以下成員之抗原的核酸序列：腺病毒科(包括例如人類腺病毒)、疱疹病毒科(包括例如HSV-1、HSV-2、EBV、CMV及VZV)、乳多空病毒科(包括例如HPV)、痘病毒科(包括例如天花及牛痘)、細小病毒科(包括例如小病毒B 19)、呼腸孤病毒科(包括例如輪狀病毒)、冠狀病毒科(包括例如SARS)、黃病毒科(包括例如黃熱病、西尼羅河病毒、登革熱、C型肝炎及蜱媒腦炎)、小核糖核酸病毒科(包括脊髓灰質炎、鼻病毒及A型肝炎)、披膜病毒科(包括例如風疹病毒)、絲狀病毒科(包括例如馬爾堡及埃博拉)、副黏病毒科(包括例如副流感病毒、呼吸道合胞病毒、腮腺炎及麻疹)、彈狀病毒科(包括例如狂犬病病毒)、布尼亞病毒科(包括例如漢坦病毒)、正黏病毒科(包括例如A型、B型及C型流感病毒)、反轉錄病毒科(包括例如HIV及HTLV)及肝DNA病毒科(包括例如B型肝炎)。A DNA vaccine may comprise a nucleic acid sequence encoding an antigen for use in the treatment or prevention of a number of conditions including, but not limited to, cancer, allergy, toxicity, and pathogen infection, such as, but not limited to, fungi; viruses, including human papillomavirus (HPV). , HIV, HSV2/HSV1, influenza virus (types A, B and C), poliovirus, RSV virus, rhinovirus, rotavirus, hepatitis A virus, norovirus group, enterovirus, astrovirus, Measles virus, parainfluenza virus, mumps virus, varicella-zoster virus, giant cell virus, Epstein-Barr virus, adenovirus, rubella virus, human T-cell lymphoma type I virus (HTLV-I), B Hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, poxvirus, Marburg and Ebola; bacteria, including Mycobacterium tuberculosis, Chlamydia, Neisseria gonorrhoeae, Shiga Bacillus, Salmonella, Vibrio cholerae, Treponema pallidum, Pseudomonas, Bordetella pertussis, Brucella, Francia serrata, Helicobacter pylori, Leptospira, pulmonaryophilic veterans Rod Yersinia pestis, Streptococcus (types A and B), pneumococcal, meningococcus, Haemophilus influenzae (type b), Toxoplasma gondii, Campylobacter, catarrh Lactobacillus, Donovan and actinomycosis; fungal pathogens, including candidiasis and rickets; parasitic pathogens, including aphids, trematodes, aphids, amebiasis, piriflagellate, cryptosides Insects, schistosomiasis, pneumocystis pneumoniae, trichomoniasis and trichinosis. A DNA vaccine may comprise a nucleic acid sequence encoding an antigen from a member of the family: adenoviridae (including, for example, human adenovirus), herpesviridae (including, for example, HSV-1, HSV-2, EBV, CMV, and VZV), papovavirus Sections (including, for example, HPV), poxviridae (including, for example, smallpox and vaccinia), parvoviridae (including, for example, small virus B 19), reoviridae (including, for example, rotavirus), coronavirus (including, for example, SARS) ), Flaviviridae (including, for example, yellow fever, West Nile virus, dengue fever, hepatitis C and sputum encephalitis), picornavirus (including polio, rhinovirus, and hepatitis A), togaviridae (including, for example, rubella virus), Filoviridae (including, for example, Marlborough and Ebola), Paramyxoviridae (including, for example, parainfluenza virus, respiratory syncytial virus, mumps and measles), Rhabdoviridae (including For example, rabies virus), Bunia virus family (including, for example, Hantavirus), Orthomyxoviridae (including, for example, influenza A, B, and C viruses), retroviridae (including, for example, HIV and HTLV), and liver DNA virus family (including, for example, hepatitis B).

抗原可來自造成獸醫學疾病之病原體，且尤其可來自病毒病原體，包括例如呼腸孤病毒(諸如非洲馬病或藍舌病毒)及疱疹病毒(包括馬疱疹)。抗原可為來自***病毒、蜱媒腦炎病毒、登革熱病毒、SARS、西尼羅河病毒及漢坦病毒之抗原。抗原可來自免疫缺陷病毒，且可例如來自SIV或貓免疫缺陷病毒。The antigen may be from a pathogen causing a veterinary disease, and may in particular be derived from a viral pathogen, including, for example, a reovirus (such as African horse disease or bluetongue virus) and a herpes virus (including horse herpes). The antigen may be an antigen derived from foot-and-mouth disease virus, tick-borne encephalitis virus, dengue virus, SARS, West Nile virus, and Hantavirus. The antigen can be from an immunodeficiency virus and can be, for example, from a SIV or feline immunodeficiency virus.

DNA疫苗亦可包含編碼腫瘤抗原或腫瘤相關抗原之核酸序列。腫瘤相關抗原之實例包括但不限於新抗原(諸如由來自癌症/腫瘤之突變基因編碼的抗原，新抗原引發T細胞反應以殺死呈現此類新抗原之癌症/腫瘤細胞)、癌症-睪丸抗原(諸如MAGE家族之成員(MAGE 1、2、3等)、NY-ESO-1及SSX-2)、分化抗原(諸如酪胺酸酶、gp1OO、PSA、Her-2及CEA)、突變的自身抗原及病毒性腫瘤抗原(諸如來自致癌HPV類型之E6及/或E7)。特定腫瘤抗原之其他實例包括MART-I、Melan-A、p97、β-HCG、GaINAc、MAGE-I、MAGE-2、MAGE-4、MAGE-12、MUC1、MUC2、MUC3、MUC4、MUC18、CEA、DDC、P1A、EpCam、黑素瘤抗原gp75、Hker 8、高分子量黑素瘤抗原、K1 9、Tyr1、Tyr2、pMel 17基因家族之成員、c-Met、PSM (***黏蛋白抗原)、PSMA (***特異性膜抗原)、***分泌蛋白、α-胎蛋白、CA 125、CA 19.9、TAG-72、BRCA-I及BRCA-2抗原。The DNA vaccine may also comprise a nucleic acid sequence encoding a tumor antigen or a tumor associated antigen. Examples of tumor-associated antigens include, but are not limited to, new antigens (such as antigens encoded by mutant genes from cancer/tumors, new antigens elicit T cell responses to kill cancer/tumor cells presenting such new antigens), cancer-test capsule antigens (such as members of the MAGE family (MAGE 1, 2, 3, etc.), NY-ESO-1 and SSX-2), differentiation antigens (such as tyrosinase, gp1OO, PSA, Her-2, and CEA), mutations themselves Antigens and viral tumor antigens (such as E6 and/or E7 from oncogenic HPV types). Other examples of specific tumor antigens include MART-I, Melan-A, p97, β-HCG, GaINAc, MAGE-I, MAGE-2, MAGE-4, MAGE-12, MUC1, MUC2, MUC3, MUC4, MUC18, CEA , DDC, P1A, EpCam, melanoma antigen gp75, Hker 8, high molecular weight melanoma antigen, K1 9, Tyr1, Tyr2, members of the pMel 17 gene family, c-Met, PSM (prostate mucin antigen), PSMA (Prostate specific membrane antigen), prostate secretory protein, alpha-fetoprotein, CA 125, CA 19.9, TAG-72, BRCA-I and BRCA-2 antigens.

在某些實施例中，所關注之DNA片段編碼新抗原，其係由癌症或腫瘤中之突變基因編碼的抗原，且可僅存在於癌症或腫瘤中。此類新抗原可充當癌症治療之個人化疫苗。In certain embodiments, the DNA fragment of interest encodes a new antigen, which is an antigen encoded by a mutated gene in a cancer or tumor, and may be present only in a cancer or tumor. Such new antigens can serve as personalized vaccines for cancer treatment.

在某些實施例中，所關注之DNA片段為治療性DNA分子，例如用於基因療法之治療性DNA分子。應注意，本文所述之本發明之組合物及方法為通常適用於基因療法之平台技術，包括可藉由提供編碼引起疾病之缺陷內源性基因及作為基因療法之靶標的所關注之功能性DNA片段的一或多個複本來校正的所有單基因人類疾病。In certain embodiments, the DNA fragment of interest is a therapeutic DNA molecule, such as a therapeutic DNA molecule for gene therapy. It should be noted that the compositions and methods of the invention described herein are platform technologies that are generally applicable to gene therapy, including functionality that can be exploited by providing endogenous genes encoding defects that cause disease and as targets for gene therapy. One or more copies of the DNA fragment to correct for all single-gene human diseases.

舉例而言，此類DNA分子可用於表現功能基因，其中個體患有由該基因之功能異常形式引起的基因病症。此類疾病之實例包括杜興氏肌肉萎縮症(DMD)、囊腫性纖維化(CF)、高歇氏疾病及腺苷脫胺酶(ADA)缺乏症。For example, such DNA molecules can be used to express a functional gene in which the individual has a genetic disorder caused by a dysfunctional form of the gene. Examples of such diseases include Duchenne Muscular Dystrophy (DMD), Cystic Fibrosis (CF), Gaucher's Disease, and Adenosine Deaminase (ADA) Deficiency.

基因療法可能有用之其他疾病包括發炎疾病、自體免疫、慢性及感染性疾病，包括諸如AIDS、癌症、神經疾病、心血管疾病、高膽固醇血症、各種血液病症(包括各種貧血症、地中海貧血症及血友病)及肺氣腫之病症。Other diseases that may be useful for gene therapy include inflammatory diseases, autoimmune, chronic and infectious diseases, including diseases such as AIDS, cancer, neurological diseases, cardiovascular diseases, hypercholesterolemia, various blood disorders (including various anemias, thalassemia). Symptoms and hemophilia) and emphysema.

為了治療實體腫瘤，可表現編碼毒性肽(亦即化學治療劑，諸如蓖麻毒素、白喉毒素及眼鏡蛇毒因子)之基因、腫瘤抑制基因(諸如p53)、編碼轉化致癌基因之反義mRNA序列的基因、諸如腫瘤壞死因子(TNF)及其他細胞介素之抗腫瘤肽、或轉化致癌基因之反式顯性陰性突變體。For the treatment of solid tumors, genes encoding toxic peptides (ie, chemotherapeutic agents such as ricin, diphtheria toxin, and cobra venom factor), tumor suppressor genes (such as p53), and antisense mRNA sequences encoding transformed oncogenes can be expressed. Genes, anti-tumor peptides such as tumor necrosis factor (TNF) and other interleukins, or trans-dominant negative mutants that convert oncogenes.

亦考慮其他類型之治療性DNA分子，包括例如轉錄成活性RNA形式(例如小干擾RNA，諸如siRNA、shRNA、miRNA或小活化RNA(saRNA))的DNA分子；或編碼CRISPR/Cas組分(諸如Cas9酶或sgRNA)之DNA。Other types of therapeutic DNA molecules are also contemplated, including, for example, DNA molecules transcribed into an active RNA form (eg, small interfering RNA, such as siRNA, shRNA, miRNA, or small activating RNA (saRNA)); or encoding a CRISPR/Cas component (such as DNA of Cas9 enzyme or sgRNA).

在涉及生產具有治療效用之DNA分子的實施例中，所關注之DNA片段通常包含表現卡匣，該表現卡匣包含一或多個啟動子或增強子元件及編碼所關注之mRNA或蛋白質的基因或其他編碼序列。在涉及用於基因療法之DNA疫苗分子或DNA分子之產生的具體實施例中，DNA模板包含由可操作地連接於編碼所關注之蛋白質的序列的真核啟動子及視情況選用之強化子及/或真核轉錄終止序列組成的表現卡匣。In embodiments involving the production of therapeutically useful DNA molecules, the DNA fragment of interest typically comprises a performance cassette comprising one or more promoter or enhancer elements and a gene encoding the mRNA or protein of interest. Or other coding sequence. In a specific embodiment involving the production of a DNA vaccine molecule or DNA molecule for gene therapy, the DNA template comprises a eukaryotic promoter operably linked to a sequence encoding a protein of interest and optionally a enhancer and / or the expression of the eukaryotic transcriptional termination sequence consists of a cassette.

3. LTR及ITR
儘管本文所述之本發明之組合物及方法不依賴於任何病毒載體來遞送所關注之DNA片段，但本發明之DNA構築體可含有一些病毒載體中常見的某些元件，包括ITR (反向末端重複序列)及LTR (長末端重複序列)。此類病毒元件可用作本發明之末端序列，其可充當所關注之DNA片段的抗分解元件。其他適合之末端序列可包括端粒序列或其功能衍生物或等效物。3. LTR and ITR
Although the compositions and methods of the invention described herein are not dependent on any viral vector for delivery of a DNA fragment of interest, the DNA constructs of the invention may contain some of the elements commonly found in viral vectors, including ITR (reverse) End repeats) and LTR (long terminal repeats). Such viral elements can be used as end sequences of the invention, which can serve as anti-decomposition elements of the DNA fragments of interest. Other suitable end sequences can include telomere sequences or functional derivatives or equivalents thereof.

一旦本發明之cdsDNA被靶細胞攝取，本發明之末端序列使得cdsDNA能夠作為長期穩定的染色體外基因元件存在。具有末端序列之cdsDNA可穩定6小時、12小時、1天、3天、5天、10天、2週、3週、4週、1個月、2個月、3個月、4個月、5個月、6個月、9個月、12個月、2年、3年、5年、10年或更長時間，或可在諸如骨骼、心臟或平滑肌細胞之靶細胞的整個生命週期中穩定。Once the cds DNA of the present invention is taken up by target cells, the terminal sequences of the present invention enable cdsDNA to exist as long-term stable extrachromosomal genetic elements. The cdsDNA having the terminal sequence can be stable for 6 hours, 12 hours, 1 day, 3 days, 5 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 2 years, 3 years, 5 years, 10 years or longer, or throughout the life cycle of target cells such as bone, heart or smooth muscle cells stable.

在某些實施例中，所關注之DNA片段側接一對ITR (例如雙股DNA病毒諸如AAV，包括AAV1-AAV10之ITR)，或LTR或內部重複序列(例如DNA病毒之LTR，諸如HSV之LTR)。在某些實施例中，ITR為AAV1-AAV10中之任一者的ITR。在某些實施例中，LTR為HSV之LTR。In certain embodiments, the DNA fragment of interest is flanked by a pair of ITRs (eg, a double-stranded DNA virus such as AAV, including the ITR of AAV1-AAV10), or an LTR or an internal repeat (eg, an LTR of a DNA virus, such as HSV) LTR). In certain embodiments, the ITR is an ITR of any of AAV1-AAV10. In certain embodiments, the LTR is the LTR of the HSV.

在某些實施例中，該對LTR為來自γ-反轉錄病毒或慢病毒之元件。LTR可包含在逆轉錄病毒原病毒之任一側發現的U3-R-U5區。In certain embodiments, the pair of LTRs are elements from gamma-retroviral or lentivirus. The LTR can comprise the U3-R-U5 region found on either side of the retroviral provirus.

在某些實施例中，U3為病毒基因組RNA之3'端的獨特3'區(但在原病毒之5'及3'端均發現)，其含有活化病毒基因組RNA轉錄所必需之序列。In certain embodiments, U3 is the unique 3' region of the 3' end of the viral genomic RNA (but found at the 5' and 3' ends of the provirus), which contains sequences necessary for activation of viral genomic RNA transcription.

在某些實施例中，R為在反轉錄病毒/慢病毒載體之5'及3' LTR內均發現之重複序列。Tat蛋白結合於此區。In certain embodiments, R is a repeat sequence found in both the 5' and 3' LTR of a retroviral/lentiviral vector. The Tat protein binds to this region.

在某些實施例中，U5為病毒基因組RNA之5'端的獨特5'區(但在原病毒之5'及3'端均發現)。In certain embodiments, U5 is the unique 5' region of the 5' end of the viral genomic RNA (but found at both the 5' and 3' ends of the provirus).

在某些實施例中，所關注之DNA片段的一端或兩端具有5' LTR區，其可充當RNA pol II啟動子。轉錄物可在其R區之起點開始，封端，且前進經過U5及所關注之DNA片段的其餘部分。在某些實施例中，雜合5' LTR可與諸如CMV或RSV啟動子之組成型啟動子一起使用。In certain embodiments, the DNA fragment of interest has a 5' LTR region at one or both ends that can serve as an RNA pol II promoter. The transcript can begin at the beginning of its R region, capped, and progress through U5 and the remainder of the DNA fragment of interest. In certain embodiments, a hybrid 5' LTR can be used with a constitutive promoter such as a CMV or RSV promoter.

在某些實施例中，LTR之R區可包含轉活化反應元件TAR，且充當Tat (結合TAR以活化自LTR啟動子之轉錄的轉活化因子)之結合位點。In certain embodiments, the R region of the LTR can comprise a transactivation response element TAR and serve as a binding site for Tat (a transactivation factor that binds TAR to activate transcription from the LTR promoter).

在某些實施例中，所關注之DNA片段的一端或兩端具有3' LTR區，其可藉由恰好在其R序列後添加poly A段來終止在所關注之DNA片段的另一端由5' LTR開始的轉錄。In certain embodiments, the DNA fragment of interest has a 3' LTR region at one or both ends, which can be terminated by the addition of a poly A segment just after its R sequence by the other end of the DNA fragment of interest. 'LTR begins transcription.

在某些實施例中，所關注之DNA片段的轉錄由5' LTR引發。In certain embodiments, transcription of a DNA fragment of interest is initiated by a 5' LTR.

在某些實施例中，所關注之DNA片段的轉錄由3' LTR引發。In certain embodiments, transcription of a DNA fragment of interest is initiated by a 3' LTR.

在某些實施例中，替代地或另外地，所關注之DNA片段本身可攜帶其自身的啟動子、強化子、其他轉錄/轉譯調節元件、聚腺苷酸化信號及/或轉譯終止位點。In certain embodiments, alternatively or additionally, the DNA fragment of interest may itself carry its own promoter, enhancer, other transcription/translation regulatory elements, polyadenylation signals, and/or translation termination sites.

在某些實施例中，轉譯調節元件包含WPRE或土拔鼠肝炎病毒轉錄後調節元件，其經由增加的核輸出刺激轉殖基因之表現。In certain embodiments, the translational regulatory element comprises a WPRE or a hamster hepatitis virus post-transcriptional regulatory element that stimulates expression of the transgene via increased nuclear export.

在某些實施例中，該對ITR為來自腺相關病毒(AAV)之元件。AAV ITR各約145個鹼基。ITR形成T形髮夾，其通常充當病毒DNA複製之起源。其含有封裝所需之D區。In certain embodiments, the pair of ITRs are elements from an adeno-associated virus (AAV). The AAV ITRs are each approximately 145 bases. The ITR forms a T-shaped hairpin that typically serves as the origin of viral DNA replication. It contains the D zone required for the package.

4. 原核端粒酶及原核端粒酶識別序列
本發明之方法包含藉由在促進cdsDNA產生之條件下使擴增的DNA構築體與釋放嵌段之原核端粒酶接觸來產生cdsDNA的步驟。4. Prokaryotic telomerase and prokaryotic telomerase recognition sequence The method of the present invention comprises the step of producing cdsDNA by contacting the amplified DNA construct with a prokaryotic telomerase releasing the block under conditions which promote cdsDNA production.

用於本發明之原核端粒酶為能夠裂解及重新連接包含原核端粒酶靶序列之模板以產生共價cdsDNA分子的任何多肽。具有原核端粒酶活性之酶亦描述為端粒解離酶(例如在伯氏疏螺旋體中)。原核端粒酶靶序列之要求如下所述。給定多肽催化自包含原核端粒酶靶序列之模板產生cdsDNA的能力可使用此項技術中描述之任何適合之分析來確定。A protelomerase useful in the present invention is any polypeptide capable of cleaving and religating a template comprising a prokaryotic telomerase target sequence to produce a covalent cds DNA molecule. An enzyme having protelomerase activity is also described as a telomere dissociation enzyme (e.g., in Borrelia burgdorferi). The requirements for the protelomerase target sequence are as follows. The ability of a given polypeptide to catalyze the production of cds DNA from a template comprising a prokaryotic telomerase target sequence can be determined using any suitable assay described in the art.

已在噬菌體中描述原核端粒酶。在一些溶源性細菌中，噬菌體作為染色體外DNA存在，其包含具有共價封閉末端之線性雙股。此DNA之複製及共價封閉末端(或端粒末端)之維持視原核端粒酶之活性而定。此催化活性之實例係由來自感染大腸桿菌之噬菌體N15的酶TelN提供。TelN識別環狀雙股DNA中之特定核苷酸序列。此序列為略微不完美的反向回文結構，稱為telRL，包含兩個半部telR及telL，側接22個鹼基對的反向完美重複序列(telO)。藉由作用於線性原噬菌體DNA之特定DNA聚合酶的初始活性，在環狀雙股DNA中形成兩個telRL位點。TelN將此環狀DNA轉化為兩個相同的線性原噬菌體DNA分子，從而完成複製週期。telR及telL包含線性原噬菌體DNA之封閉末端，使DNA能夠以相同的方式進一步複製。Prokaryotic telomerase has been described in phage. In some lysogenic bacteria, phage exist as extrachromosomal DNA, which comprises a linear double strand with a covalently closed end. The replication of this DNA and the maintenance of covalently blocked ends (or telomere ends) depend on the activity of the protelomerase. An example of this catalytic activity is provided by the enzyme TelN from phage N15 infected with E. coli. TelN recognizes a specific nucleotide sequence in circular double-stranded DNA. This sequence is a slightly imperfect reverse palindrome, called telRL, containing two halves of telR and telL, flanked by 22 base pairs of inverted perfect repeats (telO). Two telRL sites are formed in the circular double-stranded DNA by the initial activity of a specific DNA polymerase acting on linear prophage DNA. TelN converts this circular DNA into two identical linear prophage DNA molecules, completing the replication cycle. telR and telL contain the closed ends of linear prophage DNA, allowing DNA to be replicated in the same manner.

適合之原核端粒酶的實例包括來自以下噬菌體之原核端粒酶，諸如來自海水鹽單胞菌(Halomonas aquamarina )之phiHAP-1 (SEQ ID NO: 7)、來自小腸結腸炎耶爾森氏菌(Yersinia enterolytica )之PY54 (SEQ ID NO: 9)、來自產酸克雷伯氏菌(Klebsiella oxytoca )之phiKO2 (SEQ ID NO: 11)、來自弧菌屬之VP882 (SEQ ID NO: 13)及來自大腸桿菌之N15 (SEQ ID NO: 15)或其任何變體。在某些實施例中，使用噬菌體N15原核端粒酶(SEQ ID NO: 15)或其變體。Examples of suitable protelomerases include prokaryotic telomerase from the following phage, such as phiHAP-1 (SEQ ID NO: 7) from Halomonas aquamarina , from Yersinia enterocolitica ( Yersinia enterolytica ) PY54 (SEQ ID NO: 9), phiKO2 (SEQ ID NO: 11) from Klebsiella oxytoca , VP882 (SEQ ID NO: 13) from Vibrio and N15 from E. coli (SEQ ID NO: 15) or any variant thereof. In certain embodiments, phage N15 prokaryotic telomerase (SEQ ID NO: 15) or variants thereof are used.

SEQ ID NO: 7、9、11、13及15之變體包括其同源物或突變體。突變體包括相對於天然序列之截短、取代或缺失。變體必須自包含如上所述之原核端粒酶靶位點的模板產生封閉的線性DNA。Variants of SEQ ID NO: 7, 9, 11, 13 and 15 include homologs or mutants thereof. Mutants include truncations, substitutions or deletions relative to the native sequence. The variant must produce a closed linear DNA from a template comprising a prokaryotic telomerase target site as described above.

本文提及之任何同源物通常為功能同源物，且通常與天然蛋白質之相關區域至少40%同源。可使用已知方法量測同源性。舉例而言，UWGCG套件提供BESTFIT程式，其可用於計算同源性(例如在其預設設置下使用) (Devereux等人 (1984) Nucleic Acids Research 12, 387-395)。PILEUP及BLAST算法可用於計算同源性或對齊序列(通常在其預設設置下)，如Altschul S. F. (1993) J Mol Evol 36:290-300；Altschul, S, F等人 (1990) J Mol Biol 215:403-10中所述。進行BLAST分析之軟體可經由國家生物技術資訊中心(National Center for Biotechnology Information)公開獲得。Any homologue referred to herein is typically a functional homolog and is typically at least 40% homologous to the relevant region of the native protein. The homology can be measured using known methods. For example, the UWGCG kit provides a BESTFIT program that can be used to calculate homology (eg, used under its default settings) (Devereux et al. (1984) Nucleic Acids Research 12, 387-395). The PILEUP and BLAST algorithms can be used to calculate homology or alignment sequences (usually under their default settings), eg Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al. (1990) J Mol Biol 215: 403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

BLAST算法進行兩個序列之間相似性的統計分析；參見例如Karlin及Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787。BLAST算法提供的一種相似性量度係最小和概率(P(N))，其提供兩個核苷酸或胺基酸序列之間偶然發生匹配之概率的指示。舉例而言，若第一序列與第二序列之比較中的最小和概率小於約1，較佳小於約0.1，更佳小於約0.01且最佳小於約0.001，則認為序列與另一序列相似。The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One similarity measure provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability of an accidental match between two nucleotide or amino acid sequences. For example, a sequence is considered similar to another sequence if the minimum sum probability in the comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

「原核端粒酶靶序列」或「原核端粒酶識別序列」(在本文中可互換使用)係由原核端粒酶識別之聚核苷酸(例如雙股DNA)序列，用於藉由原核端粒酶裂解及重新連接以形成共價封閉末端dsDNA。藉由原核端粒酶消化原核端粒酶靶/識別序列產生兩個「半原核端粒酶靶/識別序列」。"Prokaryotic telomerase target sequence" or "prokaryotic telomerase recognition sequence" (used interchangeably herein) is a sequence of polynucleotide (eg, double stranded DNA) recognized by protelomerase, used for prokaryotic Telomerase cleaves and religates to form covalently blocked terminal dsDNA. Two "semi-prokaryotic telomerase target/recognition sequences" are produced by prokaryotic telomerase digestion of prokaryotic telomerase target/recognition sequences.

在某些實施例中，原核端粒酶靶序列包含完美的回文序列，亦即具有雙重旋轉對稱性之雙股DNA序列，在本文中亦描述為完美的反向重複序列。In certain embodiments, the prokaryotic telomerase target sequence comprises a perfect palindromic sequence, ie, a double stranded DNA sequence having dual rotational symmetry, also described herein as a perfect inverted repeat sequence.

來自各種嗜溫性噬菌體及細菌質體之原核端粒酶靶序列均具有包含完美反向重複序列之共同特徵。完美反向重複序列之長度視特定生物體而不同。Prokaryotic telomerase target sequences from various mesophilic bacteriophages and bacterial plastids all have the common feature of containing perfect inverted repeats. The length of a perfect inverted repeat differs depending on the particular organism.

在某些實施例中，完美反向重複序列來自伯氏疏螺旋體，且長度為14 bp。In certain embodiments, the perfect inverted repeat sequence is from Borrelia burgdorferi and is 14 bp in length.

在某些實施例中，完美反向重複序列來自嗜溫性噬菌體，且長度為22 bp或更長。In certain embodiments, the perfect inverted repeat sequence is derived from a mesophilic phage and is 22 bp or longer in length.

在某些實施例中，原核端粒酶識別序列來自大腸桿菌N15，其中中央完美反向回文序列側接反向重複序列，亦即形成較大的不完美反向回文序列的一部分(參見US9109250B2中之圖2及3，以引用的方式併入本文中；加下劃線的鹼基表示反向重複序列之對稱性被中斷處)。In certain embodiments, the prokaryotic telomerase recognition sequence is from E. coli N15, wherein the central perfect inverted palindrome sequence is flanked by inverted repeats, ie, forms part of a larger imperfect inverted palindrome sequence (see Figures 2 and 3 of US 9109250 B2 are incorporated herein by reference; the underlined bases indicate that the symmetry of the inverted repeat is interrupted).

在某些實施例中，原核端粒酶靶序列包含至少14個鹼基對長度之雙股回文(完美反向重複)序列。在某些實施例中，完美反向重複序列包括SEQ ID NO: 11至16之序列及其變體。In certain embodiments, the prokaryotic telomerase target sequence comprises a double stranded palindrome (perfect inverted repeat) sequence of at least 14 base pairs in length. In certain embodiments, the perfect inverted repeat sequence comprises the sequences of SEQ ID NOs: 11 to 16 and variants thereof.

SEQ ID NO: 11 (NCATNNTANNCGNNTANNATGN)為用於嗜溫性噬菌體完美反向重複序列之22個鹼基的共同序列。完美反向重複序列之鹼基對在不同噬菌體之間的某些位置為保守的，而在其他位置序列可能為靈活的。因此，SEQ ID NO: 11為在本發明之方法中與噬菌體原核端粒酶一起使用之完美反向重複序列的最小共同序列。SEQ ID NO: 11 (NCATNNTANNCGNNTANNATGN) is a 22 base common sequence for a mesophilic phage perfect inverted repeat. Base pairs of perfect inverted repeats are conserved at certain positions between different phage, while sequences at other positions may be flexible. Thus, SEQ ID NO: 11 is the minimal common sequence of the perfect inverted repeats used with the bacteriophage protelomerase in the methods of the invention.

在由SEQ ID NO: 11定義之共同序列內，SEQ ID NO: 12 (CCATTATACGCGCGTATAATGG)係與大腸桿菌噬菌體N15 (SEQ ID NO: 10)及克雷伯氏菌噬菌體Phi KO2 (SEQ ID NO: 6)原核端粒酶一起使用的完美反向重複序列。Within the common sequence defined by SEQ ID NO: 11, SEQ ID NO: 12 (CCATTATACGCGCGTATAATGG) is linked to Escherichia coli bacteriophage N15 (SEQ ID NO: 10) and Klebsiella phage Phi KO2 (SEQ ID NO: 6). The perfect inverted repeat sequence used with protelomerase.

亦在由SEQ ID NO: 11定義之共同序列內，SEQ ID NO: 13至15：
GCATACTACGCGCGTAGTATGC (SEQ ID NO: 13)，
CCATACTATACGTATAGTATGG (SEQ ID NO: 14)，
GCATACTATACGTATAGTATGC (SEQ ID NO: 15)，
為分別與來自耶爾森氏菌噬菌體PY54 (SEQ ID NO: 4)、鹽單胞菌噬菌體phiHAP-1 (SEQ ID NO: 2)及弧菌噬菌體VP882 (SEQ ID NO: 8)之原核端粒酶一起使用的完美反向重複序列。Also within the common sequence defined by SEQ ID NO: 11, SEQ ID NOS: 13 to 15:
GCATACTACGCGCGTAGTATGC (SEQ ID NO: 13),
CCATACTATACGTATAGTATGG (SEQ ID NO: 14),
GCATACTATACGTATAGTATGC (SEQ ID NO: 15),
For prokaryotic telomeres from Yersinia phage PY54 (SEQ ID NO: 4), S. cerevisiae phage phiHAP-1 (SEQ ID NO: 2) and Vibrio phage VP882 (SEQ ID NO: 8), respectively The perfect inverted repeat sequence used with the enzyme.

SEQ ID NO: 16 (ATTATATATATAAT)係與伯氏疏螺旋體原核端粒酶一起使用的完美反向重複序列。此完美反向重複序列來自線性共價封閉質體，亦即伯氏疏螺旋體中所包含之lpB31.16。此14-bp序列短於噬菌體之22-bp共同完美反向重複序列(SEQ ID NO: 11)，表明細菌原核端粒酶可能對噬菌體原核端粒酶之特定靶序列要求不同。然而，所有原核端粒酶靶序列共有完美反向重複序列之共同結構基元。SEQ ID NO: 16 (ATTATATATATAAT) is a perfect inverted repeat sequence for use with Borrelia burgdorferi protelomerase. This perfect inverted repeat is derived from a linear covalently closed plastid, lpB31.16 contained in Borrelia burgdorferi. This 14-bp sequence is shorter than the 22-bp common perfect inverted repeat of phage (SEQ ID NO: 11), indicating that bacterial protelomerase may have different target sequence requirements for phage protelomerase. However, all protelomerase target sequences share a common structural motif of a perfect inverted repeat.

完美反向重複序列之長度可大於22 bp，視本發明方法中使用之特定原核端粒酶的要求而定。因此，在一些實施例中，完美反向重複序列的長度可為至少30、至少40、至少60、至少80或至少100個鹼基對。此類完美反向重複序列之實例包括SEQ ID NO: 17至19及其變體。
GGCATACTATACGTATAGTATGCC (SEQ ID NO: 17)
ACCTATTTCAGCATACTACGCGCGTAGTATGCTGAAATAGGT (SEQ ID NO: 18)
CCTATATTGGGCCACCTATGTATGCACAGTTCGCCCATACTATACGTATAGTATGGGCGAACTGTGCATACATAGGTGGCCCAATATAGG (SEQ ID NO: 19)The perfect inverted repeat sequence can be greater than 22 bp in length, depending on the requirements of the particular protelomerase used in the methods of the invention. Thus, in some embodiments, the perfect inverted repeat sequence can be at least 30, at least 40, at least 60, at least 80, or at least 100 base pairs in length. Examples of such perfect inverted repeat sequences include SEQ ID NOS: 17 to 19 and variants thereof.
GGCATACTATACGTATAGTATGCC (SEQ ID NO: 17)
ACCTATTTCAGCATACTACGCGCGTAGTATGCTGAAATAGGT (SEQ ID NO: 18)
CCTATATTGGGCCACCTATGTATGCACAGTTCGCCCATACTATACGTATAGTATGGGCGAACTGTGCATACATAGGTGGCCCAATATAGG (SEQ ID NO: 19)

SEQ ID NO: 17至19及其變體分別與來自弧菌噬菌體VP882 (SEQ ID NO: 8)、耶爾森氏菌噬菌體PY54 (SEQ ID NO: 4)及鹽單胞菌噬菌體phi HAP-1 (SEQ ID NO: 2)之原核端粒酶一起使用。SEQ ID NOS: 17 to 19 and variants thereof, respectively, from Vibrio phage VP882 (SEQ ID NO: 8), Yersinia phage PY54 (SEQ ID NO: 4), and the phage phi HAP-1 The prokaryotic telomerase of (SEQ ID NO: 2) was used together.

完美反向重複序列可側接額外的反向重複序列。側接反向重複序列可為完美或不完美的重複序列，亦即可為完全對稱或部分對稱的。側接反向重複序列可與中央回文序列鄰接或非鄰接。A perfect inverted repeat can be flanked by additional inverted repeats. The flanked inverted repeats can be perfect or imperfect repeats, either completely symmetrical or partially symmetrical. The flanked inverted repeats may be contiguous or non-contiguous with the central palindrome sequence.

在某些實施例中，原核端粒酶靶序列可包含不完美的反向重複序列，其包含至少14個鹼基對長的完美反向重複序列。一個實例為SEQ ID NO: 24。In certain embodiments, a prokaryotic telomerase target sequence can comprise an imperfect inverted repeat sequence comprising a perfect inverted repeat of at least 14 base pairs long. An example is SEQ ID NO: 24.

在某些實施例中，不完美的反向重複序列可包含至少22個鹼基對長的完美反向重複序列。一個實例為SEQ ID NO: 20。In certain embodiments, an imperfect inverted repeat sequence can comprise a perfect inverted repeat of at least 22 base pairs long. An example is SEQ ID NO: 20.

在某些實施例中，原核端粒酶靶序列包含SEQ ID NO: 20-24中之任一者的序列或其變體。
TATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGTGCTGATA (SEQ ID NO: 20)
ATGCGCGCATCCATTATACGCGCGTATAATGGCGATAATACA (SEQ ID NO: 21)
TAGTCACCTATTTCAGCATACTACGCGCGTAGTATGCTGAAATAGGTTACTG (SEQ ID NO: 22)
GGGATCCCGTTCCATACATACATGTATCCATGTGGCATACTATACGTATAGTATGCCGATGTTACATATGGTATCATTCGGGATCCCGTT (SEQ ID NO: 23)
TACTAAATAAATATTATATATATAATTTTTTATTAGTA (SEQ ID NO: 24)In certain embodiments, the protelomerase target sequence comprises the sequence of any one of SEQ ID NOs: 20-24, or variants thereof.
TATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGTGCTGATA (SEQ ID NO: 20)
ATGCGCGCATCCATTATACGCGCGTATAATGGCGATAATACA (SEQ ID NO: 21)
TAGTCACCTATTTCAGCATACTACGCGCGTAGTATGCTGAAATAGGTTACTG (SEQ ID NO: 22)
GGGATCCCGTTCCATACATACATGTATCCATGTGGCATACTATACGTATAGTATGCCGATGTTACATATGGTATCATTCGGGATCCCGTT (SEQ ID NO: 23)
TACTAAATAAATATTATATATATAATTTTTTATTAGTA (SEQ ID NO: 24)

SEQ ID NO: 20至24之序列包含如上所述之完美反向重複序列，且另外包含來自相關生物體之側接序列。The sequences of SEQ ID NOS: 20 to 24 comprise a perfect inverted repeat as described above, and additionally comprise flanking sequences from related organisms.

在某些實施例中，包含SEQ ID NO: 20之序列或其變體的原核端粒酶靶序列係與SEQ ID NO: 10之大腸桿菌N15 TelN原核端粒酶及其變體組合使用。In certain embodiments, a prokaryotic telomerase target sequence comprising the sequence of SEQ ID NO: 20 or a variant thereof is used in combination with E. coli N15 TelN protelomerase of SEQ ID NO: 10 and variants thereof.

在某些實施例中，包含SEQ ID NO: 21之序列或其變體的原核端粒酶靶序列係與SEQ ID NO: 6之克雷伯氏菌噬菌體Phi K02原核端粒酶及其變體組合使用。In certain embodiments, the prokaryotic telomerase target sequence comprising the sequence of SEQ ID NO: 21 or a variant thereof and the Klebsiella phage Phi K02 prokaryotic telomerase of SEQ ID NO: 6 and variants thereof Used in combination.

在某些實施例中，包含SEQ ID NO: 22之序列或其變體的原核端粒酶靶序列係與SEQ ID NO: 4之耶爾森氏菌噬菌體PY54原核端粒酶及其變體組合使用。In certain embodiments, the prokaryotic telomerase target sequence comprising the sequence of SEQ ID NO: 22 or a variant thereof is combined with the Yersinia phage PY54 prokaryotic telomerase of SEQ ID NO: 4 and variants thereof use.

在某些實施例中，包含SEQ ID NO: 23之序列或其變體的原核端粒酶靶序列係與SEQ ID NO: 8之弧菌噬菌體VP882原核端粒酶及其變體組合使用。In certain embodiments, a prokaryotic telomerase target sequence comprising the sequence of SEQ ID NO: 23 or a variant thereof is used in combination with Vibrio phage VP882 prokaryotic telomerase of SEQ ID NO: 8 and variants thereof.

在某些實施例中，包含SEQ ID NO: 24之序列或其變體的原核端粒酶靶序列係與伯氏疏螺旋體原核端粒酶組合使用。In certain embodiments, a prokaryotic telomerase target sequence comprising the sequence of SEQ ID NO: 24 or a variant thereof is used in combination with Borrelia burgdorferi protelomerase.

上述回文序列或原核端粒酶靶序列中之任一者的變體包括其同源物或突變體。突變體包括相對於天然序列之截短、取代或缺失。變體序列為當存在於DNA模板中時允許其藉由原核端粒酶之酶活性轉化為封閉的線性DNA的任何序列。此點很容易藉由使用針對所形成封閉的線性DNA之適當分析法來確定。可使用此項技術中描述之任何合適分析法。一個合適分析法實例描述於Deneke等人, PNAS 97:7721-7726, 2000 (以引用的方式併入本文)中。在某些實施例中，變體可允許原核端粒酶之結合性及活性與天然序列觀察到的活性相當。Variants of any of the above palindrome sequences or prokaryotic telomerase target sequences include homologs or mutants thereof. Mutants include truncations, substitutions or deletions relative to the native sequence. A variant sequence is any sequence that, when present in a DNA template, is allowed to be converted to a closed linear DNA by the enzymatic activity of protelomerase. This is easily determined by using an appropriate assay for the blocked linear DNA formed. Any suitable assay described in this technique can be used. An example of a suitable assay is described in Deneke et al., PNAS 97:7721-7726, 2000 (incorporated herein by reference). In certain embodiments, the variant may allow for the binding and activity of prokaryotic telomerase to be comparable to that observed for the native sequence.

本文所述之回文序列之變體的實例包括截短的回文序列，其保留完美的重複結構，且仍能夠允許形成封閉的線性DNA。Examples of variants of the palindromic sequences described herein include truncated palindromic sequences that retain a perfect repeating structure and are still capable of allowing the formation of a closed linear DNA.

在某些實施例中，變體原核端粒酶靶序列經修飾以使其不再保留完美的回文序列，其限制條件為其能夠充當原核端粒酶活性之受質。In certain embodiments, the variant protelomerase target sequence is modified such that it no longer retains a perfect palindromic sequence, the restriction being that it is capable of acting as a substrate for protelomerase activity.

應理解，熟習此項技術者將能夠基於上述結構原理容易地鑑定適用於本發明之原核端粒酶靶序列。It will be appreciated that those skilled in the art will be able to readily identify prokaryotic telomerase target sequences suitable for use in the present invention based on the above structural principles.

在某些實施例中，可使用上述分析篩選候選原核端粒酶靶序列促進封閉末端DNA形成的能力。In certain embodiments, the above assay can be used to screen for the ability of a candidate protelomerase target sequence to facilitate blocking of terminal DNA formation.

下面列出可用於本發明之組合物及方法的某些例示性原核端粒酶序列。Certain exemplary prokaryotic telomerase sequences useful in the compositions and methods of the invention are listed below.

鹽單胞菌噬菌體phiHAP-1原核端粒酶核酸序列：
Protease phage phiHAP-1 prokaryotic telomerase nucleic acid sequence:

鹽單胞菌噬菌體phiHAP-1原核端粒酶胺基酸序列：
Protease phage phiHAP-1 prokaryotic telomerase amino acid sequence:

耶爾森氏菌噬菌體PY54原核端粒酶核酸序列：
Yersinia phage PY54 prokaryotic telomerase nucleic acid sequence:

耶爾森氏菌噬菌體PY54原核端粒酶胺基酸序列：
Yersinia phage PY54 prokaryotic telomerase amino acid sequence:

克雷伯氏菌噬菌體phiKO2原核端粒酶核酸序列：
Klebsiella phage phiKO2 prokaryotic telomerase nucleic acid sequence:

克雷伯氏菌噬菌體phiKO2原核端粒酶胺基酸序列：
Prokaryotic telomerase amino acid sequence of Klebsiella phage phiKO2:

弧菌噬菌體VP882原核端粒酶核酸序列：
Vibrio phage VP882 prokaryotic telomerase nucleic acid sequence:

弧菌噬菌體VP882原核端粒酶胺基酸序列：
Vibrio phage VP882 prokaryotic telomerase amino acid sequence:

大腸桿菌噬菌體N15端粒酶(telN)及二級免疫阻遏物(cA)核酸序列：
E. coli phage N15 telomerase (telN) and secondary immunorepressor (cA) nucleic acid sequences:

大腸桿菌噬菌體N15端粒酶胺基酸序列：
E. coli bacteriophage N15 telomerase amino acid sequence:

擴增之DNA構築體可在促進封閉cdsDNA產生的條件下與至少一種原核端粒酶一起培育。促進dsDNA裂解及重新連接之條件包含原核端粒酶靶序列，以形成具有髮夾末端之共價封閉的末端DNA。促進封閉末端DNA產生之條件包含使用允許產生封閉末端DNA之任何溫度，通常在20至90℃之範圍內。溫度可在25至40℃之範圍內，諸如約25至約35℃，或約30℃。可根據上文關於DNA聚合酶之溫度條件概述的原理選擇特定原核端粒酶的適當溫度。與SEQ ID NO: 10之大腸桿菌噬菌體TelN原核端粒酶一起使用的適合溫度為約25至約35℃，諸如約30℃。The amplified DNA construct can be incubated with at least one protelomerase under conditions that promote the production of blocked cdsDNA. Conditions that facilitate cleavage and religation of dsDNA comprise a prokaryotic telomerase target sequence to form a covalently blocked terminal DNA having a hairpin end. Conditions for promoting blocked terminal DNA production include the use of any temperature that permits the production of blocked terminal DNA, typically in the range of 20 to 90 °C. The temperature can range from 25 to 40 °C, such as from about 25 to about 35 °C, or about 30 °C. The appropriate temperature for a particular prokaryotic telomerase can be selected according to the principles outlined above with respect to the temperature conditions of the DNA polymerase. Suitable temperatures for use with the E. coli bacteriophage TelN protelomerase of SEQ ID NO: 10 are from about 25 to about 35 °C, such as about 30 °C.

促進封閉末端DNA產生之條件亦包含存在原核端粒酶及適合之緩衝劑/pH及酶效能或穩定性所需之其他因素。適合之條件包括用於提供此項技術中已知的原核端粒酶活性的任何條件。舉例而言，在使用大腸桿菌噬菌體TelN原核端粒酶之情況下，適合之緩衝液可為20 mM TrisHCl，pH 7.6；5 mM CaCl2；50 mM麩胺酸鉀；0.1 mM EDTA；1 mM二硫蘇糖醇(DTT)。維持最佳活性及穩定性之藥劑及條件亦可選自關於DNA聚合酶所列之藥劑及條件。Conditions that promote closed-end DNA production also include other factors required for the presence of prokaryotic telomerase and suitable buffer/pH and enzyme potency or stability. Suitable conditions include any conditions for providing prokaryotic telomerase activity as known in the art. For example, in the case of using E. coli bacteriophage TelN protelomerase, a suitable buffer may be 20 mM TrisHCl, pH 7.6; 5 mM CaCl2; 50 mM potassium glutamate; 0.1 mM EDTA; 1 mM disulfide Threitol (DTT). The agents and conditions for maintaining optimal activity and stability may also be selected from the agents and conditions listed for the DNA polymerase.

用於本發明方法之所有酶及蛋白質可例如在細菌中重組產生。可使用熟習此項技術者已知的任何允許重組表現的方式。可將包含編碼所關注蛋白質之核酸序列的質體或其他形式之表現載體引入細菌中，以使其表現所編碼之蛋白質。舉例而言，對於SEQ ID NO: 2、4、6、8或10之表現，載體可分別包含SEQ ID NO: 1、3、5、7或9之序列。所表現之蛋白質隨後通常例如藉由使用足量的親和標籤來純化，且以適用於本發明方法之形式提供。用於重組蛋白生產之此類方法為熟習此項技術者基於其一般知識常規可用的。以上論述適用於提供本文所論述之任何蛋白質。All of the enzymes and proteins used in the methods of the invention can be produced recombinantly, for example, in bacteria. Any means known to those skilled in the art that allows for recombinant performance can be used. A plastid or other form of expression vector comprising a nucleic acid sequence encoding a protein of interest can be introduced into the bacterium to express the encoded protein. For example, for the expression of SEQ ID NO: 2, 4, 6, 8, or 10, the vector may comprise the sequence of SEQ ID NO: 1, 3, 5, 7, or 9, respectively. The expressed protein is then typically purified, for example, by using a sufficient amount of affinity tag, and is provided in a form suitable for use in the methods of the invention. Such methods for recombinant protein production are routinely available to those skilled in the art based on their general knowledge. The above discussion is applicable to providing any of the proteins discussed herein.

在某些實施例中，擴增之DNA在與原核端粒酶接觸之前經純化。因此，本發明之方法可進一步包含純化擴增之DNA的步驟。在某些實施例中，擴增之DNA在與原核端粒酶接觸之前未經純化。在一些實施例中，該方法包含添加提供原核端粒酶活性之緩衝液，亦即提供促進封閉末端DNA形成之條件。In certain embodiments, the amplified DNA is purified prior to contact with prokaryotic telomerase. Thus, the method of the invention may further comprise the step of purifying the amplified DNA. In certain embodiments, the amplified DNA is not purified prior to contact with prokaryotic telomerase. In some embodiments, the method comprises adding a buffer that provides protelomerase activity, that is, providing conditions that promote the formation of blocked terminal DNA.

5. DNA構築體
本發明之DNA構築體可包含不止一種原核端粒酶靶序列，例如2、3、4、5、6、7、8、9、10或更多種原核端粒酶靶序列。多種原核端粒酶靶序列允許自較大的DNA分子(諸如質體或自我複製的染色體外基因元件)切除各自包含所關注之DNA片段的短封閉末端DNA。舉例而言，一或多個嵌段，各自包含側接其自身的一對末端序列的所關注之DNA片段，可各在任一側(末端序列之外)進一步側接原核端粒酶靶序列。可在兩個相鄰嵌段之間共享相同的原核端粒酶靶序列。兩個側接原核端粒酶序列可隨後介導自擴增之較大DNA分子切除各嵌段作為封閉末端DNA，經受原核端粒酶之作用。5. DNA constructs The DNA constructs of the invention may comprise more than one protelomerase target sequence, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more protelomerase target sequences. . A variety of protelomerase target sequences allow for the excision of short closed-end DNA, each containing a DNA fragment of interest, from a larger DNA molecule, such as a plastid or self-replicating extra-chromosomal genetic element. For example, one or more of the blocks, each comprising a DNA fragment of interest flanked by its own pair of end sequences, can be further flanked by prokaryotic telomerase target sequences on either side (outside the end sequence). The same prokaryotic telomerase target sequence can be shared between two adjacent blocks. The two flanking protelomerase sequences can then be mediated by the larger DNA molecules amplified from the amplified blocks as closed-end DNA, subjected to protelomerase.

在一個實施例中，DNA構築體包含具有所關注之DNA片段(諸如表現卡匣)的嵌段，其在任一側側接原核端粒酶靶序列。在一個實施例中，所關注之DNA片段(例如表現卡匣)可包含可操作地連接於所關注之編碼序列的真核啟動子及視情況選用之真核轉錄終止序列。在此實施例中，在擴增DNA構築體且與原核端粒酶接觸後，所關注之DNA片段(例如表現卡匣)作為封閉末端DNA自擴增的DNA構築體釋放。In one embodiment, the DNA construct comprises a block having a DNA fragment of interest (such as a representational cassette) flanked by a prokaryotic telomerase target sequence on either side. In one embodiment, a DNA fragment of interest (eg, a representational cassette) can comprise a eukaryotic promoter operably linked to a coding sequence of interest and, optionally, a eukaryotic transcription termination sequence. In this embodiment, after amplification of the DNA construct and contact with the protelomerase, the DNA fragment of interest (eg, the expression cassette) is released as a closed-end DNA from the amplified DNA construct.

由於原核端粒酶消化，DNA構築體中之其他序列同時缺失。此類序列通常為細菌或載體序列，其可包括細菌複製起點、細菌選擇標記(例如抗生素抗性基因)及未甲基化之CpG二核苷酸。此類序列之缺失產生「最小的」所關注之DNA片段(例如表現卡匣)，其不含外來基因物質。Due to protelomerase digestion, other sequences in the DNA construct are simultaneously deleted. Such sequences are typically bacterial or vector sequences which may include bacterial origins of replication, bacterial selectable markers (eg, antibiotic resistance genes), and unmethylated CpG dinucleotides. Deletion of such sequences results in a "minimum" DNA fragment of interest (eg, a performance cassette) that is free of foreign genetic material.

在一些實施例中，尤其在封閉末端DNA產物為DNA疫苗時，CpG基元可保留在產物之序列中。In some embodiments, particularly when the blocked terminal DNA product is a DNA vaccine, the CpG motif can remain in the sequence of the product.

因此，本發明之一個態樣提供用於生產醫藥組合物之活體外方法，該醫藥組合物包含側接末端序列之封閉末端雙股DNA。此方法包含：a)在宿主細胞(例如真核或原核宿主細胞)中擴增DNA構築體，其包含側接一對末端序列(諸如在宿主細胞之生命週期中促進cdsDNA穩定性之ITR、LTR或端粒序列)且進一步側接一對原核端粒酶靶序列的至少一種所關注之DNA片段(例如表現卡匣)；及b)在促進該對原核端粒酶靶序列裂解之條件下，使a)中產生的擴增之DNA構築體與一或多個特異性針對該對原核端粒酶靶序列之原核端粒酶接觸，以形成包含所關注之DNA片段的封閉末端雙股DNA。Accordingly, one aspect of the invention provides an in vitro method for the production of a pharmaceutical composition comprising a blocked terminal double stranded DNA flanked by a terminal sequence. The method comprises: a) amplifying a DNA construct in a host cell (eg, a eukaryotic or prokaryotic host cell) comprising a pair of terminal sequences flanking (such as an ITR, LTR that promotes cdsDNA stability during the life cycle of the host cell) Or a telomere sequence) and further flanked by at least one DNA fragment of interest (eg, a cassette) of a prokaryotic telomerase target sequence; and b) under conditions that promote cleavage of the prokaryotic telomerase target sequence, The amplified DNA construct produced in a) is contacted with one or more prokaryotic telomerase specific for the pair of prokaryotic telomerase target sequences to form a closed terminal double stranded DNA comprising the DNA fragment of interest.

在某些實施例中，cdsDNA包含以下、由以下組成或基本上由以下組成：可操作地連接於所關注之編碼序列的真核啟動子及視情況選用之真核轉錄終止序列，其全部側接一對末端序列。In certain embodiments, the cdsDNA comprises, consists of, or consists essentially of: a eukaryotic promoter operably linked to a coding sequence of interest, and optionally a eukaryotic transcription termination sequence, all sides of which Take a pair of end sequences.

在某些實施例中，cdsDNA缺乏細菌或載體序列，諸如(i)細菌複製起點；(ii)細菌選擇標記(通常為抗生素抗性基因)；及(iii)未甲基化之CpG基元。In certain embodiments, the cds DNA lacks a bacterial or vector sequence, such as (i) a bacterial origin of replication; (ii) a bacterial selectable marker (typically an antibiotic resistance gene); and (iii) an unmethylated CpG motif.

所關注之DNA片段可包含DNA疫苗，或編碼可校正靶細胞缺陷之基因產物的治療性DNA分子。The DNA fragment of interest may comprise a DNA vaccine, or a therapeutic DNA molecule encoding a gene product that corrects for defects in the target cell.

DNA構築體可呈質體或通常用於容納細菌繁殖基因之其他載體的形式，包括任何商業上可獲得的質體或其他載體，諸如商業上可獲得的DNA藥品，其經工程改造以包括側接末端序列及原核端粒酶識別序列。The DNA construct can be in the form of a plastid or other vector typically used to contain bacterial breeding genes, including any commercially available plastid or other vector, such as a commercially available DNA drug, engineered to include a side. The terminal sequence and the prokaryotic telomerase recognition sequence are ligated.

在某些實施例中，DNA構築體不含滾環擴增(RCA)所需之任何序列元件。In certain embodiments, the DNA construct does not contain any of the sequence elements required for rolling circle amplification (RCA).

6. 醫藥調配物及遞送
在藉由原核端粒酶之作用產生cdsDNA後，本發明之方法可進一步包含純化cdsDNA產物之步驟。6. Pharmaceutical Formulations and Delivery After the cdsDNA is produced by the action of protelomerase, the method of the present invention may further comprise the step of purifying the cdsDNA product.

在某些實施例中，純化移除不需要的副產物或雜質或兩者。In certain embodiments, purification removes unwanted by-products or impurities or both.

純化可藉由此項技術中已知的任何適合之方式來進行。舉例而言，純化可包括苯酚/氯仿核酸提取，或使用選擇性結合核酸之管柱，諸如可自Qiagen購得之彼等管柱。熟習此項技術者可常規地鑑定用於分離DNA之適合的純化技術。Purification can be carried out by any suitable means known in the art. For example, purification can include phenol/chloroform nucleic acid extraction, or use of a column that selectively binds nucleic acids, such as those available from Qiagen. Suitable purification techniques for isolating DNA can be routinely identified by those skilled in the art.

一旦已產生且純化足夠量的cdsDNA，該方法可進一步包含其作為DNA組合物之調配物，例如治療性DNA組合物或包含cdsDNA之醫藥組合物。Once a sufficient amount of cds DNA has been produced and purified, the method can further comprise its formulation as a DNA composition, such as a therapeutic DNA composition or a pharmaceutical composition comprising cdsDNA.

治療性DNA組合物包含上文提及之類型的治療性DNA分子，例如包含治療功能的所關注之DNA片段，其側接一對末端序列，且進一步側接一對原核端粒酶半識別序列。一旦cdsDNA (例如治療性DNA分子)在靶細胞內遞送，尤其當cdsDNA作為未整合至宿主細胞基因組/染色體中之染色體外基因元件存在時，末端序列可用以防止DNA分解。此類組合物將包含治療有效量之所關注之DNA片段，其呈適用於藉由所需途徑投與之形式，例如氣溶膠、可注射組成物或適用於非經腸(i . v . 、i . m . 等)、經口、黏膜或局部投藥之調配物。Therapeutic DNA composition comprises a therapeutic DNA molecule of the type mentioned above, such as a DNA fragment of interest comprising a therapeutic function flanked by a pair of terminal sequences and further flanked by a pair of prokaryotic telomerase semi-recognition sequences . Once cdsDNA (eg, a therapeutic DNA molecule) is delivered within a target cell, particularly when cdsDNA is present as an extrachromosomal gene element that is not integrated into the host cell genome/chromosome, the end sequence can be used to prevent DNA breakdown. Such compositions will contain a therapeutically effective amount of the DNA fragment of interest which was suitable for the desired route of administration therewith by form, e.g. an aerosol, or an injectable composition suitable for parenteral (i. V., i. m. and the like), the oral formulations, the mucous membrane or topical administration.

可使用熟習此項技術者可用之標準醫藥調配物化學及方法將DNA調配為習知醫藥製劑。可使用任何適合之醫藥學上可接受之載劑或賦形劑。輔助物質，諸如濕潤劑或乳化劑、pH緩衝物質及其類似物，可存在於賦形劑或媒劑中。此等賦形劑、媒劑及輔助物質一般為醫藥劑，其可投與而無過度毒性，且在疫苗組合物之情況下，在接收組合物之個體中不會誘導免疫反應。適合之載劑可為脂質體。The DNA can be formulated into a conventional pharmaceutical preparation using standard pharmaceutical formulation chemistry and methods available to those skilled in the art. Any suitable pharmaceutically acceptable carrier or excipient can be used. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the vehicle or vehicle. Such excipients, vehicles and auxiliary substances are generally pharmaceutical agents which can be administered without undue toxicity and, in the case of a vaccine composition, do not induce an immune response in the individual receiving the composition. Suitable carriers can be liposomes.

醫藥學上可接受之賦形劑包括但不限於液體，諸如水、鹽水、聚乙二醇、玻尿酸、甘油及乙醇。Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol.

醫藥學上可接受之鹽亦可包括於其中，例如無機酸鹽，諸如鹽酸鹽、氫溴酸鹽、磷酸鹽、硫酸鹽及其類似物；及有機酸鹽，諸如乙酸鹽、丙酸鹽、丙二酸鹽、苯甲酸鹽及其類似物。Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and organic acid salts such as acetates, propionates , malonate, benzoate and the like.

在某些實施例中，製劑亦含有醫藥學上可接受之賦形劑，其充當尤其肽、蛋白質或其他類似分子(若其包括於組合物中)之穩定劑。亦充當肽穩定劑之適合載劑的實例包括但不限於醫藥級右旋糖、蔗糖、乳糖、海藻糖、甘露糖醇、山梨糖醇、肌醇、葡聚糖及其類似物。其他適合之載劑包括但不限於澱粉、纖維素、磷酸鈉或磷酸鈣、檸檬酸、酒石酸、甘胺酸、高分子量聚乙二醇(PEG)及其組合。醫藥學上可接受之賦形劑、媒劑及輔助物質之透徹論述可在REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991)中獲得，其以引用的方式併入本文中。In certain embodiments, the formulation also contains a pharmaceutically acceptable excipient that acts as a stabilizer for particular peptides, proteins, or other similar molecules if included in the composition. Examples of suitable carriers that also function as peptide stabilizers include, but are not limited to, pharmaceutical grade dextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran, and the like. Other suitable carriers include, but are not limited to, starch, cellulose, sodium or calcium phosphate, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycol (PEG), and combinations thereof. A thorough discussion of pharmaceutically acceptable excipients, vehicles, and auxiliary substances is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991), which is incorporated herein by reference.

可使用非病毒遞送系統將主題cdsDNA遞送至任何所關注之靶細胞。非病毒基因遞送系統係基於藉由DNA或RNA之帶負電荷磷酸骨架與陽離子脂質、肽或其他聚合物之間的靜電相互作用將基因物質壓縮成奈米粒子(Erbacher等人,Gene Therapy 6:138-145, 1999)。與病毒相反，使用包括脂質之非病毒轉染載體可導致較低的毒性，尤其較低的免疫原性、更高的安全性、降低的成本、合理有效的靶向性及增強的封裝能力，例如處理核酸材料之大片段的能力。The subject cdsDNA can be delivered to any target cell of interest using a non-viral delivery system. Non-viral gene delivery systems are based on the compression of genetic material into nanoparticle by electrostatic interaction between a negatively charged phosphate backbone of DNA or RNA and a cationic lipid, peptide or other polymer (Erbacher et al, Gene Therapy 6: 138-145, 1999). In contrast to viruses, the use of non-viral transfection vectors including lipids can result in lower toxicity, especially lower immunogenicity, higher safety, reduced cost, reasonable and effective targeting, and enhanced encapsulation. For example the ability to process large fragments of nucleic acid material.

在某些實施例中，主題cdsDNA係使用任何適合之非病毒基因療法載體來遞送，諸如Yin等人,Non - viral vectors for gene - based therapy .Nature reviews Genetics , 15:541-555, 2014；或Schroeder等人,Lipid - based nanotherapeutics for siRNA delivery .J Intern Med . 267:9-21, 2010；或Zhao及Huang, Lipid nanoparticles for gene delivery.Adv Genet . 88: 13-36, 2014(全部以引用的方式併入本文中)中所述之彼等非病毒基因療法載體。In certain embodiments, the subject cdsDNA is delivered using any suitable non-viral gene therapy vector, such as Yin et al, Non - viral vectors for gene - based therapy . Nature reviews Genetics , 15:541-555, 2014; Schroeder et al, Lipid - based nanotherapeutics for siRNA delivery . J Intern Med . 267:9-21, 2010; or Zhao and Huang, Lipid nanoparticles for gene delivery. Adv Genet . 88: 13-36, 2014 (all cited The modes are incorporated into their non-viral gene therapy vectors as described herein.

在某些實施例中，cdsDNA係使用脂複合體遞送用於基於脂質之核酸複合物(參見Feigner等人,Human Gene Therapy 8:511-512, 1997，以引用的方式併入)。術語「LPD」為脂多聚複合體之一種形式，代表包含脂質、整合素(或其他受體)結合肽及DNA (或其他核酸)之調配物。LPD複合物經由整合素介導或其他受體介導之途徑實現轉染。其不一定需要具有總體正電荷，因此可減少不期望的血清相互作用。LPD之肽組分提供核酸封裝功能，保護DNA或RNA免於細胞內或細胞外核內體或以其他方式降解。脂質組分藉由膜融合或滲透介導與核內體脂質雙層之相互作用，減少核內體或溶酶體降解且允許核酸負荷物運輸至細胞質。In certain embodiments, the cdsDNA is delivered using a lipid complex for a lipid-based nucleic acid complex (see Feigner et al, Human Gene Therapy 8: 511-512, 1997, incorporated by reference). The term "LPD" is a form of a lipid multimeric complex that represents a formulation comprising a lipid, an integrin (or other receptor) binding peptide, and DNA (or other nucleic acid). LPD complexes are transfected via integrin-mediated or other receptor-mediated pathways. It does not necessarily need to have an overall positive charge, thus reducing undesirable serum interactions. The peptide component of LPD provides a nucleic acid encapsulation function that protects DNA or RNA from intracellular or extracellular endosomes or otherwise degrades. The lipid component mediates interaction with the endosomal lipid bilayer by membrane fusion or osmosis, reduces endosomal or lysosomal degradation and allows nucleic acid load to be transported to the cytoplasm.

在某些實施例中，肽組分設計為細胞類型特異性或細胞表面受體特異性的。舉例而言，整合素或其他受體之特異性程度可賦予LPD複合物一定程度的細胞特異性。特異性由靶向細胞表面受體(例如整合素受體)產生，且可實現與一些腺病毒載體相當之轉染效率。(參見Du等人, The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations. Sci Rep. 4:7107, 2014；Welser等人, Gene delivery using ternary lipopolyplexes incorporating branched cationic peptides: the role of Peptide sequence and branching. Mol Pharm. 10:127-41, 2013；Meng等人, Inhibition of neointimal hyperplasia in a rabbit vein graft model following non-viral transfection with human iNOS cDNA. Gene Ther. 20:979-86, 2013；Manunta等人, Airway deposition of nebulized gene delivery nanocomplexes monitored by radioimaging agents. Am J Respir Cell Mol Biol. 49:471-80, 2013；Kenny等人, Multifunctional receptor-targeted nanocomplexes for the delivery of therapeutic nucleic acids to the Brain. Biomaterials. 34:9190-200, 2013；Tagalakis等人, Receptor-targeted liposome -peptide nanocomplexes for siRNA delivery. Biomaterials. 32:6302-15, 2011；Tagalakis等人, Integrin-targeted nanocomplexes for tumor specific delivery and therapy by systemic administration. Biomaterials. 32: 1370-6, 2011；Manunta等人, Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium. PLoS One. 6:e26768, 2011；Grosse等人, Tumor-specific gene transfer with receptor-mediated nanocomplexes modified by polyethylene glycol shielding and endosomally cleavable lipid and peptide linkers. F ASEB J. 24:2301-13, 2010)。In certain embodiments, the peptide component is designed to be cell type specific or cell surface receptor specific. For example, the degree of specificity of integrin or other receptors can confer a certain degree of cellular specificity to the LPD complex. Specificity is produced by targeting cell surface receptors (eg, integrin receptors) and achieves transfection efficiencies comparable to some adenoviral vectors. (See Du et al, The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations. Sci Rep. 4:7107, 2014;Welser et al, Gene delivery using ternary lipopolyplexes incorporating branched etching peptides: the role of Peptide sequence and Mol Pharm. 10:127-41, 2013;Meng P., Inhibition of neointimal hyperplasia in a rabbit vein graft model following non-viral transfection with human iNOS cDNA. Gene Ther. 20:979-86, 2013; Manunta et al. Amway Respir Cell Mol Biol. 49:471-80, 2013; Kenny et al, Multifunctional receptor-targeted nanocomplexes for the delivery of therapeutic nucleic acids to the Brain. Biomaterials 34:9190-200, 2013; Tagalakis et al, Receptor-targeted liposome -peptide nanocomplexes for siRNA delivery. Biomaterials. 32:6302-15, 2011; Tagalakis et al, Integrin-targeted nanocomplexes for tumor specific delivery and therapy by systemic Ad Ministration. Biomaterials. 32: 1370-6, 2011; Manunta et al., Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium. PLoS One. 6:e26768, 2011; Grosse et al., Tumor-specific gene transfer with receptor -mediated nanocomplexes modified by polyethylene glycol shielding and endosomally cleavable lipid and peptide linkers. F ASEB J. 24:2301-13, 2010).

在某些實施例中，非病毒遞送包含靶向人類氣管上皮細胞之肽。參見WO02/072616 (以引用的方式併入)。In certain embodiments, the non-viral delivery comprises a peptide that targets human tracheal epithelial cells. See WO 02/072616 (incorporated by reference).

在某些實施例中，非病毒遞送包含靶向樹突狀細胞之肽。參見WO2004/108938 (以引用的方式併入)。In certain embodiments, the non-viral delivery comprises a peptide that targets dendritic cells. See WO2004/108938 (incorporated by reference).

在某些實施例中，非病毒遞送為脂質/肽載體，其以高效率及低毒性轉染一系列細胞株及初級細胞培養物，包括上皮細胞(40%效率)、血管平滑肌細胞(50%效率)、內皮細胞(30%效率)、造血細胞(10%效率)、小鼠支氣管上皮細胞(參見Manunta等人, Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium. PLoS One. 2011;6:e26768；Tagalakis等人, A receptor-targeted nanocomplex vector system optimized for respiratory gene transfer. Mol Ther. 2008; 16:907-15；Jenkins等人, Formation of LID vector complexes in water alters physicochemical properties and enhances pulmonary gene expressionin vivo , Gene Therapy 2003, 10, 1026-34)、大鼠肺(Jenkins等人, An integrin-targeted non-viral vector for pulmonary gene therapy, Gene Therapy 2000, 7, 393-400)及豬肺(Manunta等人, Airway deposition of nebulized gene delivery nanocomplexes monitored by radioimaging agents. Am J Respir Cell Mol Biol. 2013;49:471-80；Cunningham等人, Evaluation of a porcine model for pulmonary gene transfer using a novel synthetic vector, J Gene Med 2002, 4, 438-46)，效率與腺病毒載體相當(Jenkins等人, 2000, 如上所述)。In certain embodiments, the non-viral delivery is a lipid/peptide carrier that transfects a range of cell lines and primary cell cultures, including epithelial cells (40% efficiency), vascular smooth muscle cells (50%) with high efficiency and low toxicity. Efficiency), endothelial cells (30% efficiency), hematopoietic cells (10% efficiency), mouse bronchial epithelial cells (see Manunta et al, Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium. PLoS One. 2011; :e26768;Tagalakis et al, A receptor-targeted nanocomplex vector system optimized for respiratory gene transfer. Mol Ther. 2008; 16:907-15; Jenkins et al, Formation of LID vector complexes in water alters physicochemical properties and enhances pulmonary gene expression In vivo , Gene Therapy 2003, 10, 1026-34), rat lung (Jenkins et al, An integrin-targeted non-viral vector for pulmonary gene therapy, Gene Therapy 2000, 7, 393-400) and pig lung (Manunta Airway deposition of nebulized gene delivery nanocomplexes monitored by radioimaging agents. Am J Respir Cell Mol Biol. 20 13; 49: 471-80; Cunningham et al, Evaluation of a porcine model for pulmonary gene transfer using a novel synthetic vector, J Gene Med 2002, 4, 438-46), efficiency comparable to adenoviral vectors (Jenkins et al, 2000, as mentioned above).

在某些實施例中，用於此類LPD複合物或脂質/肽複合物之肽具有兩種功能：含有細胞表面受體(例如整合素)識別序列之「頭端」及可非共價結合DNA之「尾部」。在某些實施例中，肽之此兩種組分經由間隔子以不干擾其各自功能之方式共價連接。在某些實施例中，肽具有「尾部」，其為聚陽離子核酸結合組分，諸如WO96/15811中所述之肽。In certain embodiments, peptides for such LPD complexes or lipid/peptide complexes have two functions: a "head end" containing a cell surface receptor (eg, integrin) recognition sequence and non-covalent binding The "tail" of DNA. In certain embodiments, the two components of the peptide are covalently linked via a spacer in a manner that does not interfere with their respective functions. In certain embodiments, the peptide has a "tail" which is a polycationic nucleic acid binding component, such as the peptide described in WO 96/15811.

在某些實施例中，LPD複合物之脂質組分為Proc. Natl. Acad. Sci. USA 84, 7413-7417, 1987及US 5,264,618 (兩者均以引用的方式併入)中報導之陽離子脂質。商標為「Lipofectin」之市售陽離子脂質體，其由細胞轉染素、DOTMA 1及中性脂質DOPE 2以1:1之比率組成，為此類脂質之一個實例。In certain embodiments, the lipid component of the LPD complex is a cationic lipid as reported in Proc. Natl. Acad. Sci. USA 84, 7413-7417, 1987 and US 5,264,618 (both incorporated by reference) . A commercially available cationic liposome of the trademark "Lipofectin" consisting of cell transfectin, DOTMA 1 and neutral lipid DOPE 2 in a ratio of 1:1 is an example of such a lipid.

在某些實施例中，陽離子脂質體調配物組合合成的陽離子細胞轉染素及中性脂質。舉例而言，一些基於甘油骨架(諸如DOTMA)或膽固醇，諸如DC-Chol 3。In certain embodiments, the cationic liposome formulation combines the synthesized cationic cell transfectin and neutral lipid. For example, some are based on a glycerol backbone (such as DOTMA) or cholesterol, such as DC-Chol 3.

在某些實施例中，主題cdsDNA調配於脂質奈米粒子(LNP)中，諸如SNALP (穩定核酸-脂質粒子)。此類脂質奈米粒子通常包含一或多種可電離脂質、磷脂、膽固醇及PEG-脂質作為包含在核酸(諸如RNAi劑(siRNA、miRNA、shRNA)、反義寡核苷酸(ASO)、CRISPR/Cas系統組分(諸如編碼Cas9酶之核酸及sgRNA)、治療性mRNA、基因疫苗或傳統DNA載體諸如質體)內之載劑材料。In certain embodiments, the subject cdsDNA is formulated in a lipid nanoparticle (LNP), such as SNALP (stabilized nucleic acid-lipid particle). Such lipid nanoparticles typically comprise one or more ionizable lipids, phospholipids, cholesterol, and PEG-lipids as nucleic acids (such as RNAi agents (siRNA, miRNA, shRNA), antisense oligonucleotides (ASO), CRISPR/ A carrier material within a Cas system component (such as a nucleic acid encoding a Cas9 enzyme and sgRNA), a therapeutic mRNA, a genetic vaccine, or a conventional DNA vector such as a plastid.

在某些實施例中，主題cdsDNA調配於脂質體中，該等脂質體通常包含一或多種磷脂、膽固醇及PEG-脂質作為包含在核酸(諸如上述核酸)內之載劑材料。In certain embodiments, the subject cdsDNA is formulated in a liposome that typically comprises one or more phospholipids, cholesterol, and PEG-lipids as a carrier material contained within a nucleic acid, such as the nucleic acid described above.

在某些實施例中，主題cdsDNA調配於基於聚合物之奈米粒子(NP)中，其通常包含一或多種聚丙交酯(例如PLGA)、嵌段共聚物(諸如PEG-b-PLGA)及多糖(諸如聚葡萄胺糖及纖維素)作為包含在核酸(諸如上述核酸)內之載劑材料。In certain embodiments, the subject cdsDNA is formulated in a polymer-based nanoparticle (NP), which typically comprises one or more polylactide (eg, PLGA), a block copolymer (such as PEG-b-PLGA), and Polysaccharides such as polyglucosamine and cellulose are used as carrier materials for inclusion in nucleic acids such as the nucleic acids described above.

下文舉例說明某些特定類型之LNP、脂質體及聚合物NP。Some specific types of LNPs, liposomes, and polymer NPs are exemplified below.

SNALP
在某些實施例中，主題cdsDNA調配於SNALP (穩定核酸-脂質粒子)中，其為具有非層狀結構之穩定脂質粒子。SNALP包含可電離之脂質、屏蔽脂質(例如聚乙二醇(PEG))、膽固醇及內源性或外源性靶向配體，諸如ApoE脂蛋白。其為在生理pH下中性帶電且藉由PEG穩定之單層(單脂質雙層)脂質體，其將核酸囊封在SNALP內(諸如cdsDNA)。SNALP適用於遞送至各種組織及細胞類型。 SNALP
In certain embodiments, the subject cdsDNA is formulated in SNALP (stable nucleic acid-lipid particles), which are stable lipid particles having a non-lamellar structure. SNALPs comprise ionizable lipids, barrier lipids (eg, polyethylene glycol (PEG)), cholesterol, and endogenous or exogenous targeting ligands, such as ApoE lipoproteins. It is a monolayer (single lipid bilayer) liposome that is neutrally charged at physiological pH and stabilized by PEG, which encapsulates the nucleic acid within SNALP (such as cdsDNA). SNALP is suitable for delivery to a variety of tissues and cell types.

在某些實施例中，SNALP包含可電離的陽離子脂質DLinDMA (1,2-二亞油氧基-3-二甲基胺基丙烷)。在某些實施例中，SNALP包含DLin-KC2-DMA及DLin-MC3-DMA，且藉由結構-活性關係(SAR)研究產生，其最佳化陽離子脂質部分之可電離胺基脂質頭端的pKa。在某些實施例中，SNALP為reLNP (快速消除的LNP)，諸如L319，其為現有的SNALP平台提供生物可降解性且自血漿及組織中快速消除。In certain embodiments, the SNALP comprises an ionizable cationic lipid DLinDMA (1,2-dilinoleoxy-3-dimethylaminopropane). In certain embodiments, the SNALP comprises DLin-KC2-DMA and DLin-MC3-DMA and is produced by a structure-activity relationship (SAR) study that optimizes the pKa of the ionizable amine lipid head end of the cationic lipid moiety . In certain embodiments, the SNALP is reLNP (rapidly eliminated LNP), such as L319, which provides biodegradability to existing SNALP platforms and is rapidly eliminated from plasma and tissue.

在某些實施例中，將主題cdsDNA調配為複數個核酸-脂質粒子，其中該複數個粒子中之各粒子包含：(a)核酸(例如cdsDNA，其可編碼蛋白質、RNAi、反義寡核苷酸或mRNA)；(b)陽離子脂質，佔粒子中存在之總脂質的約50-85 mol% (例如約50-65 mol%；約56.5-66.5 mol%；約52-62 mol%)；(c)非陽離子脂質(諸如膽固醇或其衍生物，或磷脂(諸如二棕櫚醯磷脂醯膽鹼(DPPC)、二硬脂醯磷脂醯膽鹼(DSPC)或其混合物)，或磷脂及膽固醇或其衍生物之混合物)，佔粒子中存在之總脂質的約13-49.5 mol% (例如約31.5-42.5 mol%之膽固醇或其衍生物；或4-10 mol%之磷脂及30-40 mol%膽固醇；或5-9 mol%之磷脂及32-36 mol%膽固醇；或10-30 mol%磷脂及10-30 mol%膽固醇)；及(d)抑制粒子聚集之結合脂質(例如PEG-脂質結合物，諸如PEG-二醯基甘油(PEG-DAG)結合物、PEG-二烷氧基丙基(PEG-DAA)結合物或其混合物)，佔粒子中存在之總脂質的約0.5-10 mol% (例如約0.5-2 mol%或約1-2 mol%)，視情況其中該複數個粒子中至少約95%之粒子具有非層狀形態。在某些實施例中，核酸-脂質粒子包含約61.5 mol%陽離子脂質、約36.9%膽固醇或其衍生物及約1.5 mol% PEG-脂質結合物。在某些實施例中，核酸-脂質粒子包含約57.1 mol%陽離子脂質、約7.1 mol%磷脂、約34.3 mol%膽固醇或其衍生物及約1.4 mol% PEG-脂質結合物。In certain embodiments, the subject cdsDNA is formulated into a plurality of nucleic acid-lipid particles, wherein each of the plurality of particles comprises: (a) a nucleic acid (eg, cds DNA, which encodes a protein, RNAi, antisense oligonucleoside) (b) a cationic lipid, comprising from about 50 to 85 mol% (eg, from about 50 to 65 mol%; from about 56.5 to 66.5 mol%; from about 52 to 62 mol%) of the total lipid present in the particle; c) a non-cationic lipid such as cholesterol or a derivative thereof, or a phospholipid such as dipalmitoside phospholipid choline (DPPC), distearyl phospholipid choline (DSPC) or a mixture thereof, or phospholipids and cholesterol or a mixture of derivatives), comprising about 13-49.5 mol% of total lipid present in the particles (eg, about 31.5-42.5 mol% of cholesterol or a derivative thereof; or 4-10 mol% of phospholipids and 30-40 mol% of cholesterol) Or 5-9 mol% phospholipids and 32-36 mol% cholesterol; or 10-30 mol% phospholipids and 10-30 mol% cholesterol); and (d) binding lipids that inhibit particle aggregation (eg PEG-lipid conjugates) , such as PEG-dimercaptoglycerol (PEG-DAG) conjugate, PEG-dialkoxypropyl (PEG-DAA) conjugate or mixtures thereof, accounting for about 0 of the total lipid present in the particle .5-10 mol% (e.g., about 0.5-2 mol% or about 1-2 mol%), wherein at least about 95% of the plurality of particles have a non-lamellar morphology, as appropriate. In certain embodiments, the nucleic acid-lipid particle comprises about 61.5 mol% cationic lipid, about 36.9% cholesterol or a derivative thereof, and about 1.5 mol% PEG-lipid conjugate. In certain embodiments, the nucleic acid-lipid particle comprises about 57.1 mol% cationic lipid, about 7.1 mol% phospholipid, about 34.3 mol% cholesterol or a derivative thereof, and about 1.4 mol% PEG-lipid conjugate.

在某些實施例中，將主題cdsDNA調配為複數個核酸-脂質粒子，其中該複數個粒子中之各粒子包含：(a)核酸；(b)陽離子脂質，佔粒子中存在之總脂質的約50 mol%至約85 mol% (例如約50 mol%至約65 mol%，或約50 mol%至約60 mol%)；(c)非陽離子脂質，佔粒子中存在之總脂質的約13 mol%至約49.5 mol% (例如磷脂及膽固醇或膽固醇衍生物之混合物，其中磷脂佔粒子中存在之總脂質的約3 mol%至約15 mol% (或約4 mol%至約12 mol%)；且其中膽固醇或其衍生物佔粒子中存在之總脂質的約30 mol%至約40 mol%)；及(d)抑制粒子聚集之結合脂質，佔粒子中存在之總脂質的約0.5 mol%至約10 mol% (例如約0.5 mol%至約2 mol%或約5 mol%至約10 mol%)，其中該複數個粒子中至少約95%之粒子具有非層狀形態。In certain embodiments, the subject cdsDNA is formulated into a plurality of nucleic acid-lipid particles, wherein each of the plurality of particles comprises: (a) a nucleic acid; (b) a cationic lipid that occupies about the total lipid present in the particle 50 mol% to about 85 mol% (eg, about 50 mol% to about 65 mol%, or about 50 mol% to about 60 mol%); (c) non-cationic lipids, about 13 mol of total lipid present in the particles % to about 49.5 mol% (eg, a mixture of phospholipids and cholesterol or cholesterol derivatives, wherein the phospholipids comprise from about 3 mol% to about 15 mol% (or from about 4 mol% to about 12 mol%) of the total lipid present in the particles; And wherein the cholesterol or its derivative accounts for from about 30 mol% to about 40 mol% of the total lipid present in the particle; and (d) the binding lipid which inhibits aggregation of the particles, which accounts for about 0.5 mol% of the total lipid present in the particle to About 10 mol% (e.g., from about 0.5 mol% to about 2 mol% or from about 5 mol% to about 10 mol%), wherein at least about 95% of the plurality of particles have a non-lamellar morphology.

在某些實施例中，將主題cdsDNA調配為複數個核酸-脂質粒子，其中該複數個粒子中之各粒子包含：(a)核酸(諸如cdsDNA，其可編碼蛋白質、RNAi、反義寡核苷酸或mRNA)；(b)陽離子脂質(例如粒子中存在之總脂質的約10-50 mol%、約20-50 mol%或約20-40 mol%)；(c)非陽離子脂質(例如粒子中存在之總脂質的約10-60 mol%、約20-55 mol%或約25-50 mol%)；及(d)抑制粒子聚集之結合脂質(例如粒子中存在之總脂質的約0.5-20 mol%、約2-20 mol%或約1.5-18 mol%)，其中該複數個粒子中至少約95%之粒子具有非層狀形態，或其中該複數個粒子中至少約95%之粒子為電子密集。In certain embodiments, the subject cdsDNA is formulated into a plurality of nucleic acid-lipid particles, wherein each of the plurality of particles comprises: (a) a nucleic acid (such as cds DNA, which encodes a protein, RNAi, antisense oligonucleoside) Acid or mRNA); (b) cationic lipid (eg, about 10-50 mol%, about 20-50 mol%, or about 20-40 mol% of total lipid present in the particles); (c) non-cationic lipids (eg, particles) About 10-60 mol%, about 20-55 mol% or about 25-50 mol%) of the total lipid present; and (d) a binding lipid that inhibits aggregation of the particles (eg, about 0.5- of the total lipid present in the particle) 20 mol%, about 2-20 mol% or about 1.5-18 mol%), wherein at least about 95% of the plurality of particles have a non-lamellar morphology, or wherein at least about 95% of the plurality of particles Intensive for electronics.

cdsDNA可編碼任何所關注之基因產物，包括蛋白質、反義寡核苷酸或RNAi構築體(諸如siRNA、aiRNA、miRNA、Dicer-受質dsRNA、shRNA、ssRNAi寡核苷酸及其組合)。cdsDNA can encode any gene product of interest, including proteins, antisense oligonucleotides or RNAi constructs (such as siRNA, aiRNA, miRNA, Dicer-substrate dsRNA, shRNA, ssRNAi oligonucleotides, and combinations thereof).

在某些實施例中，陽離子脂質包含1,2-二亞油氧基-N,N-二甲基胺基丙烷(DLinDMA)、1,2-二次亞麻油氧基-N,N-二甲基胺基丙烷(DLenDMA)、l,2-二-y-次亞麻油氧基-N,N-二甲基胺基丙烷(γ-DLenDMA)、2,2-二亞油醯基-4-(2-二甲胺基乙基)-[l,3]-二氧戊環(DLin-K-C2-DMA)、2,2-二亞油醯基-4-二甲胺基甲基-[1,3]-二氧戊環(DLin-K-DMA)或其混合物。In certain embodiments, the cationic lipid comprises 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-secondary linoleyloxy-N,N-di Methylaminopropane (DLenDMA), 1,2-di-y-linolenic acid-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-diindolizin-4 -(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinole-4-ylaminomethyl -[1,3]-dioxolane (DLin-K-DMA) or a mixture thereof.

在某些實施例中，陽離子脂質包含MC3、LenMC3、CP-LenMC3、y-LenMC3、CP-y-LenMC3、MC3MC、MC2MC、MC3醚、MC4醚、MC3醯胺、Pan-MC3、Pan-MC4、Pan MC5或其混合物。In certain embodiments, the cationic lipid comprises MC3, LenMC3, CP-LenMC3, y-LenMC3, CP-y-LenMC3, MC3MC, MC2MC, MC3 ether, MC4 ether, MC3 decylamine, Pan-MC3, Pan-MC4, Pan MC5 or a mixture thereof.

在某些實施例中，陽離子脂質包含N,N-二油基-N,N-二甲基氯化銨(DODAC)、N,N-二硬脂基-N,N-二甲基溴化銨(DDAB)、N-(1-(2,3-二油醯氧基)丙基)-N,N,N-三甲基氯化銨(DOTAP)、N-(1-(2,3-二油氧基)丙基)-N,N,N-三甲基氯化銨(DOTMA)、N,N-二甲基-2,3-二油氧基丙胺(DODMA)及其組合。In certain embodiments, the cationic lipid comprises N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearoyl-N,N-dimethyl bromide Ammonium (DDAB), N-(1-(2,3-dioleoxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3) -Dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoxypropylamine (DODMA), and combinations thereof.

在某些實施例中，陽離子脂質為如美國專利第7,799,565號；第8,569,256號；第9,018,187號及第9,181,545號中所述之任一者(均以引用的方式併入本文中)。In certain embodiments, the cationic lipids are any of those described in U.S. Patent No. 7,799,565; U.S. Patent No. 8, 569, 256, No. 9, 018, 187, and No. 9, 181, 545, incorporated herein by reference.

在某些實施例中，非陽離子脂質為二棕櫚醯磷脂醯膽鹼(DPPC)、二油醯磷脂醯乙醇胺(DOPE)、棕櫚醯油醯基磷脂醯膽鹼(POPC)卵磷脂醯膽鹼(EPC)、二硬脂醯磷脂醯膽鹼(DSPC)、膽固醇及其組合。In certain embodiments, the non-cationic lipid is dipalmitoside phospholipid choline (DPPC), diolein phospholipid ethanolamine (DOPE), palmitoyl phosphatase choline (POPC) lecithin choline ( EPC), distearyl phospholipid choline (DSPC), cholesterol, and combinations thereof.

在某些實施例中，非陽離子脂質為磷脂。In certain embodiments, the non-cationic lipid is a phospholipid.

在某些實施例中，非陽離子脂質為膽固醇或膽固醇衍生物。膽固醇衍生物之實例包括但不限於膽甾烷醇、膽甾烷酮、膽甾烯酮、腎固醇、膽固醇基-2'-羥乙基醚、膽固醇基-4'-羥基丁基醚及其混合物。In certain embodiments, the non-cationic lipid is a cholesterol or cholesterol derivative. Examples of cholesterol derivatives include, but are not limited to, cholesterol, cholestyrone, cholestenone, renal sterol, cholesterol-based 2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, and Its mixture.

在某些實施例中，非陽離子脂質為磷脂及膽固醇或膽固醇衍生物之混合物。In certain embodiments, the non-cationic lipid is a mixture of phospholipids and cholesterol or cholesterol derivatives.

在某些實施例中，非陽離子脂質為選自由二棕櫚醯磷脂醯膽鹼(DPPC)、二硬脂醯磷脂醯膽鹼(DSPC)或其混合物組成之群的磷脂。In certain embodiments, the non-cationic lipid is a phospholipid selected from the group consisting of dipalmitoside phosphocholine (DPPC), distearyl phospholipid choline (DSPC), or mixtures thereof.

在某些實施例中，非陽離子脂質為DPPC。In certain embodiments, the non-cationic lipid is DPPC.

在某些實施例中，非陽離子脂質為DPPC及膽固醇之混合物。In certain embodiments, the non-cationic lipid is a mixture of DPPC and cholesterol.

在某些實施例中，抑制粒子聚集之結合脂質為聚乙二醇(PEG)-脂質結合物。舉例而言，PEG可具有約550道爾頓至約5,000道爾頓之平均分子量，或約2,000道爾頓之平均分子量，或約750道爾頓之平均分子量。In certain embodiments, the binding lipid that inhibits particle aggregation is a polyethylene glycol (PEG)-lipid conjugate. For example, the PEG can have an average molecular weight of from about 550 Daltons to about 5,000 Daltons, or an average molecular weight of about 2,000 Daltons, or an average molecular weight of about 750 Daltons.

在某些實施例中，PEG-脂質結合物為選自由以下組成之群的成員：PEG-二醯基甘油(PEG-DAG)結合物、PEG二烷氧基丙基(PEG-DAA)結合物、PEG-磷脂結合物、PEG-神經醯胺(PEG-Cer)結合物及其混合物。In certain embodiments, the PEG-lipid conjugate is a member selected from the group consisting of PEG-dimercaptoglycerol (PEG-DAG) conjugate, PEG dialkoxypropyl (PEG-DAA) conjugate , PEG-phospholipid conjugates, PEG-neura amide (PEG-Cer) conjugates, and mixtures thereof.

在某些實施例中，PEG-脂質結合物為PEG-DAA結合物，諸如美國專利第8,936,942號及第7,982,027號(以引用的方式併入)中所述之彼等結合物。In certain embodiments, the PEG-lipid conjugate is a PEG-DAA conjugate such as those described in U.S. Patent Nos. 8,936, 942 and 7, 982, 027, incorporated by reference.

在某些實施例中，PEG-DAA結合物為選自由以下組成之群的成員：PEG-二癸氧基丙基(C10 )結合物、PEG-二月桂基氧基丙基(C12 )結合物、PEG-二肉豆蔻氧基丙基(C14 )結合物、PEG-二棕櫚氧基丙基(C16 )結合物、PEG-二硬脂氧基丙基(C18 )結合物及其混合物。在某些實施例中，PEG-脂質結合物描述於美國專利第7,803,397號(以引用的方式併入)中。In certain embodiments, the PEG-DAA conjugate is a member selected from the group consisting of PEG-dimethoxypropyl (C 10 ) conjugate, PEG-dilauryloxypropyl (C 12 ) a conjugate, a PEG-dimyristyloxypropyl (C 14 ) conjugate, a PEG-dipalmitylpropyl (C 16 ) conjugate, a PEG-distearoxypropyl (C 18 ) conjugate, and Its mixture. In certain embodiments, PEG-lipid conjugates are described in U.S. Patent No. 7,803,397, incorporated herein by reference.

在某些實施例中，PEG-DAA結合物為PEG-二肉豆蔻氧基丙基(CH)結合物。In certain embodiments, the PEG-DAA conjugate is a PEG-dimyristyloxypropyl (CH) conjugate.

在某些實施例中，粒子中之核酸(例如cdsDNA)在37℃下粒子暴露於核酸酶約20分鐘或約30分鐘後基本上不降解。In certain embodiments, the nucleic acid in the particle (eg, cds DNA) does not substantially degrade after exposure of the particle to the nuclease for about 20 minutes or about 30 minutes at 37 °C.

在某些實施例中，cdsDNA完全囊封於粒子中。亦即，cdsDNA完全囊封於脂質粒子之脂質部分內，使得脂質粒子中之cdsDNA在水溶液對酶降解(例如藉由核酸酶或蛋白酶)具有抗性。在某些其他實施例中，脂質粒子對諸如人類之哺乳動物基本上無毒。In certain embodiments, the cdsDNA is completely encapsulated in the particle. That is, the cds DNA is completely encapsulated within the lipid portion of the lipid particle such that the cds DNA in the lipid particle is resistant to enzymatic degradation (eg, by nuclease or protease) in aqueous solution. In certain other embodiments, the lipid particles are substantially non-toxic to mammals such as humans.

在某些實施例中，粒子之脂質:cdsDNA質量比為約5:1至約15:1。In certain embodiments, the lipid: cdsDNA mass ratio of the particles is from about 5:1 to about 15:1.

在某些實施例中，粒子之中值直徑為約30或40 nm至約150 nm。In certain embodiments, the median diameter of the particles is from about 30 or 40 nm to about 150 nm.

在某些實施例中，陽離子脂質佔粒子中存在之總脂質的約52 mol%至約62 mol%。In certain embodiments, the cationic lipid comprises from about 52 mol% to about 62 mol% of the total lipid present in the particle.

在某些實施例中，非陽離子脂質為磷脂及膽固醇或其衍生物之混合物，佔粒子中存在之總脂質的約36 mol%至約47 mol%。In certain embodiments, the non-cationic lipid is a mixture of phospholipids and cholesterol or a derivative thereof, from about 36 mol% to about 47 mol% of the total lipid present in the particles.

在某些實施例中，抑制粒子聚集之結合脂質為PEG-脂質結合物，佔粒子中存在之總脂質的約1 mol%至約2 mol%。In certain embodiments, the binding lipid that inhibits particle aggregation is a PEG-lipid conjugate that comprises from about 1 mol% to about 2 mol% of the total lipid present in the particle.

在某些實施例中，陽離子脂質佔粒子中存在之總脂質的約56.5 mol%至約66.5 mol%。In certain embodiments, the cationic lipid comprises from about 56.5 mol% to about 66.5 mol% of the total lipid present in the particle.

在某些實施例中，非陽離子脂質為膽固醇或其衍生物，佔粒子中存在之總脂質的約31.5 mol%至約42.5 mol%。In certain embodiments, the non-cationic lipid is cholesterol or a derivative thereof, which comprises from about 31.5 mol% to about 42.5 mol% of the total lipid present in the particle.

在某些實施例中，抑制粒子聚集之結合脂質為PEG-脂質結合物，佔粒子中存在之總脂質的約1 mol%至約2 mol%。In certain embodiments, the binding lipid that inhibits particle aggregation is a PEG-lipid conjugate that comprises from about 1 mol% to about 2 mol% of the total lipid present in the particle.

在某些實施例中，陽離子脂質佔粒子中存在之總脂質的約50 mol%至約60 mol%。In certain embodiments, the cationic lipid comprises from about 50 mol% to about 60 mol% of the total lipid present in the particle.

在某些實施例中，非陽離子脂質為磷脂及膽固醇或其衍生物之混合物，佔粒子中存在之總脂質的約35 mol%至約45 mol%。In certain embodiments, the non-cationic lipid is a mixture of phospholipids and cholesterol or a derivative thereof, from about 35 mol% to about 45 mol% of the total lipid present in the particles.

在某些實施例中，抑制粒子聚集之結合脂質為PEG-脂質結合物，佔粒子中存在之總脂質的約5 mol%至約10 mol%。In certain embodiments, the binding lipid that inhibits particle aggregation is a PEG-lipid conjugate that comprises from about 5 mol% to about 10 mol% of the total lipid present in the particle.

在某些實施例中，陽離子脂質佔粒子中存在之總脂質的約55 mol%至約65 mol%。In certain embodiments, the cationic lipid comprises from about 55 mol% to about 65 mol% of the total lipid present in the particle.

在某些實施例中，非陽離子脂質為膽固醇或其衍生物，佔粒子中存在之總脂質的約30 mol%至約40 mol%。In certain embodiments, the non-cationic lipid is cholesterol or a derivative thereof, which comprises from about 30 mol% to about 40 mol% of the total lipid present in the particle.

在某些實施例中，抑制粒子聚集之結合脂質為PEG-脂質結合物，佔粒子中存在之總脂質的約5 mol%至約10 mol%。In certain embodiments, the binding lipid that inhibits particle aggregation is a PEG-lipid conjugate that comprises from about 5 mol% to about 10 mol% of the total lipid present in the particle.

在某些實施例中，大於95%、至少96%、至少97%、至少98%、至少99%或大於99%之粒子具有非層狀形態，亦即非雙層結構。在某些實施例中，大於95%、至少96%、至少97%、至少98%、至少99%或大於99%之粒子為電子密集。In certain embodiments, greater than 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% of the particles have a non-lamellar morphology, ie, a non-bilayer structure. In certain embodiments, greater than 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% of the particles are electron dense.

在某些實施例中，粒子之非層狀形態包含反六角形(HII )或立方相結構。In certain embodiments, the non-lamellar morphology of the particles comprises an anti-hexagonal (H II ) or cubic phase structure.

在某些實施例中，具有非層狀形態之粒子為電子密集。粒子之非層狀形態可藉由例如低溫透射電子顯微法(cryo-TEM)、X射線繞射或藉由差示掃描量熱法(DSC)來測定，其中在5至75℃下未觀察到熱轉變。In certain embodiments, particles having a non-layered morphology are electron dense. The non-lamellar morphology of the particles can be determined, for example, by cryo transmission electron microscopy (cryo-TEM), X-ray diffraction, or by differential scanning calorimetry (DSC), where no observation is made at 5 to 75 °C. To the heat transition.

製造SNALP及遞送SNALP之方法為此項技術中已知的，參見例如US7901708、US9492386、WO2009127060A1及WO2012000104A1 (以引用的方式併入本文中)。Methods of making SNALP and delivering SNALP are known in the art, see, for example, US7901708, US9492386, WO2009127060A1, and WO2012000104A1, herein incorporated by reference.

可以訂製優先靶向所關注之特定組織、器官或腫瘤之脂質粒子。在某些其他情況下，可能需要使靶向部分附接至脂質粒子之表面以進一步增強粒子之靶向。將靶向部分(例如抗體、蛋白質等)附接至脂質(諸如本發明粒子中使用之彼等)的方法為熟習此項技術者已知的。Lipid particles that preferentially target a particular tissue, organ or tumor of interest can be customized. In some other cases, it may be desirable to attach the targeting moiety to the surface of the lipid particle to further enhance the targeting of the particle. Methods of attaching targeting moieties (e.g., antibodies, proteins, etc.) to lipids, such as those used in the particles of the invention, are known to those skilled in the art.

一旦形成，SNALP脂質粒子可用於將活性劑或治療劑(例如cdsDNA)引入細胞中。因此，本發明亦提供將活性劑或治療劑(諸如cdsDNA)引入細胞中之方法。Once formed, SNALP lipid particles can be used to introduce an active agent or therapeutic agent (eg, cds DNA) into a cell. Accordingly, the invention also provides methods of introducing an active agent or therapeutic agent, such as cds DNA, into a cell.

在一些情況下，細胞為肌肉細胞，諸如骨骼肌細胞、心肌細胞或平滑肌細胞。在某些實施例中，肌肉細胞為脛骨前肌細胞。In some cases, the cells are muscle cells, such as skeletal muscle cells, cardiomyocytes, or smooth muscle cells. In certain embodiments, the muscle cells are tibialis anterior muscle cells.

進行該等方法時，可藉由在活體外、離體或活體內首先形成如上所述之粒子，隨後使該等粒子與細胞接觸一段足以將活性劑或治療劑(例如cdsDNA)遞送至細胞的時間。In carrying out such methods, the particles as described above may be first formed in vitro, ex vivo or in vivo, and then the particles are contacted with the cells for a sufficient amount to deliver the active agent or therapeutic agent (eg, cds DNA) to the cells. time.

SNALP脂質粒子可吸附至與其混合或接觸之幾乎任何細胞類型。一旦吸附，粒子可由一部分細胞內吞，與細胞膜交換脂質或與細胞融合。粒子之活性劑或治療劑(例如cdsDNA)部分的轉移或併入可經由此等途徑中之任一者進行。特定言之，當發生融合時，粒子膜整合至細胞膜中且粒子之內含物與細胞內流體組合。SNALP lipid particles can be adsorbed to almost any cell type with which they are mixed or contacted. Once adsorbed, the particles can be endocytosed by a portion of the cells, exchange lipids with the cell membrane, or fuse with the cells. Transfer or incorporation of a portion of the active agent or therapeutic agent (e.g., cdsDNA) of the particle can be performed via any of these routes. In particular, when fusion occurs, the particle film is integrated into the cell membrane and the contents of the particles are combined with the intracellular fluid.

SNALP脂質粒子可單獨或與醫藥學上可接受之載劑(例如生理鹽水或磷酸鹽緩衝液)混合投與，該載劑根據投藥途徑及標準醫藥實踐選擇。一般而言，正常緩衝鹽水(例如135-150 mM NaCl)將用作醫藥學上可接受之載劑。其他適合之載劑包括例如水、緩衝水、0.4%鹽水、0.3%甘胺酸及其類似物，包括用於增強穩定性之糖蛋白，諸如白蛋白、脂蛋白、球蛋白等。額外適合之載劑描述於例如REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 第17版 (1985)中。如本文所用，「載劑」包括任何及所有溶劑、分散介質、媒劑、包衣、稀釋劑、抗細菌及抗真菌劑、等張及吸收延遲劑、緩衝劑、載劑溶液、懸浮液、膠體及其類似物。片語「醫藥學上可接受」係指當投與人類時不產生過敏或類似不良反應的分子實體及組合物。The SNALP lipid particles can be administered alone or in admixture with a pharmaceutically acceptable carrier such as physiological saline or phosphate buffer, which is selected according to the route of administration and standard pharmaceutical practice. In general, normal buffered saline (eg, 135-150 mM NaCl) will be used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhancing stability, such as albumin, lipoproteins, globulins, and the like. Additional suitable carriers are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th Edition (1985). As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffering agents, carrier solutions, suspensions, Colloids and their analogues. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.

醫藥學上可接受之載劑一般在脂質粒子形成後添加。因此，在形成脂質粒子(例如SNALP)之後，可將粒子稀釋於醫藥學上可接受之載劑中，諸如正常緩衝鹽水。Pharmaceutically acceptable carriers are typically added after the lipid particles are formed. Thus, after the formation of lipid particles (eg, SNALP), the particles can be diluted in a pharmaceutically acceptable carrier, such as normal buffered saline.

醫藥調配物中之粒子濃度可廣泛變化，亦即以重量計自小於約0.05%，通常處於或至少約2至5%，至高達約10至90%，且將根據所選特定投藥模式，主要藉由流體體積、黏度等來選擇。舉例而言，可增加濃度以降低與治療相關之流體負荷。此在患有動脈粥樣硬化相關之充血性心臟衰竭或嚴重高血壓之患者中可能為特別理想的。或者，可將由刺激性脂質構成之粒子稀釋至低濃度以減輕投與部位之炎症。The concentration of the particles in the pharmaceutical formulation can vary widely, i.e., from less than about 0.05% by weight, usually at or at least about 2 to 5%, up to about 10 to 90%, and will vary depending upon the particular mode of administration selected. It is selected by fluid volume, viscosity, and the like. For example, the concentration can be increased to reduce the fluid load associated with the treatment. This may be particularly desirable in patients with atherosclerosis-related congestive heart failure or severe hypertension. Alternatively, particles composed of stimulating lipids can be diluted to a low concentration to reduce inflammation at the site of administration.

本發明之醫藥組合物可藉由眾所周知的習知滅菌技術來滅菌。水溶液可封裝使用或在無菌條件下過濾並凍乾，凍乾製劑在投與之前與無菌水溶液組合。組合物可含有接近生理條件所需之醫藥學上可接受之輔助物質，諸如pH調節及緩衝劑、張力調節劑及其類似物，例如乙酸鈉、乳酸鈉、氯化鈉、氯化鉀及氯化鈣。另外，粒子懸浮液可包括脂質保護劑，其保護脂質在儲存時免受自由基及脂質過氧化損害。親脂性自由基淬滅劑，諸如α-生育酚，及水溶性鐵特異性螯合劑，諸如鐵胺，為適合的。The pharmaceutical compositions of the present invention can be sterilized by well known conventional sterilization techniques. The aqueous solution can be packaged for use or filtered under sterile conditions and lyophilized, and the lyophilized preparation is combined with a sterile aqueous solution prior to administration. The composition may contain pharmaceutically acceptable auxiliary substances required for physiological conditions such as pH adjustment and buffering agents, tonicity adjusting agents and the like, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride and chlorination. calcium. Additionally, the particle suspension can include a lipid protectant that protects the lipid from free radicals and lipid peroxidation during storage. Lipophilic free radical quenchers, such as alpha-tocopherol, and water soluble iron specific chelating agents, such as ferric amine, are suitable.

在某些其他實施例中，可將治療有效量之脂質粒子投與哺乳動物。在其他情況下，自患者移出細胞，活體外遞送脂質粒子(例如使用本文所述之SNALP)，且將細胞重新注射至患者體內。In certain other embodiments, a therapeutically effective amount of lipid particles can be administered to a mammal. In other instances, the cells are removed from the patient, the lipid particles are delivered in vitro (eg, using the SNALP described herein), and the cells are reinjected into the patient.

用於活體內療法之全身遞送，例如經由諸如循環之身體系統將治療性核酸遞送至遠端靶細胞，已使用核酸-脂質粒子實現，諸如PCT公開案第WO 05/007196號、第WO 05/121348號、第WO 05/120152號及第WO 04/002453號中所述之核酸-脂質粒子，其揭示內容出於所有目的以全文引用之方式併入本文中。本發明亦提供完全囊封之脂質粒子，其保護核酸免受血清中之核酸酶降解，為非免疫原性的，尺寸小，且適用於重複給藥。Systemic delivery for in vivo therapy, for example delivery of therapeutic nucleic acids to distal target cells via a body system such as circulation, has been achieved using nucleic acid-lipid particles, such as PCT Publication No. WO 05/007196, WO 05/ Nucleic acid-lipid particles as described in No. 121, 348, WO 05/120152, and WO 04/002453, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. The present invention also provides fully encapsulated lipid particles which protect nucleic acids from nuclease degradation in serum, are non-immunogenic, are small in size, and are suitable for repeated administration.

對於活體內投與，投與可為此項技術中已知的任何方式，例如藉由注射、經口投與、吸入(例如鼻內或氣管內)、經皮施用或直腸投與。投與可經由單次或分次劑量來實現。醫藥組合物可非經腸投與，亦即關節內、靜脈內、腹膜內、皮下或肌肉內。在一些實施例中，醫藥組合物藉由快速注射靜脈內、肌肉內或腹膜內投與(參見例如美國專利第5,286,634號)。細胞內核酸遞送亦已論述於Straubringer等人, Methods Enzymol.,101:512 (1983)；Mannino等人, Biotechniques, 6:682 (1988)；Nicolau等人, Crit. Rev. Ther. Drug Carrier Syst., 6:239 (1989)；及Behr, Acc. Chem. Res., 26:21 (1993)中。投與基於脂質之治療劑的其他方法描述於例如美國專利第3,993,754號；第4,145,410號；第4,235,871號；第4,224,179號；第4,522,803號及第4,588,578號中。脂質粒子可藉由在疾病部位直接注射或藉由在遠離疾病部位之部位注射來投與(參見例如Culver, HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. 第70-71頁(1994))。出於所有目的，上述參考文獻之揭示內容以全文引用之方式併入本文中。For administration in vivo, administration can be by any means known in the art, such as by injection, oral administration, inhalation (e.g., intranasal or intratracheal), transdermal administration, or rectal administration. Administration can be achieved by single or divided doses. The pharmaceutical composition can be administered parenterally, that is, intra-articularly, intravenously, intraperitoneally, subcutaneously or intramuscularly. In some embodiments, the pharmaceutical composition is administered intravenously, intramuscularly, or intraperitoneally by rapid injection (see, e.g., U.S. Patent No. 5,286,634). Intracellular nucleic acid delivery has also been discussed in Straubringer et al, Methods Enzymol., 101: 512 (1983); Mannino et al, Biotechniques, 6: 682 (1988); Nicolau et al, Crit. Rev. Ther. Drug Carrier Syst. , 6:239 (1989); and Behr, Acc. Chem. Res., 26:21 (1993). Other methods of administering lipid-based therapeutic agents are described in, for example, U.S. Patent Nos. 3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803 and 4,588,578. Lipid particles can be administered by injection directly at the disease site or by injection at a site remote from the disease site (see, for example, Culver, HUMAN GENE THERAPY, Mary Ann Liebert, Inc., Publishers, New York. pp. 70-71 (1994) )). The disclosure of the above references is hereby incorporated by reference in its entirety for all purposes.

在靜脈內投與SNALP脂質粒子之實施例中，粒子之總注射劑量的至少約5%、10%、15%、20%或25%在注射後約8、12、24、36或48小時存在於血漿中。在其他實施例中，脂質粒子之總注射劑量的大於約20%、30%、40%及高達約60%、70%或80%在注射後約8、12、24、36或48小時存在於血漿中。在某些情況下，複數個粒子之大於約10%在投與後約1小時存在於哺乳動物之血漿中。在某些其他情況下，在粒子投與後至少約1小時可偵測到脂質粒子之存在。在某些實施例中，在投與後約8、12、24、36、48、60、72或96小時，在肺細胞、肝細胞、腫瘤細胞中或在炎症部位可偵測到治療劑諸如核酸之存在。In embodiments in which SNALP lipid particles are administered intravenously, at least about 5%, 10%, 15%, 20%, or 25% of the total injected dose of the particles are present at about 8, 12, 24, 36, or 48 hours after injection. In the plasma. In other embodiments, greater than about 20%, 30%, 40%, and up to about 60%, 70%, or 80% of the total injected dose of lipid particles are present at about 8, 12, 24, 36, or 48 hours after injection. In plasma. In some cases, greater than about 10% of the plurality of particles are present in the plasma of the mammal about 1 hour after administration. In some other instances, the presence of lipid particles can be detected at least about one hour after administration of the particles. In certain embodiments, a therapeutic agent such as a therapeutic agent can be detected in lung cells, hepatocytes, tumor cells, or at a site of inflammation at about 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration. The presence of nucleic acids.

本發明之組合物，單獨或與其他適合之組分組合，可製成氣溶膠調配物(亦即其可「經霧化」)以經由吸入(例如鼻內或氣管內)投與(參見Brigham等人, Am. J. Sci., 298:278 (1989))。可將氣溶膠調配物置於可接受之加壓推進劑中，諸如二氯二氟甲烷、丙烷、氮氣及其類似物。The compositions of the present invention, alone or in combination with other suitable components, can be formulated into aerosol formulations (i.e., they can be "atomized") for administration via inhalation (e.g., intranasal or intratracheal) (see Brigham). Et al, Am. J. Sci., 298:278 (1989)). The aerosol formulation can be placed into an acceptable pressurized propellant such as dichlorodifluoromethane, propane, nitrogen, and the like.

在某些實施例中，醫藥組合物可藉由鼻內噴霧、吸入及/或其他氣溶膠遞送媒劑遞送。經由經鼻氣溶膠噴霧劑將核酸組合物直接遞送至肺的方法已描述於例如美國專利第5,756,353號及第5,804,212號中。同樣，使用鼻內微粒樹脂及溶血磷脂醯甘油化合物遞送藥物(美國專利5,725,871)亦為醫藥技術中眾所周知的。類似地，以聚四氟乙烯支撐基質形式遞送經黏膜藥物描述於美國專利第5,780,045號中。出於所有目的，上述專利之揭示內容以全文引用之方式併入本文中。In certain embodiments, the pharmaceutical compositions can be delivered by intranasal spray, inhalation, and/or other aerosol delivery vehicles. A method of delivering a nucleic acid composition directly to the lung via a nasal aerosol spray is described in, for example, U.S. Patent Nos. 5,756,353 and 5,804,212. Similarly, the use of intranasal particulate resins and lysophospholipid glycerol compound delivery drugs (U.S. Patent 5,725,871) is also well known in the art. Similarly, delivery of a transmucosal drug in the form of a polytetrafluoroethylene support matrix is described in U.S. Patent No. 5,780,045. The disclosure of the above patents is hereby incorporated by reference in its entirety for all purposes.

適用於非經腸投與，諸如藉由關節內(關節中)、靜脈內、肌肉內、皮內、腹膜內及皮下途徑投與之調配物包括水性及非水性等張無菌注射溶液，其可含有抗氧化劑、緩衝劑、抑菌劑及使調配物與預定接受者之血液等張之溶質；及水性及非水性無菌懸浮液，其可包括懸浮劑、增溶劑、增稠劑、穩定劑及防腐劑。在本發明之實踐中，組合物較佳例如藉由靜脈內輸注、經口、局部、腹膜內、膀胱內或鞘內投與。Formulations suitable for parenteral administration, such as by intra-articular (in-articular), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injectable solutions, which may be a solute comprising an antioxidant, a buffering agent, a bacteriostatic agent, and an isotonic acid in the blood of the intended recipient; and an aqueous and non-aqueous sterile suspension, which may include a suspending agent, a solubilizing agent, a thickening agent, a stabilizer, and preservative. In the practice of the invention, the compositions are preferably administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.

一般而言，當靜脈內投與時，脂質粒子調配物用適合之醫藥載劑調配。許多醫藥學上可接受之載劑可用於本發明之組合物及方法。適用於本發明之調配物見於例如REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 第17版 (1985)中。可使用各種含水載劑，例如水、緩衝水、0.4%鹽水、0.3%甘胺酸及其類似物，且可包括用於增強穩定性之糖蛋白，諸如白蛋白、脂蛋白、球蛋白等。一般而言，正常的緩衝鹽水(135-150 mM NaCl)將用作醫藥學上可接受之載劑，但其他適合之載劑就足夠。此等組合物可藉由習知脂質體滅菌技術滅菌，諸如過濾。組合物可含有接近生理條件所需要之醫藥學上可接受之輔助物質，諸如pH調節劑及緩衝劑、張力調節劑、濕潤劑及其類似物，例如乙酸鈉、乳酸鈉、氯化鈉、氯化鉀、氯化鈣、脫水山梨糖醇單月桂酸酯、三乙醇胺油酸酯等。此等組合物可使用上文提及之技術滅菌，或者，其可在無菌條件下生產。所得水溶液可封裝使用或在無菌條件下過濾並凍乾，凍乾製劑在投與之前與無菌水溶液組合。In general, when administered intravenously, the lipid particle formulation is formulated with a suitable pharmaceutical carrier. Many pharmaceutically acceptable carriers are useful in the compositions and methods of the present invention. Formulations suitable for use in the present invention are found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th Edition (1985). Various aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine, and the like, and can include glycoproteins for enhancing stability, such as albumin, lipoproteins, globulins, and the like. In general, normal buffered saline (135-150 mM NaCl) will be used as a pharmaceutically acceptable carrier, but other suitable carriers will suffice. Such compositions can be sterilized by conventional liposome sterilization techniques, such as filtration. The composition may contain pharmaceutically acceptable auxiliary substances such as pH adjusting agents and buffers, tonicity adjusting agents, wetting agents and the like, such as sodium acetate, sodium lactate, sodium chloride, chlorination, which are required to be close to physiological conditions. Potassium, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like. These compositions can be sterilized using the techniques mentioned above, or they can be produced under sterile conditions. The resulting aqueous solution can be packaged for use or filtered under sterile conditions and lyophilized, the lyophilized formulation being combined with a sterile aqueous solution prior to administration.

在某些應用中，本文所揭示之脂質粒子可經由經口投與遞送至個體。粒子可與賦形劑合併且以可攝入之錠劑、頰內錠劑、糖衣錠、膠囊、丸劑、***錠、酏劑、漱口劑、懸浮液、口腔噴霧劑、糖漿、粉片及其類似物之形式使用(參見例如美國專利第5,641,515號、第5,580,579號及第5,792,451號，其揭示內容出於所有目的以全文引用之方式併入本文中)。此等經口劑型亦可含有以下：黏合劑、明膠；賦形劑、潤滑劑及/或調味劑。當單位劑型為膠囊時，除上述材料之外，其可含有液體載劑。各種其他材料可以包衣形式存在或以其他方式改變劑量單位之物理形式。當然，用於製備任何單位劑型之任何材料應為醫藥學上純的且在所使用之量下基本上無毒。In certain applications, the lipid particles disclosed herein can be delivered to an individual via oral administration. The particles may be combined with excipients and ingestible lozenges, buccal tablets, dragees, capsules, pills, troches, tinctures, mouthwashes, suspensions, mouth sprays, syrups, powders and It is used in the form of its analogs (see, for example, U.S. Patent Nos. 5,641,515, 5, 580, 579, and 5, 792, 451, the disclosures of each of These oral dosage forms may also contain the following: binders, gelatin; excipients, lubricants and/or flavoring agents. When the unit dosage form is a capsule, it may contain a liquid carrier in addition to the above materials. Various other materials may be present in the form of a coating or otherwise alter the physical form of the dosage unit. Of course, any material used to prepare any unit dosage form should be pharmaceutically pure and substantially non-toxic in the amounts employed.

通常，此等經口調配物可含有至少約0.1%的脂質粒子或更多，但粒子之百分比當然可變化且可方便地在約1%或2%及約60%或70%或更多的總調配物之重量或體積之間變化。當然，可製備各治療上有用之組合物中之粒子量，使得在任何給定單位劑量化合物中獲得適合之劑量。製備此類醫藥調配物之熟習此項技術者應考慮諸如溶解度、生物可用性、生物半衰期、投藥途徑、產物存放期以及其他藥理學考慮因素之因素，且因此，各種劑量及治療方案可為可取的。Typically, such oral formulations may contain at least about 0.1% lipid particles or more, although the percentage of particles may of course vary and may conveniently be at about 1% or 2% and about 60% or 70% or more. The weight or volume of the total formulation varies. Of course, the amount of particles in each of the therapeutically useful compositions can be prepared such that a suitable dosage is obtained in any given unit dosage of the compound. Those skilled in the art of preparing such pharmaceutical formulations should consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and thus, various dosages and treatment regimens may be desirable. .

適用於經口投與之調配物可由以下組成：(a)液體溶液，諸如懸浮於稀釋劑諸如水、鹽水或PEG 400中的有效量之封裝治療劑諸如核酸(例如cdsDNA)；(b)膠囊、藥囊或錠劑，各含有預定量之呈液體、固體、顆粒或明膠形式之治療劑，諸如核酸(例如cdsDNA)；(c)於適當液體中之懸浮液；及(d)適合之乳液。錠劑形式可包括以下中之一或多者：乳糖、蔗糖、甘露糖醇、山梨糖醇、磷酸鈣、玉米澱粉、馬鈴薯澱粉、微晶纖維素、明膠、膠態二氧化矽、滑石、硬脂酸鎂、硬脂酸，及其他賦形劑、著色劑、填充劑、黏合劑、稀釋劑、緩衝劑、濕潤劑、防腐劑、調味劑、染料、崩解劑及醫藥學上相容之載劑。***錠形式可包含調味劑例如蔗糖中之治療劑，諸如核酸(例如cdsDNA)，以及在惰性基質中包含治療劑的片劑，該惰性基質諸如明膠及甘油或蔗糖及***膠乳液、凝膠及其類似物，除治療劑之外含有此項技術中已知的載劑。Formulations suitable for oral administration can be composed of: (a) a liquid solution such as an effective amount of a packaged therapeutic agent such as a nucleic acid (e.g., cdsDNA) suspended in a diluent such as water, saline or PEG 400; (b) a capsule , sachets or lozenges, each containing a predetermined amount of a therapeutic agent in the form of a liquid, solid, granule or gelatin, such as a nucleic acid (eg, cdsDNA); (c) a suspension in a suitable liquid; and (d) a suitable emulsion . The tablet form may include one or more of the following: lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal cerium oxide, talc, hard Magnesium citrate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible Carrier. The buccal form may comprise a flavoring agent such as a therapeutic agent in sucrose, such as a nucleic acid (e.g., cds DNA), and a tablet comprising a therapeutic agent in an inert matrix such as gelatin and glycerin or sucrose and gum arabic emulsion, gel And analogs thereof, in addition to therapeutic agents, contain carriers known in the art.

在其另一個使用實例中，脂質粒子可併入多種局部劑型中。舉例而言，含有核酸-脂質粒子諸如SNALP之懸浮液可經調配且以凝膠、油、乳液、局部乳膏、糊劑、軟膏、洗劑、泡沫、慕斯及其類似物之形式投與。In another use case thereof, the lipid particles can be incorporated into a variety of topical dosage forms. For example, suspensions containing nucleic acid-lipid particles such as SNALP can be formulated and administered in the form of gels, oils, lotions, topical creams, pastes, ointments, lotions, foams, mousses and the like. .

當製備本發明之脂質粒子的醫藥製劑時，較佳使用一定量的已經純化之粒子，以減少或消除空粒子或治療劑諸如核酸與外表面締合之粒子。When preparing a pharmaceutical preparation of the lipid particle of the present invention, it is preferred to use a certain amount of purified particles to reduce or eliminate particles associated with empty particles or therapeutic agents such as nucleic acids associated with the outer surface.

本發明之方法可在各種宿主中實踐。較佳宿主包括哺乳動物物種，諸如靈長類動物(例如人類及黑猩猩以及其他非人類靈長類動物)、犬科動物、貓科動物、馬科動物、牛科動物、綿羊、山羊、嚙齒動物(例如大鼠及小鼠)、兔類動物及豬。The methods of the invention can be practiced in a variety of hosts. Preferred hosts include mammalian species such as primates (eg, humans and chimpanzees and other non-human primates), canines, felines, equines, bovines, sheep, goats, rodents (eg rat and mouse), rabbits and pigs.

投與之粒子的量將視治療劑(例如核酸)與脂質之比率、所使用之特定治療劑(例如核酸)、所治療之疾病或病症、患者之年齡、體重及病況以及臨床醫師之判斷而定，但通常每次投與(例如注射)約0.01至約50 mg/kg體重，較佳約0.1至約5 mg/kg體重。The amount of particles administered will depend on the ratio of therapeutic agent (e.g., nucleic acid) to lipid, the particular therapeutic agent (e.g., nucleic acid) used, the disease or condition being treated, the age, weight and condition of the patient, and the judgment of the clinician. Preferably, but usually administered (e.g., by injection) from about 0.01 to about 50 mg/kg body weight, preferably from about 0.1 to about 5 mg/kg body weight.

對於活體外應用，可將治療劑諸如核酸(例如cdsDNA)遞送至培養物中生長之任何細胞，不論植物或動物來源、脊椎動物或無脊椎動物以及任何組織或類型。在較佳實施例中，細胞為動物細胞，更佳哺乳動物細胞，且最佳人類細胞(例如腫瘤細胞或肝細胞)。For in vitro applications, a therapeutic agent, such as a nucleic acid (eg, cds DNA), can be delivered to any cell grown in culture, regardless of plant or animal source, vertebrate or invertebrate, and any tissue or type. In a preferred embodiment, the cells are animal cells, more preferably mammalian cells, and optimal human cells (e.g., tumor cells or hepatocytes).

當活體外進行時，細胞與脂質粒子之間的接觸在生物相容性介質中進行。粒子之濃度視具體應用而廣泛變化，但通常在約1 μmol與約10 mmol之間。用脂質粒子處理細胞一般在生理溫度(約37℃)下進行約1至48小時、較佳約2至4小時的時間段。When performed in vitro, the contact between the cells and the lipid particles is carried out in a biocompatible medium. The concentration of the particles varies widely depending on the particular application, but is typically between about 1 μmol and about 10 mmol. Treatment of the cells with the lipid particles is typically carried out at physiological temperature (about 37 ° C) for a period of from about 1 to 48 hours, preferably from about 2 to 4 hours.

在一個實施例中，將脂質粒子懸浮液添加至60-80%匯合的塗覆細胞中，細胞密度為約103 至約105 個細胞/毫升，更佳約2 × 104 個細胞/毫升。添加至細胞中之懸浮液的濃度較佳為約0.01至0.2 μg/ml，更佳約0.1 μg/ml。In one embodiment, the lipid particle suspension is added to 60-80% confluent coated cells at a cell density of from about 10 3 to about 10 5 cells/ml, more preferably about 2 x 10 4 cells/ml. . The concentration of the suspension added to the cells is preferably from about 0.01 to 0.2 μg/ml, more preferably about 0.1 μg/ml.

在可能需要細胞組織培養之程度上，其為此項技術中熟知的。舉例而言，Freshney, Culture of Animal Cells, a Manual of Basic Technique, 第3版, Wiley-Liss, New York (1994)，Kuchler等人, Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977)及其中列舉之參考文獻提供細胞培養之一般指導。培養的細胞系統通常呈單層細胞形式，但亦使用細胞懸浮液。To the extent that cell tissue culture may be required, it is well known in the art. For example, Freshney, Culture of Animal Cells, a Manual of Basic Technique, 3rd Edition, Wiley-Liss, New York (1994), Kuchler et al, Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc (1977) and the references cited therein provide general guidance for cell culture. The cultured cell system is usually in the form of a monolayer of cells, but cell suspensions are also used.

使用核內體釋放參數(ERP)分析，可最佳化本發明之SNALP或其他脂質粒子的遞送效率。ERP分析詳細描述於美國專利公開案第20030077829號中，其揭示內容出於所有目的以全文引用之方式併入本文中。更特定言之，ERP分析之目的為基於其對核內體膜之結合/攝入或融合/去穩定化之相對效應來區分SNALP或其他脂質粒子之各種陽離子脂質及輔助脂質組分之效應。此分析允許定量測定SNALP或其他脂質粒子之每種組分如何影響遞送效率，從而最佳化SNALP或其他脂質粒子。通常，ERP分析量測報導蛋白(例如螢光素酶、β-半乳糖苷酶、綠色螢光蛋白(GFP)等)之表現，且在一些情況下，針對表現質體最佳化之SNALP調配物亦適合於囊封干擾RNA。在其他情況下，ERP分析可適於在存在或不存在干擾RNA (例如siRNA)之情況下量測靶序列之轉錄或轉譯的下調。藉由比較各種SNALP或其他脂質粒子中之每一者的ERP，吾人可容易地確定經最佳化之系統，例如SNALP或在細胞中具有最大攝入之其他脂質粒子。Delivery efficiency of SNALP or other lipid particles of the invention can be optimized using endosomal release parameter (ERP) analysis. The ERP analysis is described in detail in U.S. Patent Publication No. 20030077829, the disclosure of which is hereby incorporated by reference in its entirety in its entirety in its entirety. More specifically, the purpose of ERP analysis is to distinguish the effects of various cationic lipids and helper lipid components of SNALP or other lipid particles based on their relative effects on binding/ingesting or fusion/destabilization of the endosomal membrane. This analysis allows quantitative determination of how each component of SNALP or other lipid particles affects delivery efficiency, thereby optimizing SNALP or other lipid particles. Typically, ERP assays measure the performance of reporter proteins (eg, luciferase, beta-galactosidase, green fluorescent protein (GFP), etc.) and, in some cases, SNALP blending for plastid optimization. The substance is also suitable for encapsulating interfering RNA. In other instances, ERP analysis can be adapted to measure down-regulation of transcription or translation of a target sequence in the presence or absence of interfering RNA (e.g., siRNA). By comparing the ERP of each of the various SNALP or other lipid particles, one can readily determine an optimized system, such as SNALP or other lipid particles that have the greatest uptake in the cell.

本發明之組合物及方法用於活體內及活體外治療多種細胞類型。適合之細胞包括但不限於肝細胞、網狀內皮細胞(例如單核球、巨噬細胞等)、成纖維細胞、內皮細胞、血小板細胞、感染及/或易感染病毒之其他細胞類型、造血前體(幹)細胞、角質細胞、骨骼肌細胞、心肌細胞及平滑肌細胞、成骨細胞、神經元、靜止淋巴細胞、終末分化細胞、緩慢或非循環初級細胞、實質細胞、淋巴細胞、上皮細胞、骨細胞及其類似物。The compositions and methods of the invention are useful for treating a variety of cell types in vivo and in vitro. Suitable cells include, but are not limited to, hepatocytes, reticuloendothelial cells (eg, mononuclear spheres, macrophages, etc.), fibroblasts, endothelial cells, platelet cells, infections, and/or other cell types susceptible to infection, pre-hematopoietic Body (dry) cells, keratinocytes, skeletal muscle cells, cardiomyocytes and smooth muscle cells, osteoblasts, neurons, resting lymphocytes, terminally differentiated cells, slow or non-circulating primary cells, parenchymal cells, lymphocytes, epithelial cells, Bone cells and their analogues.

在具體實施例中，將活性劑或治療劑諸如cdsDNA遞送至癌細胞(例如實體腫瘤細胞)，包括但不限於肝癌細胞、肺癌細胞、結腸癌細胞、直腸癌細胞、肛門癌細胞、膽管癌細胞、小腸癌細胞、胃癌細胞、食道癌細胞、膽囊癌細胞、胰臟癌細胞、闌尾癌細胞、乳癌細胞、卵巢癌細胞、子宮頸癌細胞、***癌細胞、腎癌細胞、中樞神經系統癌細胞、膠質母細胞瘤腫瘤細胞、皮膚癌細胞、淋巴瘤細胞、絨膜癌腫瘤細胞、頭頸癌細胞、骨原性肉瘤腫瘤細胞及血癌細胞。In a specific embodiment, the active agent or therapeutic agent, such as cds DNA, is delivered to cancer cells (eg, solid tumor cells) including, but not limited to, liver cancer cells, lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, cholangiocarcinoma cells. Small intestinal cancer cells, gastric cancer cells, esophageal cancer cells, gallbladder cancer cells, pancreatic cancer cells, appendix cancer cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, renal cancer cells, central nervous system cancer cells Glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells, and blood cancer cells.

囊封核酸(例如cdsDNA)之脂質粒子諸如SNALP的活體內遞送適於靶向任何細胞類型之細胞。該等方法及組合物可與多種脊椎動物之細胞一起使用，包括哺乳動物，諸如犬科動物、貓科動物、馬科動物、牛科動物、綿羊、山羊、嚙齒動物(例如小鼠、大鼠及天竺鼠)、兔類動物、豬及靈長類動物(例如猴、黑猩猩及人類)。In vivo delivery of lipid particles such as SNALP that encapsulates nucleic acids (eg, cds DNA) is suitable for targeting cells of any cell type. The methods and compositions can be used with a variety of vertebrate cells, including mammals, such as canines, felines, equines, bovines, sheep, goats, rodents (eg, mice, rats) And guinea pigs, rabbits, pigs and primates (eg monkeys, chimpanzees and humans).

AtuPLEX
AtuPLEX為多層(多個脂質雙層)且帶正電荷之siRNA-脂複合體，其將siRNA與三脂質脂質體組合在一起。此可經修飾以遞送主題cdsDNA用於各種目的。 AtuPLEX
AtuPLEX is a multi-layer (multiple lipid bilayer) and positively charged siRNA-lipid complex that combines siRNA with trilipid liposomes. This can be modified to deliver the subject cdsDNA for a variety of purposes.

脂質體含有專有的陽離子脂質AtuFect01、共脂質(促融合或穩定化)及聚乙二醇化脂質，以形成核酸(siRNA或cdsDNA)嵌入粒子之多個脂質雙層內的奈米粒子結構。Liposomes contain proprietary cationic lipids AtuFect01, co-lipids (pro-fusion or stabilization) and PEGylated lipids to form nanoparticle structures within the multiple lipid bilayers of nucleic acids (siRNA or cdsDNA) embedded in the particles.

因此，在某些實施例中，將主題cdsDNA調配於包含醫藥學上可接受之載劑及式(I)化合物之組合物中：

其中R1及R2各自獨立地選自包含烷基之群；
n為1至4之任何整數；
R3為選自包含離胺醯基、鳥胺醯基、2,4-二胺基丁醯基、組胺醯基及根據式(II)之醯基部分之群的醯基，

其中m為1至3之任何整數且Y- 為醫藥學上可接受之陰離子。Thus, in certain embodiments, the subject cdsDNA is formulated in a composition comprising a pharmaceutically acceptable carrier and a compound of formula (I):

Wherein R 1 and R 2 are each independently selected from the group consisting of alkyl groups;
n is any integer from 1 to 4;
R3 is a fluorenyl group selected from the group consisting of an amidoxime group, an aguanamine group, a 2,4-diaminobutanyl group, a histidine group, and a group of a fluorenyl group according to formula (II).

Wherein m is any integer from 1 to 3 and Y - is a pharmaceutically acceptable anion.

在某些實施例中，R1及R2各自獨立地選自包含月桂基、肉豆蔻基、棕櫚基及油基之群。In certain embodiments, R1 and R2 are each independently selected from the group consisting of lauryl, myristyl, palmitiferyl, and oleyl.

在某些實施例中，R1為月桂基且R2為肉豆蔻基；或R1為棕櫚基且R2為油基。In certain embodiments, R1 is lauryl and R2 is myristyl; or R1 is palmitoyl and R2 is an oily group.

在某些實施例中，m為1或2。In certain embodiments, m is 1 or 2.

在某些實施例中，Y- 係選自由鹵離子、乙酸根及三氟乙酸根組成之群。In certain embodiments, the Y - line is selected from the group consisting of halides, acetates, and trifluoroacetates.

在某些實施例中，化合物選自由以下組成之群：
In certain embodiments, the compound is selected from the group consisting of:

在某些實施例中，組合物進一步包含選自由肽、蛋白質、寡核苷酸、聚核苷酸及核酸組成之群的醫藥活性組分。In certain embodiments, the composition further comprises a pharmaceutically active component selected from the group consisting of peptides, proteins, oligonucleotides, polynucleotides, and nucleic acids.

在某些實施例中，組合物進一步包含至少一種選自由磷脂及類固醇組成之群的輔助脂質組分。舉例而言，輔助脂質組分可選自由1,2-二植烷醯基-sn-甘油基-3-磷酸乙醇胺及1,2-二油基-sn-甘油基-3-磷酸乙醇胺組成之群。在某些實施例中，輔助脂質組分之含量為組合物之總脂質含量的約20 mol%至約80 mol%。在某些實施例中，至少一種輔助脂質包含選自由PEG、HEG、聚羥乙基澱粉(polyHES)及聚丙烯組成之群的部分。在某些實施例中，包含PEG部分之輔助脂質選自由1,2-二硬脂醯基-sn-甘油基-3-磷酸乙醇胺、1,2-二烷基-sn-甘油基-3-磷酸乙醇胺及神經醯胺-PEG組成之群。In certain embodiments, the composition further comprises at least one auxiliary lipid component selected from the group consisting of phospholipids and steroids. For example, the helper lipid component can be selected from the group consisting of 1,2-diphytyl-sn-glyceryl-3-phosphoethanolamine and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine. group. In certain embodiments, the auxiliary lipid component is present in an amount from about 20 mol% to about 80 mol% of the total lipid content of the composition. In certain embodiments, the at least one helper lipid comprises a moiety selected from the group consisting of PEG, HEG, polyhydroxyethyl starch (polyHES), and polypropylene. In certain embodiments, the helper lipid comprising a PEG moiety is selected from the group consisting of 1,2-distearyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dialkyl-sn-glyceryl-3- A group consisting of phosphoethanolamine and neuropterin-PEG.

在某些實施例中，組合物包含：a) 50 mol%之β-精胺醯基-2,3-二胺基丙酸-N-棕櫚基-N-油基-醯胺三鹽酸鹽、48 mol%之1,2-二植烷醯基-sn-甘油基-3-磷酸乙醇胺及2 mol% 1,2-二硬脂醯基-sn-甘油基-3-磷酸乙醇胺-PEG2000；或b) β-精胺醯基-2,3-二胺基丙酸-N-棕櫚基-N-油基-醯胺三鹽酸鹽、49 mol% 1,2-二植烷醯基-sn-甘油基-3-磷酸乙醇胺及1 mol% 1,2-二硬脂醯基-sn-甘油基-3-磷酸乙醇胺-PEG2000。In certain embodiments, the composition comprises: a) 50 mol% of β-spermine mercapto-2,3-diaminopropionic acid-N-palmityl-N-oleyl-decylamine trihydrochloride 48 mol% of 1,2-diphytylsulfonyl-sn-glyceryl-3-phosphoethanolamine and 2 mol% 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-PEG2000; Or b) β-spermine mercapto-2,3-diaminopropionic acid-N-palmityl-N-oleyl-decylamine trihydrochloride, 49 mol% 1,2-diphytyl fluorenyl- Sn-glyceryl-3-phosphoethanolamine and 1 mol% 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-PEG2000.

在某些實施例中，組合物包含：a) 50 mol%之β-精胺醯基-2,3-二胺基丙酸-N-棕櫚基-N-油基-醯胺三鹽酸鹽、48 mol%之1,2-二植烷醯基-sn-甘油基-3-磷酸乙醇胺及2 mol% N(羰基-甲氧基聚乙二醇-2000)-1,2-二硬脂醯基-sn-甘油基-3-磷酸乙醇胺；或b) 50 mol%之β-精胺醯基-2,3-二胺基丙酸-N-棕櫚基-N-油基-醯胺三鹽酸鹽、49 mol% 1,2-二植烷醯基-sn-甘油基-3-磷酸乙醇胺及1 mol% N(羰基-甲氧基聚乙二醇-2000)-1,2-二硬脂醯基-sn-甘油基-3-磷酸乙醇胺。In certain embodiments, the composition comprises: a) 50 mol% of β-spermine mercapto-2,3-diaminopropionic acid-N-palmityl-N-oleyl-decylamine trihydrochloride 48 mol% of 1,2-diphytyl-sn-glyceryl-3-phosphoethanolamine and 2 mol% of N (carbonyl-methoxypolyethylene glycol-2000)-1,2-distearyl Mercapto-sn-glyceryl-3-phosphoethanolamine; or b) 50 mol% of β-spermine mercapto-2,3-diaminopropionic acid-N-palmityl-N-oleyl-decylamine Hydrochloride, 49 mol% 1,2-diphytyl-sn-glyceryl-3-phosphoethanolamine and 1 mol% N (carbonyl-methoxy polyethylene glycol-2000)-1,2-di Stearyl-sn-glyceryl-3-phosphoethanolamine.

在某些實施例中，組合物包含：a) 50 mol%之β-L-精胺醯基-2,3-L-二胺基丙酸-N-棕櫚基-N-油基-醯胺三鹽酸鹽、48 mol%之1,2-二植烷醯基-sn-甘油基-3-磷酸乙醇胺及2 mol% N(羰基-甲氧基聚乙二醇-2000)-1,2-二硬脂醯基-sn-甘油基-3-磷酸乙醇胺；或b) 50 mol%之β-L-精胺醯基-2,3-L-二胺基丙酸-N-棕櫚基-N-油基-醯胺三鹽酸鹽、49 mol% 1,2-二植烷醯基-sn-甘油基-3-磷酸乙醇胺及1 mol% N(羰基-甲氧基聚乙二醇-2000)-1,2-二硬脂醯基-sn-甘油基-3-磷酸乙醇胺。In certain embodiments, the composition comprises: a) 50 mol% of β-L-spermine mercapto-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-decylamine Trihydrochloride, 48 mol% 1,2-diphytylsulfonyl-sn-glyceryl-3-phosphoethanolamine and 2 mol% N (carbonyl-methoxypolyethylene glycol-2000)-1,2 - distearyl-sn-glyceryl-3-phosphoethanolamine; or b) 50 mol% of β-L-spermine mercapto-2,3-L-diaminopropionic acid-N-palmityl- N-Oryl-decylamine trihydrochloride, 49 mol% 1,2-diphytyl-sn-glyceryl-3-phosphoethanolamine and 1 mol% N (carbonyl-methoxypolyethylene glycol- 2000)-1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine.

用於生產粒子之多個脂質雙層的其他適合之脂質組合物描述於美國專利第8,357,722號、第8,017,804號及第9,486,538號中(均以引用的方式併入)。Other suitable lipid compositions for the production of a plurality of lipid bilayers of the particles are described in U.S. Patent Nos. 8,357,722, 8,017,804, and 9, 486, 538, each incorporated by reference.

DACC
DACC為包括AtuFect01 (參見上文)之脂質遞送系統，且用於將siRNA嵌入多脂質雙層結構中。此系統亦可經修飾以遞送主題cdsDNA用於各種目的。 DACC
DACC is a lipid delivery system comprising AtuFect01 (see above) and is used to embed siRNA in a multi-lipid bilayer structure. This system can also be modified to deliver the subject cdsDNA for a variety of purposes.

雖然與AtuPLEX系統密切相關，但DACC具有顯著不同的生物醫藥特性，且將核酸遞送至肺血管內皮。Although closely related to the AtuPLEX system, DACCs have significantly different biomedical properties and deliver nucleic acids to the pulmonary vascular endothelium.

DBTC
DBTC為將siRNA遞送至肝細胞及肝實質之肝血管系統，而非僅靶向肝臟肝細胞的脂質遞送系統。此系統亦可經修飾以遞送主題cdsDNA用於各種目的。 DBTC
DBTC is a lipid delivery system that delivers siRNA to the hepatic vasculature of hepatocytes and liver parenchyma, rather than only liver hepatocytes. This system can also be modified to deliver the subject cdsDNA for a variety of purposes.

DBTC奈米粒子由合成遞送系統組成，其含有(i)基於環糊精之線性聚合物(CDP)，(ii)呈現於奈米粒子之外部上的人類轉鐵蛋白(hTf)靶向配體以接合癌細胞表面上之Tf受體(hTfR)，(iii)親水性聚合物(用於促進生物流體中之奈米粒子穩定性的聚乙二醇(PEG))及(iv)經設計以降低靶基因表現之siRNA。參見Davis等人,Nature 464(7291):1067-1070, 2010 (以引用的方式併入)。可藉由用主題cdsDNA置換siRNA組分來修飾此系統。亦可藉由置換靶向部分來修飾該系統，從而可實現向其他特定組織部位之遞送。舉例而言，為遞送至骨骼肌或心肌細胞，TfR可用骨骼或心血管細胞上之受體的天然配體或對骨骼或心血管細胞上之標記具有特異性的抗體或其片段置換。靶向部分亦可衍生自具有所需天然趨向性之病毒(例如用於心肌細胞遞送之腺病毒5型旋鈕蛋白)。相同策略可用於本文所述之奈米粒子之任何其他靶向遞送。The DBTC nanoparticle consists of a synthetic delivery system containing (i) a cyclodextrin-based linear polymer (CDP), (ii) a human transferrin (hTf) targeting ligand presented on the exterior of the nanoparticle. To bind the Tf receptor (hTfR) on the surface of cancer cells, (iii) a hydrophilic polymer (polyethylene glycol (PEG) for promoting the stability of nanoparticles in biological fluids) and (iv) are designed to siRNA that reduces the performance of the target gene. See Davis et al, Nature 464 (7291): 1067-1070, 2010 (incorporated by reference). This system can be modified by replacing the siRNA component with the subject cdsDNA. The system can also be modified by replacing the targeting moiety so that delivery to other specific tissue sites can be achieved. For example, for delivery to skeletal muscle or cardiomyocytes, TfR can be replaced with a natural ligand for a receptor on bone or cardiovascular cells or an antibody or fragment thereof that is specific for a marker on bone or cardiovascular cells. The targeting moiety can also be derived from a virus having the desired natural tropism (eg, an adenovirus type 5 knob protein for cardiomyocyte delivery). The same strategy can be used for any other targeted delivery of the nanoparticles described herein.

已顯示此等奈米粒子在非人類靈長類動物之多劑量研究中具有良好耐受性，且可全身遞送以治療例如實體腫瘤。These nanoparticles have been shown to be well tolerated in multi-dose studies of non-human primates and can be delivered systemically to treat, for example, solid tumors.

RONDEL
RONDEL表示RNAi/寡核苷酸奈米粒子遞送。其為已用於在臨床試驗中遞送siRNA用於治療實體腫瘤之平台。RONDEL為針對活體內siRNA遞送再最佳化之基於聚合物之非脂質體奈米粒子。其具有四種自組裝成奈米粒子之組分：(i) siRNA股(其可用本發明之cdsDNA置換)，(ii)含有環糊精之聚合物(CDP)，(iii)作為空間穩定劑之聚乙二醇(PEG)及(iv)人類轉鐵蛋白(Tf)。Tf為與轉鐵蛋白受體(TfR)結合之靶向配體，其通常在癌細胞上上調。但此組分可經任何靶向部分置換，諸如對肌肉細胞或其他靶組織具有特異性之抗體或抗體片段。CDP為含有帶正電荷之脒基團與糖(環糊精)部分交替的線性聚陽離子寡聚物。帶正電荷之CDP聚合物與帶負電荷之核酸(諸如cdsDNA)骨架締合，形成直徑小於100 nm之奈米粒子，使核酸在其核心且環糊精基團在其表面上。組分(iii)及(iv)經由與PEG之一個末端共價結合的疏水性金剛烷基團與CDP中之疏水性核心非共價締合。所得複合物為包覆有PEG (穩定劑)及PEG-靶向配體(諸如TfR)之含有核酸的奈米粒子。 RONDEL
RONDEL represents RNAi/oligonucleotide nanoparticle delivery. It is a platform that has been used to deliver siRNA in clinical trials for the treatment of solid tumors. RONDEL is a polymer based non-liposomal nanoparticle optimized for in vivo siRNA delivery. It has four components that self-assemble into nanoparticle: (i) siRNA strands (which can be replaced with cdsDNA of the invention), (ii) cyclodextrin-containing polymers (CDP), (iii) as steric stabilizers Polyethylene glycol (PEG) and (iv) human transferrin (Tf). Tf is a targeting ligand that binds to the transferrin receptor (TfR), which is typically up-regulated on cancer cells. However, this component can be replaced by any targeting moiety, such as an antibody or antibody fragment specific for muscle cells or other target tissues. CDP is a linear polycationic oligomer containing alternating positively charged sulfonium groups with sugar (cyclodextrin) moieties. The positively charged CDP polymer is associated with a negatively charged nucleic acid (such as cdsDNA) backbone to form nanoparticles having a diameter of less than 100 nm such that the nucleic acid is at its core and the cyclodextrin group is on its surface. Components (iii) and (iv) are non-covalently associated with the hydrophobic core in the CDP via a hydrophobic adamantyl group covalently bonded to one end of the PEG. The resulting complex is a nucleic acid-containing nanoparticle coated with PEG (stabilizer) and a PEG-targeting ligand (such as TfR).

DPC
DPC (Dynamic PolyConjugates)為一類基於聚合物結合物之非脂質體核酸(例如siRNA或cdsDNA)遞送平台。DPC係尺寸為5-20 nm之小奈米粒子，其含有兩性核內體裂解聚合物聚(丁基胺基乙烯基醚) (PBAVE)，可逆地連接屏蔽劑(例如PEG)及靶向配體，且經由可水解之二硫化物連接子連接核酸。雖然破壞膜之PBAVE聚合物經PEG側鏈掩蔽，但PEG及靶向配體在核內體之酸性環境中釋放以觸發核內體釋放。一旦進入細胞質的還原環境，就裂解二硫鍵以釋放核酸(例如cdsDNA)。參見美國專利第8,501,930號(以引用的方式併入本文中)。 DPC
DPC (Dynamic PolyConjugates) is a class of polymer conjugate-based non-liposome nucleic acid (eg, siRNA or cdsDNA) delivery platforms. DPC is a small nanoparticle with a size of 5-20 nm, which contains an amphoteric endosomal cleavage polymer poly(butylamino vinyl ether) (PBAVE), reversibly attaching a shielding agent (such as PEG) and targeting The nucleic acid is linked via a hydrolyzable disulfide linker. Although the PBAVE polymer that disrupts the membrane is masked by the PEG side chain, the PEG and targeting ligand are released in the acidic environment of the endosome to trigger endosome release. Upon entry into the cytoplasmic reducing environment, the disulfide bond is cleaved to release nucleic acids (eg, cds DNA). See U.S. Patent No. 8,501,930, incorporated herein by reference.

DPC系統之改良版利用原子轉移自由基聚合(ATRP)及可逆加成-斷裂鏈轉移(RAFT)來生產均勻且易於大規模製造的聚合物。其中之核酸不附接至DPC聚合物。相反，核酸-膽固醇結合物與類似於PBAVE聚合物之具有經可逆掩蔽之核內體裂解特性的蜂毒素樣肽共注射。A modified version of the DPC system utilizes atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer (RAFT) to produce polymers that are uniform and easy to manufacture on a large scale. The nucleic acid therein is not attached to the DPC polymer. In contrast, the nucleic acid-cholesterol conjugate is co-injected with a melittin-like peptide similar to the PBAVE polymer with reversible masked endosome cleavage properties.

SMARTICLES
SMARTICLES為兩性脂質體，其為pH依賴性電荷轉換粒子，藉由局部或全身投與向細胞提供治療性核酸(例如cdsDNA)。SMARTICLES在血液中穩定且以與習知脂質體相同的方式分佈，但當其穿過細胞膜時變成帶正電荷，從而導致在炎症部位、腫瘤、肝臟及脾臟內遞送核酸有效負載。 SMARTICLES
SMARTICLES are amphiphilic liposomes that are pH dependent charge-switching particles that provide therapeutic nucleic acids (eg, cds DNA) to cells by local or systemic administration. SMARTICLES are stable in the blood and are distributed in the same manner as conventional liposomes, but become positively charged as they pass through the cell membrane, resulting in the delivery of nucleic acid payloads in the site of inflammation, tumors, liver and spleen.

DiLA2
DiLA2 (二烷基化胺基酸)為用於自二烷基化胺基酸產生脂質體調配物以遞送治療性核酸(諸如cdsDNA)的平台。DiLA2 為基於含有組胺酸之二烷基化胺基酸系統，其允許吾人修改遞送系統之關鍵態樣，諸如電荷、連接子及醯基鏈以最佳化脂質體之特性。舉例而言，DiLA2 允許吾人最佳化向所關注之靶組織的遞送，且允許包含肽以改良多種遞送特徵，諸如奈米粒子之囊封、細胞攝取、核內體釋放及細胞/組織靶向。參見Adami等人, An amino acid-based amphoteric liposomal delivery system for systemic administration of siRNA.Mol . Ther . 19:1141-1151, 2011 (以引用的方式併入)。亦參見美國專利第8,501,824號及第7,959,505號(以引用的方式併入本文中)。 DiLA 2
DiLA 2 (dialkylated amino acid) is a platform for the production of liposome formulations from dialkylated amino acids to deliver therapeutic nucleic acids, such as cds DNA. DiLA 2 is based on a dialkylated amino acid system containing histidine, which allows us to modify key aspects of the delivery system, such as charge, linker and thiol chains to optimize liposome properties. For example, DiLA 2 allows us to optimize delivery to target tissues of interest and allows peptides to be included to improve various delivery characteristics, such as encapsulation of nanoparticles, cellular uptake, endosomal release, and cell/tissue targets. to. See Adami et al, An amino acid-based amphoteric liposomal delivery system for systemic administration of siRNA. Mol . Ther . 19: 1141-1151, 2011 (incorporated by reference). See also U.S. Patent Nos. 8,501,824 and 7,959,505 each incorporated herein by reference.

EnCore
EnCore為開發用於遞送核酸有效負載(諸如cdsDNA)之奈米粒子系統，用於遞送至肝臟及實體腫瘤。EnCore含有由不同脂質混合物之包膜包圍的脂質-核酸核心，其介導核酸有效負載之積聚、內化及釋放至靶細胞中。此亞結構粒子經設計以用於在腫瘤中優先積聚且提供高水準之有效負載遞送。參見美國專利第7,371,404號(以引用的方式併入本文中)。 EnCore
EnCore is a nanoparticle system developed for the delivery of nucleic acid payloads, such as cds DNA, for delivery to liver and solid tumors. EnCore contains a lipid-nucleic acid core surrounded by an envelope of different lipid mixtures that mediates the accumulation, internalization and release of nucleic acid payload into target cells. This substructured particle is designed for preferential accumulation in the tumor and provides a high level of payload delivery. See U.S. Patent No. 7,371,404, incorporated herein by reference.

PRX
聚輪烷或PRX為一種由多個α-環糊精環、PEG鏈及兩個龐大的塞子構成之互鎖大分子，用於物理防止環解開。PRX可自組裝且將核酸縮合成奈米結構。已在mdx小鼠中證明PRX成功遞送質體，且資料顯示在大多數肌肉組織中有豐富的質體分佈。因此，PRX亦可用於遞送主題cdsDNA。 PRX
Polyrotaxane or PRX is an interlocking macromolecule composed of a plurality of α-cyclodextrin rings, PEG chains and two bulky plugs for physically preventing ring unwinding. PRX can self-assemble and condense nucleic acids into nanostructures. PRX has been shown to successfully deliver plastids in mdx mice, and the data show a rich plastid distribution in most muscle tissues. Therefore, PRX can also be used to deliver the subject cdsDNA.

本發明之方法部分在活體外無細胞環境中進行，諸如原核端粒酶消化及下游純化及調配步驟。因此，該方法部分在不存在宿主細胞之情況下進行，且通常包含使用經純化之酶組分。因此，藉由原核端粒酶加工及其他下游操作通常藉由使溶液中之反應組分在適合之容器中接觸來進行。視情況，特定組分可以固定化形式提供，諸如附接至固體支撐物。The methods of the invention are carried out in part in an in vitro cell-free environment, such as protelomerase digestion and downstream purification and formulation steps. Thus, the method is carried out in part in the absence of host cells and typically involves the use of purified enzyme components. Thus, protelomerase processing and other downstream operations are typically performed by contacting the reaction components in solution in a suitable container. Optionally, specific components may be provided in an immobilized form, such as attached to a solid support.

應理解，本發明之方法可在任何規模下進行。然而，較佳進行該方法以商業或工業規模生產任何所關注之DNA片段，亦即以mg或更大量產生所關注之擴增DNA片段。在某些實施例中，該方法產生至少1 mg、至少10 mg、至少20 mg、至少50 mg或至少100 mg所關注之DNA片段。最終cdsDNA產物亦可以mg或更大量產生。在某些實施例中，該方法產生至少1 mg、至少2 mg、至少5 mg、至少10 mg、至少20 mg、至少50 mg或至少100 mg之cdsDNA。It should be understood that the process of the invention can be carried out at any scale. Preferably, however, the method produces any DNA fragment of interest on a commercial or industrial scale, i.e., produces amplified DNA fragments of interest in mg or greater. In certain embodiments, the method produces at least 1 mg, at least 10 mg, at least 20 mg, at least 50 mg, or at least 100 mg of the DNA fragment of interest. The final cdsDNA product can also be produced in mg or more. In certain embodiments, the method produces at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 50 mg, or at least 100 mg of cds DNA.

7. 大規模製造
包含主題cdsDNA之本發明之LNP及聚合物NP可使用任何此項技術中公認的方法製造。在某些實施例中，LNP及聚合物LP可使用市售服務及/或設備製造，諸如由Precision Nanosystems (Vancouver, British Columbia)提供之彼等服務及/或設備。7. Large scale manufacture of LNPs and polymer NPs of the invention comprising the subject cdsDNA can be made using any method recognized in the art. In certain embodiments, LNPs and polymer LPs can be manufactured using commercially available services and/or equipment, such as those provided by Precision Nanosystems (Vancouver, British Columbia).

在某些實施例中，使用Precision Nanosystems之NanoAsseblr平台製備LNP及NP。NanoAsseblr平台利用微流體混合進行受控制、經調整且完全可擴展之奈米藥物製造，包括本發明之LNP及聚合物NP。其能夠經由定製工程改造之微流體混合筒快速且受控地製造奈米藥物(脂質體、脂質奈米粒子、聚合物奈米粒子)，該等混合筒允許在奈升規模下進行奈米粒子之毫秒級混合。該平台允許使用者藉由方法及組成來控制尺寸，且調節諸如混合比、流動速率及脂質組成之參數以微調奈米粒子尺寸及囊封效率。In certain embodiments, LNP and NP are prepared using Precision Nanosystems' NanoAsseblr platform. The NanoAsseblr platform utilizes microfluidic mixing for controlled, tailored, and fully scalable nanopharmaceutical manufacturing, including the LNPs and polymer NPs of the present invention. It is capable of rapidly and controlled production of nano drugs (liposomes, lipid nanoparticles, polymer nanoparticles) via a custom engineered microfluidic mixing cartridge that allows for nanonization at nanoliter scale Millisecond mixing of particles. The platform allows the user to control the size by method and composition, and adjust parameters such as mixing ratio, flow rate and lipid composition to fine tune the nanoparticle size and encapsulation efficiency.

此系統已與Neuro9TM 轉染套組(Precision Nanosystems, Vancouver, British Columbia之非病毒轉染套組)結合使用，以活體外及活體內將siRNA及mRNA及pDNA遞送至原代神經元，實現95%之細胞轉染效率及在經轉染細胞中實現90%之阻斷基因表現，且無可觀察的毒性。Use of this system has been combined with Neuro9 TM transfection kit (Precision Nanosystems, Vancouver, British Columbia the non-viral transfection kit), in vitro and in vivo the siRNA and mRNA and pDNA delivery to primary neuron, to achieve 95 % cell transfection efficiency and 90% blocking gene expression in transfected cells with no observable toxicity.

Neuro9™將核酸囊封且保護在模擬內源性低密度脂蛋白(LDL)之合成脂質奈米粒子(LNP)中，其由如神經元、星形膠質細胞及iPSC之細胞天然攝取。Neuro9具有pH敏感性且經工程改造以將其內含物釋放至細胞質中。Neuro9TM 轉染套組及相關產品亦可用於將主題cdsDNA遞送至特定靶細胞，諸如神經元。Neuro9TM encapsulates and protects nucleic acids in synthetic lipid nanoparticles (LNP) that mimic endogenous low density lipoprotein (LDL), which are naturally taken up by cells such as neurons, astrocytes, and iPSCs. Neuro9 is pH sensitive and engineered to release its contents into the cytoplasm. Neuro9 TM Transfection kit and related products can be used for the topic cdsDNA delivered to specific target cells, such as neurons.

在某些實施例中，本發明之LNP及聚合物NP以規模擴大之量生產約10 mL至1 L，或多達5 L，或多達10 L，或多達15 L，或多達20 L，或多達25 L，或多達50 L或更多的LNP及NP調配物。規模擴大可經由微流體反應器並行化，類似於整合晶片上之電晶體陣列。在某些實施例中，製造過程受軟體控制或半/全自動化。In certain embodiments, the LNPs and polymer NPs of the invention are produced in an amount of about 10 mL to 1 L, or up to 5 L, or up to 10 L, or up to 15 L, or up to 20 in an expanded amount. L, or up to 25 L, or up to 50 L or more of LNP and NP formulations. Scale up can be parallelized via a microfluidic reactor, similar to integrating a transistor array on a wafer. In some embodiments, the manufacturing process is software controlled or semi/fully automated.

在某些實施例中，製造在NanoAssemblr Spark中進行，其具有25-250 μL之操作奈米粒子調配物體積。In certain embodiments, the fabrication is performed in a NanoAssemblr Spark having a 25-250 μL manipulated nanoparticle formulation volume.

在某些實施例中，製造在NanoAssemblr Benchtop系統中進行，其經設計用於1至15 mL之新奈米粒子調配物的快速原型製作，且具有軟體控制，其能夠控制輸入混合參數以最佳化粒子特徵，諸如尺寸及囊封效率。In certain embodiments, fabrication is performed in a NanoAssemblr Benchtop system designed for rapid prototyping of 1 to 15 mL of new nanoparticle formulations with software control that is capable of controlling input mixing parameters for optimal Particle characteristics such as size and encapsulation efficiency.

在某些實施例中，製造在適於臨床開發之NanoAssemblr Blaze系統中進行。該系統可在10 mL與1 L之間製造。In certain embodiments, the fabrication is performed in a NanoAssemblr Blaze system suitable for clinical development. The system can be manufactured between 10 mL and 1 L.

在某些實施例中，製造在NanoAssemblr Scale-Up系統中進行，該系統經開發用於在cGMP環境中製造臨床試驗奈米粒子材料。其提供在NanoAssemblr Benchtop及Blaze平台上開發之調配物的無縫規模擴大。使用相同的並聯連接的微流體混合器將在臨床前儀器上開發的製程直接轉移至規模擴大。並行混合器可在較短的時間內執行相同的製程，從而生產單個大批量批次。具有8個並行運行之微流體混合器的規模擴大系統可生產適合於早期臨床試驗之奈米粒子調配物體積。In certain embodiments, fabrication is performed in a NanoAssemblr Scale-Up system that was developed for the manufacture of clinical trial nanoparticle materials in a cGMP environment. It offers a seamless scale expansion of formulations developed on the NanoAssemblr Benchtop and Blaze platforms. The process developed on preclinical instruments was directly transferred to scale up using the same parallel-connected microfluidic mixer. The parallel mixer can perform the same process in a short period of time to produce a single high volume batch. A scale-up system with 8 microfluidic mixers operating in parallel produces a nanoparticle formulation volume suitable for early clinical trials.

8. 套組
本發明進一步提供一種套組，其包含實施本發明方法所需之組分。此套組包含至少一種原核端粒酶及視情況用於本文所述方法之說明書。該套組可包含兩種、三種、四種、五種或更多種不同的原核端粒酶。原核端蛋白酶可選自SEQ ID NO: 2、4、6、8或10中之任一者或其任何變體。在某些實施例中，該套組包含大腸桿菌N15 TelN (SEQ ID NO: 10)或其變體。8. Kits The invention further provides a kit comprising the components required to carry out the method of the invention. This kit contains at least one protelomerase and instructions for use in the methods described herein as appropriate. The kit can comprise two, three, four, five or more different protelomerases. The protelomerase may be selected from any one of SEQ ID NOs: 2, 4, 6, 8, or 10, or any variant thereof. In certain embodiments, the kit comprises E. coli N15 TelN (SEQ ID NO: 10) or a variant thereof.

該套組亦可包含適合之緩衝劑及如上所述之原核端粒酶效能或穩定性所需的其他因子。
實例
實例1 包含所關注基因之cdsDNA的產生
製備Puc19骨架質體構築體用於擴增GFP報導體基因，該構築體包括在哺乳動物啟動子控制下之GFP編碼序列且包含polyA編碼序列。在質體構築體中，GFP報導體基因側接一對AAV2 ITR序列，分別為ITR1及ITR2。由於ITR與GFP報導體基因之間存在Not I識別序列，所以各ITR可經由Not I消化與GFP報導體基因分離。側接ITR之GFP報導體基因進一步側接兩個TelN識別序列TelN-左及TelN-右。The kit may also contain suitable buffers and other factors required for prokaryotic telomerase efficacy or stability as described above.
EXAMPLES Example 1 Generation of cds DNA containing a gene of interest A Puc19 backbone plastid construct was prepared for amplification of a GFP reporter gene comprising a GFP coding sequence under the control of a mammalian promoter and comprising a polyA coding sequence. In the plastid construct, the GFP reporter gene is flanked by a pair of AAV2 ITR sequences, which are ITR1 and ITR2, respectively. Since the Not I recognition sequence exists between the ITR and the GFP reporter gene, each ITR can be separated from the GFP reporter gene via Not I digestion. The GFP reporter gene flanked by ITR is further flanked by two TelN recognition sequences TelN-left and TelN-right.

在擴增及收穫包含GFP構築體之質體後，用N6原核端粒酶TelN消化質體以將GFP構築體拆分成兩個cdsDNA，一個基本上包含側接ITR且進一步側接TelN識別序列之兩個半位點的GFP報導體基因。另一個cdsDNA包含質體骨架之其餘部分，側接TelN識別序列之兩個半位點。參見圖3中之凝膠電泳結果。After amplification and harvesting of the plastid containing the GFP construct, the plastid is digested with N6 protelomerase TelN to split the GFP construct into two cdsDNA, one comprising a flanking ITR and further flanking the TelN recognition sequence. The two half-site GFP reporter genes. Another cdsDNA contains the rest of the plastid backbone, flanked by two half-sites of the TelN recognition sequence. See the gel electrophoresis results in Figure 3.

含有GFP之cdsDNA可自凝膠中回收，或直接自TelN消化反應中純化以供進一步使用。The cds DNA containing GFP can be recovered from the gel or purified directly from the TelN digestion reaction for further use.

實例2 cdsDNA編碼之所關注基因的表現
使用標準分子生物學技術將包含GFP報導體基因之經純化之cdsDNA (圖4A)轉染至C2C12肌管中，且隨時間推移監測C2C12肌管中之GFP表現(參見圖4B)。Example 2 Expression of the gene of interest encoded by cdsDNA The purified cdsDNA containing the GFP reporter gene (Fig. 4A) was transfected into C2C12 myotubes using standard molecular biology techniques, and GFP in C2C12 myotubes was monitored over time. Performance (see Figure 4B).

作為對照，將經Not I消化之cdsDNA類似地轉染至C2C12肌管中，具有未側接ITR之相同GFP報導體基因的pCK8-GFP構築體亦是如此。As a control, Not I digested cds DNA was similarly transfected into C2C12 myotubes, as was the pCK8-GFP construct with the same GFP reporter gene not flanked by ITR.

儘管經轉染細胞在相差顯微鏡下呈現相同的形態，但FITC影像顯示轉染至C2C12細胞之cdsDNA編碼之GFP的強表現，而經Not I消化之GFP dsDNA轉染細胞具有弱表現。與經cdsDNA轉染之細胞相比，pCK8 GFP對照亦產生不良表現。確定cdsDNA編碼之GFP的轉染效率為約60%，且隨著肌管成熟，GFP表現變得更強。參見圖4C。Although transfected cells showed the same morphology under phase contrast microscopy, FITC images showed strong expression of GFP encoded by cdsDNA transfected into C2C12 cells, whereas GFP dsDNA transfected cells digested with Not I showed weak expression. The pCK8 GFP control also produced poor performance compared to cells transfected with cdsDNA. The transfection efficiency of cds DNA-encoded GFP was determined to be about 60%, and GFP appeared to become stronger as the myotubes matured. See Figure 4C.

實例3 保護cdsDNA編碼之所關注基因免受核酸外切酶消化
為了測試cdsDNA之完整性，在數種條件下用TelN及/或核酸外切酶消化實例1之質體構築體，且在凝膠電泳上解析消化產物。參見圖5A及5B。Example 3 Protection of the gene of interest encoded by cdsDNA from exonuclease digestion To test the integrity of cdsDNA, the plastid construct of Example 1 was digested with TelN and/or exonuclease under several conditions, and in a gel The digestion product was analyzed by electrophoresis. See Figures 5A and 5B.

在圖5A中，在尺寸標記右側之第一泳道中，在不存在核酸外切酶之情況下用TelN消化4 μg環狀質體DNA，結果顯示僅存在兩種預期的cdsDNA產物，一種代表包含GFP報導體基因之cdsDNA。In Figure 5A, in the first lane to the right of the size marker, 4 μg of circular plastid DNA was digested with TelN in the absence of exonuclease, and the results showed that only two expected cdsDNA products were present, one representative containing GFP reports the cdsDNA of the conductor gene.

在其右側的泳道中，進行相同的實驗，不同之處在於添加核酸外切酶。結果顯示，兩種cdsDNA產物均對核酸外切酶消化具有抗性，與TelN消化產物之封閉末端保護cdsDNA免受核酸外切酶消化的期望一致。In the lane on the right side, the same experiment was performed except that an exonuclease was added. The results show that both cdsDNA products are resistant to exonuclease digestion, consistent with the expectation that the blocked ends of the TelN digestion products protect cdsDNA from exonuclease digestion.

然而，當輸入DNA並非環狀而是在原核端粒酶消化之前經線性化時，經線性化之質體骨架對核酸外切酶消化不具有抗性，而cdsDNA (在圖中標記為SLiD)仍對核酸外切酶具有抗性。However, when the input DNA is not circular but linearized prior to protelomerase digestion, the linearized plastid backbone is not resistant to exonuclease digestion, whereas cdsDNA (labeled SLiD in the figure) Still resistant to exonucleases.

在一組類似的實驗中，其結果顯示在圖5B中，改為使用2 μg輸入質體DNA及增加量之核酸外切酶，不同之處在於輸入質體DNA首先用Xba I線性化，Xba I一旦在GFP構築體及TelN識別位點之外就消化實例1之質體構築體。作為TelN消化之結果，包含GFP構築體之cdsDNA為完整的，而包含質體骨架之其餘部分的准cdsDNA藉由Xba I消化等分，產生具有僅一個封閉末端之兩個線性dsDNA。此類消化產物易受核酸外切酶消化。參見圖5B中尺寸標記右側之第3至第5泳道。In a similar set of experiments, the results are shown in Figure 5B, using 2 μg of input plastid DNA and increasing amounts of exonuclease, except that the input plastid DNA was first linearized with Xba I, Xba I. The plastid construct of Example 1 was digested once outside the GFP construct and the TelN recognition site. As a result of TelN digestion, the cdsDNA containing the GFP construct is intact, while the quasi-cdsDNA containing the rest of the plastid backbone is digested by Xba I to produce two linear dsDNAs with only one closed end. Such digestion products are susceptible to digestion by exonucleases. See lanes 3 through 5 on the right side of the dimension mark in Figure 5B.

此實驗證明，在圖6中概述之製造過程中，在原核端粒酶消化後，核酸外切酶可用於移除污染性質體骨架cdsDNA，從而促進包含GOI之cdsDNA的純化。This experiment demonstrates that in the manufacturing process outlined in Figure 6, exonuclease can be used to remove the contaminating plastid backbone cdsDNA after protelomerase digestion, thereby facilitating the purification of cds DNA comprising GOI.

實例4 cdsDNA編碼之微型肌縮蛋白在細胞中之活體外表現
此實例證明，本發明之cdsDNA構築體可用於在根據製造商之推薦使用VIAFECTTM 轉染劑(Promega Corp., WI)由稱為SLiD-MD44之cdsDNA構築體轉染的C2C12細胞中活體外表現外源性轉殖基因。Example 4 cdsDNA micro muscle contraction encoded proteins in a cell in vitro This example demonstrates the performance, cdsDNA the present invention may be used to construct referred VIAFECT TM using transfection reagent (Promega Corp., WI) according to the recommendations by the manufacturer & Exogenous transgenic genes were expressed in vitro in C2C12 cells transfected with the cdsDNA construct of SLiD-MD44.

SLiD-MD44編碼5重複微型肌縮蛋白，其自N端至C端含有N端肌動蛋白結合結構域、鉸鏈區1 (H1)、血影蛋白樣重複序列R1、R16、R17、R23及R24、鉸鏈區4 (H4)及人類全長肌縮蛋白之C端肌縮聚糖結合結構域。此5重複微型肌縮蛋白之蛋白質序列及相關肌縮蛋白小基因描述於WO2016/115543 (以引用的方式併入本文中)中。SLiD-MD44 encodes a 5-repeat micro-muscle protein with an N-terminal actin-binding domain from the N-terminus to the C-terminus, hinge region 1 (H1), and spectrin-like repeats R1, R16, R17, R23 and R24 The hinge region 4 (H4) and the C-terminal muscle glycan binding domain of human full length muscle protein. The protein sequence of this 5-repeat minimuscular protein and related myosin minigenes are described in WO 2016/115543 (incorporated herein by reference).

圖7A顯示主題cdsDNA對核酸外切酶III消化具有抗性。包含5重複微型肌縮蛋白編碼序列(SLiD-MD44)之質體的限制性及原核端粒酶消化產生在電泳上解析的多個條帶，僅一個匹配主題cdsDNA (亦即MD44)之預期尺寸的條帶似乎對核酸外切酶III消化具有抗性，證實原核端粒酶消化產物中共價連接之末端結構。Figure 7A shows that the subject cdsDNA is resistant to exonuclease III digestion. Restriction of the plastid containing the 5-repeat minimuscular protein coding sequence (SLiD-MD44) and prokaryotic telomerase digestion yielded multiple bands resolved electrophoretically, with only one expected size of the matching cdsDNA (ie MD44) The band appears to be resistant to exonuclease III digestion, confirming the covalently linked terminal structure in the prokaryotic telomerase digestion product.

隨後使用VIAFECTTM 轉染劑將經分離之cdsDNA SLiD-MD44轉染至培養的C2C12細胞中，且使用免疫螢光染色顯露細胞骨架(抗F-肌動蛋白抗體染色)、細胞核(蘇木精染色)及任何表現之5重複微型肌縮蛋白(抗DysB抗體)。顯而易見的是，由編碼5重複微型肌縮蛋白之主題cdsDNA轉染的C2C12細胞具有5重複微型肌縮蛋白之穩固表現。同時，當使用相同的抗DysB抗體時，無DNA之對照轉染不產生信號。VIAFECT TM transfection agent is then used to isolated the cdsDNA SLiD-MD44 transfected into cultured C2C12 cells, and using immunohistochemical stains revealed cytoskeleton (F- anti-actin antibody staining), cell nuclei (hematoxylin staining ) and any of the 5 repeat microcreature proteins (anti-DysB antibodies). It is apparent that C2C12 cells transfected with the subject cdsDNA encoding the 5-repeat minimuscular protein have a robust expression of the 5-repeat minimuscular protein. At the same time, DNA-free control transfection did not produce a signal when the same anti-DysB antibody was used.

實例5 cdsDNA編碼之微型肌縮蛋白在mdx小鼠中之活體內表現
此實例證明，本發明之cdsDNA構築體可用於活體內表現外源性轉殖基因，尤其在DMD之mdx 小鼠模型中表現外源性微型肌縮蛋白基因。Example 5 In vivo expression of cdsDNA-encoded microtenosin in mdx mice This example demonstrates that the cdsDNA construct of the present invention can be used to express exogenous transgenic genes in vivo, especially in the mdx mouse model of DMD. Exogenous micro-muscle protein gene.

如實例4中製備稱為SLiD-MD4之cdsDNA構築體以編碼5重複微型肌縮蛋白。隨後將約40 μg SLiD-MD4 cdsDNA經由肌肉內電穿孔(IM電穿孔)引入mdx 小鼠中。作為對照，使用鹽水溶液代替SLiD-MD4 cdsDNA構築體。A cdsDNA construct designated SLiD-MD4 was prepared as in Example 4 to encode a 5-repeat minimuscular protein. About 40 μg of SLiD-MD4 cds DNA was subsequently introduced into mdx mice via intramuscular electroporation (IM electroporation). As a control, a saline solution was used instead of the SLiD-MD4 cdsDNA construct.

將經純化之SLiD-MD4 cdsDNA構築體以2 μg/μL調配於150 mM NaCl中，且儲存在-20℃下直至使用。將玻尿酸酶(Sigma，H-4272)以2 mg/mL調配於PBS中，且將1 mL等分試樣儲存在-20℃下直至使用。The purified SLiD-MD4 cdsDNA construct was formulated in 2 μg/μL in 150 mM NaCl and stored at -20 °C until use. Hyaluronic acidase (Sigma, H-4272) was formulated in PBS at 2 mg/mL and 1 mL aliquots were stored at -20 °C until use.

對於電穿孔，解凍SLiD-MD4及玻尿酸酶原料。經由吸入2-3%異氟醚麻醉mdx 小鼠。在脛骨前肌的位置對小鼠後腿進行刮毛。隨後藉由用0.3 mL結核菌素注射器進入肌肉中部，將50 μL玻尿酸酶直接注射至脛骨前肌中。緩慢注射材料，注射後針緩慢縮回。在玻尿酸酶注射後約2小時，藉由用結核菌素注射器進入肌肉中部的針頭，將30 μL之60 μg含SLiD-MD4 cdsDNA之構築體直接注射至脛骨前肌中。緩慢注射材料，注射後針緩慢縮回。在DNA注射後，立即使用BTX AgilePulse ID電穿孔器及5 mm 4針陣列電極(BTX，47-0045)對mdx 小鼠進行電穿孔。DNA注射部位位於2個平行針陣列之間，將其***穿過皮膚且進入脛骨前肌，直至估計之負荷讀數＜3000 ohms。使用以下設置進行電穿孔。
For electroporation, SLiD-MD4 and hyaluronic acid materials were thawed. Mdx mice were anesthetized by inhalation of 2-3% isoflurane. The hind legs of the mouse were shaved at the position of the tibialis anterior muscle. 50 μL of hyaluronan was then injected directly into the tibialis anterior muscle by entering the middle of the muscle with a 0.3 mL tuberculin syringe. The material is injected slowly and the needle is slowly retracted after injection. About 2 hours after the hyaluronan injection, 30 μL of 60 μg of the construct containing SLiD-MD4 cdsDNA was directly injected into the tibialis anterior muscle by using a tuberculin syringe into the needle in the middle of the muscle. The material is injected slowly and the needle is slowly retracted after injection. Immediately after DNA injection, mdx mice were electroporated using a BTX AgilePulse ID electroporator and a 5 mm 4-needle array electrode (BTX, 47-0045). The DNA injection site is located between 2 parallel needle arrays, inserted through the skin and into the tibialis anterior muscle until the estimated load reading is <3000 ohms. Use the following settings for electroporation.

注射後7天收集脛骨前肌進行分析。The tibialis anterior muscles were collected 7 days after injection for analysis.

隨後使用抗DysB小鼠單株抗體(Leica Biosystems，產品代碼：NCL-DYSB)進行免疫螢光染色，用於驗證自SLiD-MD4 cdsDNA構築體電穿孔之mdx 小鼠分離的肌肉組織中之微型肌縮蛋白表現。針對人類肌縮蛋白之殘基321-494產生抗體。Subsequently, immunofluorescence staining was performed using an anti-DysB mouse monoclonal antibody (Leica Biosystems, product code: NCL-DYSB) for the verification of micromuscles in muscle tissue isolated from mdx mice electroporated with SLiD-MD4 cdsDNA construct. Reduced protein performance. Antibodies are raised against residues 321-494 of human myosin.

圖8B展示廣譜螢光信號，其代表經由IM電穿孔接受SLiD-MD4 cdsDNA構築體之mdx 小鼠中的穩固肌縮蛋白表現。圖8B之放大部分展示在圖9B中，其中沿著質膜周圍的正確亞細胞定位觀察到肌縮蛋白表現，表明外源性微型肌縮蛋白不僅在mdx 小鼠之肌肉組織中成功且廣泛地表現，而且由於正確的摺疊和正確的亞細胞定位，亦可能為功能性的。Figure 8B shows a broad spectrum fluorescent signal representing robust myosin expression in mdx mice receiving SLiD-MD4 cdsDNA constructs via IM electroporation. An enlarged portion of Figure 8B is shown in Figure 9B, where fibronectin expression was observed along the correct subcellular localization around the plasma membrane, indicating that exogenous microtenosin is successful not only in muscle tissue of mdx mice but also extensively Performance, and because of proper folding and correct subcellular localization, may also be functional.

相反，經由相同程序接受鹽水對照之對照小鼠僅具有背景水準信號。參見圖8A及圖9A中之類似放大部分。In contrast, control mice that received saline control via the same procedure had only background level signals. See similar enlarged portions in Figures 8A and 9A.

圖10顯示在IM電穿孔後7天後外源性微型肌縮蛋白基因之表現持續存在。在此實驗中，經由IM電穿孔在mdx 小鼠中使用60 μg的相同SLiD-MD4 cdsDNA構築體。在電穿孔後7天，使用由紅色螢光信號標記之抗DysB單株抗體及由綠色螢光信號標記之抗層黏連蛋白單株抗體進行免疫染色。Figure 10 shows the persistence of the expression of the exogenous microtenosin gene 7 days after IM electroporation. In this experiment, 60 μg of the same SLiD-MD4 cdsDNA construct was used in mdx mice via IM electroporation. Seven days after electroporation, immunostaining was performed using an anti-DysB monoclonal antibody labeled with a red fluorescent signal and an anti-laminin monoclonal antibody labeled with a green fluorescent signal.

圖1為展示囊封封閉末端dsDNA (cdsDNA)之奈米粒子之非病毒生產的例示性實施例的示意圖，該等奈米粒子可用於例如基因療法中以將所關注之基因(GOI)在靶宿主生物體活體外、離體或活體內遞送至靶細胞。cdsDNA之中心區域由中間最長的雙線表示，代表GOI。緊鄰GOI側翼之一對短桿代表病毒基因組之反向末端重複(ITR)序列，諸如來自腺相關病毒(AAV)基因組。緊鄰ITR封端之GOI側翼的一對短條代表原核端粒酶之識別或靶序列，諸如56 bp TelN原核端粒酶識別序列。一旦cdsDNA經原核端粒酶消化，dsDNA之兩端就轉變為封閉末端單股DNA環，作為原核端粒酶消化之產物，從而形成cdsDNA分子。隨後將cdsDNA分子封裝/組裝/製備/製造於適合材料之奈米粒子中，可將奈米粒子引入靶細胞或靶生物體內之細胞中以表現GOI。1 is a schematic diagram showing an exemplary embodiment of non-viral production of encapsulated closed-end dsDNA (cdsDNA) nanoparticles, which can be used, for example, in gene therapy to target a gene of interest (GOI) at a target The host organism is delivered to the target cells in vitro, ex vivo or in vivo. The central region of cdsDNA is represented by the longest double line in the middle, representing the GOI. The pair of short rods next to the GOI flanks represent the reverse terminal repeat (ITR) sequence of the viral genome, such as from the adeno-associated virus (AAV) genome. A pair of short strands flanking the ITR-terminated GOI represent a prokaryotic telomerase recognition or target sequence, such as a 56 bp TelN protelomerase recognition sequence. Once the cds DNA is digested by protelomerase, the ends of the dsDNA are converted into a closed-end single-stranded DNA loop, which is the product of protelomerase digestion, thereby forming a cdsDNA molecule. The cds DNA molecules are then encapsulated/assembled/prepared/manufactured in nanoparticles of suitable materials, and the nanoparticles can be introduced into cells of the target cells or target organisms to express GOI.

圖2說明TelN原核端粒酶裂解dsDNA受質以產生包含圖1中之GOI的cdsDNA的過程。56-bp TelN原核端粒酶識別序列(TelN位點)展示在圖的頂部。在TelN位點中間藉由TelN裂解後，裂解位點左側頂部股中之G的3'與裂解位點左側底部股中之C的5'藉由TelN連接以形成封閉末端單股環，TelN裂解位點右側亦是如此。因此，在包含側接ITR序列(ITR1及ITR2)且進一步側接一對TelN位點(亦即TelN-左及TelN-右)之GOI (例如GFP)的dsDNA質體中，原核端粒酶消化釋放線性雙螺旋體，其包含側接ITR且進一步側接具有封端末端單股環之TelN半位點的GOI以形成cdsDNA。Figure 2 illustrates the process by which TelN protelomerase cleaves dsDNA to produce cdsDNA comprising the GOI of Figure 1. The 56-bp TelN protelomerase recognition sequence (TelN site) is shown at the top of the figure. After cleavage by TelN in the middle of the TelN site, the 3' of G in the top strand on the left side of the cleavage site and the 5' of C in the bottom bottom strand of the cleavage site are joined by TelN to form a closed-end single-stranded loop, TelN cleavage The same is true to the right side of the site. Thus, in a dsDNA plastid containing a GOI (eg, GFP) flanked by an ITR sequence (ITR1 and ITR2) and further flanked by a pair of TelN sites (ie, TelN-left and TelN-Right), prokaryotic telomerase digestion A linear double helix is released comprising a GOI flanked by an ITR and further flanked by a TelN half site having a single terminal loop to form a cds DNA.

圖3展示使用本發明之方法產生cdsDNA藥物物質。各泳道自上而下之三個條帶為包含GFP作為GOI的未消化質體；剩餘質體骨架；及由原核端粒酶消化產生之側接具有閉環末端之ITR的GOI (GFP)。Figure 3 shows the production of cdsDNA drug substance using the method of the invention. The three bands from top to bottom of each lane are undigested plastids containing GFP as GOI; remaining plastid backbone; and GOI (GFP) flanked by prokaryotic telomerase digestion with ITR with closed loop ends.

圖4A及4B展示如此產生之cdsDNA具有生物學活性，以活體外指導靶肌肉細胞中之GFP表現。圖4A之左圖為展示環狀質體(pCK8-GFP)經原核端粒酶消化以產生編碼GFP報導體基因之cdsDNA(「SLiD/非病毒AAV」)的示意圖。圖4A之右圖展示凝膠影像，其中具有核酸外切酶III消化產物之泳道形成單一條帶(cdsDNA)，而另外兩個條帶可能由於核酸外切酶III消化而消失。隨後將包含GFP編碼序列之cdsDNA引入C2C12肌管中，之後監測GFP表現。圖4B中之合併影像組包括GFP信號(由cdsDNA表示)、DAPI核染色及F-肌動蛋白染色。Figures 4A and 4B show that the cds DNA so produced is biologically active to direct GFP expression in target muscle cells in vitro. The left panel of Figure 4A is a schematic diagram showing the circular plastid (pCK8-GFP) digested by protelomerase to generate cdsDNA ("SLiD/non-viral AAV") encoding the GFP reporter gene. The right panel of Figure 4A shows a gel image in which the lane with the exonuclease III digestion product forms a single band (cdsDNA), while the other two bands may disappear due to exonuclease III digestion. The cdsDNA containing the GFP coding sequence was then introduced into the C2C12 myotubes, after which the GFP expression was monitored. The combined image set in Figure 4B includes the GFP signal (represented by cdsDNA), DAPI nuclear staining, and F-actin staining.

圖4C展示，作為對照，Not I消化之相同質體釋放基本上由GOI (GFP)組成但不包括側接的ITR之dsDNA片段。另一個對照為pCK8 GPF構築體，其為具有側接相同GFP報導體基因之ITR序列的親本質體構築體。雖然經轉染之細胞在相差顯微鏡下顯示相同的形態，但FITC影像展示轉染C2C12細胞之cdsDNA編碼之GFP的強表現，而經Not I消化之GFP dsDNA (不含ITR的開放末端DNA)轉染之細胞具有弱表現。與經cdsDNA轉染之細胞相比，pCK8 GFP對照亦產生不良表現。確定cdsDNA編碼之GFP的轉染效率為約60%，且隨著肌管成熟，GFP表現變得更強。Figure 4C shows that, as a control, the same plastids that Not I digested released essentially consisted of GOI (GFP) but did not include the dsDNA fragment of the flanking ITR. Another control is the pCK8 GPF construct, which is a pro-essential construct with an ITR sequence flanked by the same GFP reporter gene. Although the transfected cells showed the same morphology under phase contrast microscopy, FITC images showed strong expression of GFP encoded by cdsDNA transfected with C2C12 cells, whereas Not I digested GFP dsDNA (open-end DNA without ITR) The stained cells have weak performance. The pCK8 GFP control also produced poor performance compared to cells transfected with cdsDNA. The transfection efficiency of cds DNA-encoded GFP was determined to be about 60%, and GFP appeared to become stronger as the myotubes matured.

圖5A顯示由原核端粒酶消化產生之cdsDNA的閉環末端保護cdsDNA免於核酸外切酶消化。Figure 5A shows that the bls end of the cds DNA produced by prokaryotic telomerase digestion protects the cds DNA from exonuclease digestion.

圖5B展示質體骨架用核酸內切酶(諸如XbaI)線性化使得原核端粒酶消化保護包含對核酸外切酶消化敏感的質體骨架。Figure 5B shows that the plastid backbone is linearized with an endonuclease such as XbaI such that prokaryotic telomerase digestion protects the plastid backbone that is susceptible to exonuclease digestion.

圖6為說明無細胞cdsDNA藥物物質製造過程的示意圖。Figure 6 is a schematic diagram showing the manufacturing process of a cell-free cdsDNA drug substance.

圖7A展示編碼5-重複微型肌縮蛋白之主題cdsDNA (SLiD-MD44)對核酸外切酶III消化具有抗性，證實在原核端粒酶消化後存在封閉末端。Figure 7A shows that the subject cdsDNA (SLiD-MD44) encoding a 5-repeat minimuscular protein is resistant to exonuclease III digestion, confirming the presence of a closed end after protelomerase digestion.

圖7B展示主題cdsDNA可在C2C12細胞中活體外表現編碼的5-重複微型肌縮蛋白。Figure 7B shows that the subject cdsDNA can express the encoded 5-repeat minimuscular protein in vitro in C2C12 cells.

圖8A及8B展示外源微型肌縮蛋白在mdx 小鼠肌肉組織中之活體內表現。經由肌內電穿孔(IM電穿孔)向小鼠投與40 μg編碼微型肌縮蛋白小基因之主題cdsDNA (SLiD-MD4)或對照鹽水。微型肌縮蛋白在肌肉中之表現藉由使用針對微型肌縮蛋白小基因中發現的血影蛋白重複序列升高之抗DysB單株抗體進行免疫染色來驗證。Figures 8A and 8B show in vivo expression of exogenous mini-muscle proteins in mdx mouse muscle tissue. Mice were dosed with 40 μg of the subject cdsDNA (SLiD-MD4) encoding the mini-muscle protein minigene or control saline via intramuscular electroporation (IM electroporation). The expression of microtenosin in muscle was verified by immunostaining with an anti-DysB monoclonal antibody raised against the spectrin repeats found in the mini-muscle protein minigene.

圖9A及9B分別為圖8A及8B中所示之影像的放大部分。展示藉由表現之外源微型肌縮蛋白之正確的亞細胞(以及質膜)定位。9A and 9B are enlarged portions of the images shown in Figs. 8A and 8B, respectively. The display demonstrates the correct subcellular (and plasma membrane) localization by exogenous microfibrominin.

圖10展示在IM/電穿孔後7天，在接受60 μg cdsDNA構築體SLiD-MD4之mdx 小鼠中持續的微型肌縮蛋白小基因表現。微型肌縮蛋白表現顯示為紅色螢光信號，而內源性層黏連蛋白表現顯示為綠色螢光信號。Figure 10 shows the persistence of mini-fibronectin minigenes in mdx mice receiving 60 μg of cdsDNA construct SLiD-MD4 7 days after IM/electroporation. Microtrophin expression is shown as a red fluorescent signal, while endogenous laminin expression is shown as a green fluorescent signal.

Claims (20)

一種DNA構築體，其包含： (1) 骨架序列，其包含在真核(例如哺乳動物)或原核細胞中支持自我複製之序列； (2) 嵌段，其包含： (a) 所關注之DNA片段； (b) 側接該所關注之DNA片段的一對末端序列，其中該等末端序列為雙股DNA病毒之反向末端重複序列(ITR)、DNA病毒(諸如HSV)之長末端重複序列(LTR)或內部重複序列、或端粒序列；及 (c) 側接該一對ITR或LTR之一對原核端粒酶識別序列。 A DNA construct comprising: (1) a backbone sequence comprising a sequence that supports self-replication in eukaryotic (eg, mammalian) or prokaryotic cells; (2) Block, which contains: (a) a DNA fragment of interest; (b) a pair of terminal sequences flanked by the DNA fragment of interest, wherein the terminal sequences are the inverted terminal repeats (ITR) of the double-stranded DNA virus, and the long terminal repeats of the DNA virus (such as HSV) (LTR) Or an internal repeat sequence, or a telomere sequence; (c) flank the prokaryotic telomerase recognition sequence of one of the pair of ITRs or LTRs. 如請求項1之DNA構築體，其中該等原核端粒酶識別序列中之至少一者包含至少14 bp長度之完美反向重複DNA序列或其變體。The DNA construct of claim 1, wherein at least one of the prokaryotic telomerase recognition sequences comprises a perfect inverted repeat DNA sequence of at least 14 bp in length or a variant thereof. 如請求項1之DNA構築體，其中該等原核端粒酶識別序列中之至少一者包含嗜溫性噬菌體完美反向重複序列之22 bp共同序列。The DNA construct of claim 1, wherein at least one of the prokaryotic telomerase recognition sequences comprises a 22 bp common sequence of a mesophilic phage perfect inverted repeat. 如請求項1之DNA構築體，其中該等原核端粒酶識別序列中之至少一者來自大腸桿菌噬菌體N15 (諸如由大腸桿菌N15 TelN原核端粒酶識別之大腸桿菌噬菌體)、土壤桿菌克雷伯氏菌(Klebsiella )噬菌體Phi KO2、耶爾森氏菌(Yersinia )噬菌體PY54、鹽單胞菌(Halomonas )噬菌體phiHAP-1及弧菌(Vibrio )噬菌體VP882，或伯氏疏螺旋體(Borrelia burgdorferi )。The DNA construct of claim 1, wherein at least one of the prokaryotic telomerase recognition sequences is derived from Escherichia coli bacteriophage N15 (such as Escherichia coli recognized by E. coli N15 TelN protel telomerase), Agrobacterium Cray Borrelia bacteria (Klebsiella) bacteriophage Phi KO2, Yersinia (Yersinia) phage PY54, Halomonas (of Halomonas) phage phiHAP-1 and Vibrio (Vibrio) phage VP882, or Borrelia burgdorferi (Borrelia burgdorferi) . 如請求項1之DNA構築體，其中該等原核端粒酶識別序列中之至少一者包含至少30個、至少40個、至少60個、至少80個或至少100個鹼基對長度之完美反向重複序列。The DNA construct of claim 1, wherein at least one of the prokaryotic telomerase recognition sequences comprises a perfect inverse of at least 30, at least 40, at least 60, at least 80 or at least 100 base pairs in length. To the repeat sequence. 如請求項1之DNA構築體，其中該所關注之DNA片段包含在真核啟動子及/或增強子控制下/可操作地連接於真核啟動子及/或增強子之所關注之編碼序列，及視情況選用之真核轉錄終止序列。The DNA construct of claim 1, wherein the DNA fragment of interest comprises a coding sequence of interest under the control of a eukaryotic promoter and/or enhancer/operably linked to a eukaryotic promoter and/or enhancer. And the eukaryotic transcription termination sequence selected as appropriate. 如請求項6之DNA構築體，其中該所關注之編碼序列包含編碼以下抗原之DNA疫苗： (1) 用於治療或預防諸如癌症、過敏症、毒性及病原體感染之病況(例如，真菌，病毒，諸如人類乳頭瘤病毒(HPV)、HIV、HSV2/HSV1、流感病毒(A、B及C型)、脊髓灰質炎病毒、RSV病毒、鼻病毒、輪狀病毒、A型肝炎病毒、諾沃克病毒組(Norwalk Virus Group)、腸病毒、星狀病毒、麻疹病毒、副流感病毒、腮腺炎病毒、水痘-帶狀疱疹病毒、細胞巨大病毒、埃-巴二氏病毒(Epstein-Barr virus)、腺病毒、風疹病毒、人類T細胞淋巴瘤I型病毒(HTLV-I)、B型肝炎病毒(HBV)、C型肝炎病毒(HCV)、D型肝炎病毒、痘病毒、馬爾堡(Marburg)及埃博拉(Ebola))；細菌(諸如結核分枝桿菌(Mycobacterium tuberculosis)、披衣菌屬(Chlamydia)、淋病奈瑟氏菌(Neisseria gonorrhoeae)、志賀桿菌屬(Shigella)、沙門氏菌屬(Salmonella)、霍亂弧菌(Vibrio cholerae)、梅毒螺旋體(Treponema pallidum)、假單胞菌屬(Pseudomonas)、百日咳博德特氏菌(Bordetella pertussis)、布氏桿菌屬(Brucella)、土拉熱弗朗西絲菌(Francisella tularensis)、幽門螺旋桿菌(Helicobacter pylori)、鉤端螺旋體(Leptospira interrogans)、嗜肺性退伍軍人桿菌(Legionella pneumophila)、鼠疫耶爾森氏菌(Yersinia pestis)、鏈球菌屬(Streptococcus)(A型及B型)、肺炎球菌屬(Pneumococcus)、腦膜炎雙球菌屬(Meningococcus)、流感嗜血桿菌(Haemophilus influenza)(b型)、剛地弓形蟲(Toxoplasma gondii)、彎曲桿菌病(Campylobacteriosis)、卡他莫拉菌(Moraxella catarrhalis)、多諾萬病(Donovanosis)及放線菌病(Actinomycosis))；真菌病原體(諸如念珠菌病(Candidiasis)及麴菌病(Aspergillosis))；寄生蟲病原體(諸如絛蟲屬(Taenia)、吸蟲、蛔蟲、阿米巴病(Amoebiasis)、梨形鞭毛蟲病(Giardiasis)、隱胞子蟲屬(Cryptosporidium)、住血吸蟲屬(Schistosoma)、肺炎肺囊蟲(Pneumocystis carinii)、滴蟲病(Trichomoniasis)及旋毛蟲病(Trichinosis)； (2) 來自腺病毒科(adenoviridae)(例如人類腺病毒)、疱疹病毒科(herpesviridae)(例如HSV-1、HSV-2、EBV、CMV及VZV)、乳多空病毒科(papovaviridae)(例如HPV)、痘病毒科(poxyiridae)(例如天花及牛痘)、細小病毒科(parvoviridae)(例如小病毒B19)、呼腸孤病毒科(reoviridae)(例如輪狀病毒)、冠狀病毒科(coronaviridae)(例如SARS)、黃病毒科(flaviviridae)(例如黃熱病、西尼羅河病毒、登革熱、C型肝炎及蜱媒腦炎)、小核糖核酸病毒科(picornaviridae)(例如脊髓灰質炎、鼻病毒及A型肝炎)、披膜病毒科(togaviridae)(例如風疹病毒)、絲狀病毒科(filoviridae)(例如馬爾堡及埃博拉)、副黏病毒科(paramyxoviridae)(例如副流感病毒、呼吸道合胞病毒、腮腺炎及麻疹)、彈狀病毒科(rhabdoviridae)(例如狂犬病病毒)、布尼亞病毒科(bunyaviridae)(例如漢坦病毒(Hantaan virus))、正黏病毒科(orthomyxoviridae)(例如A型、B型及C型流感病毒)、反轉錄病毒科(retroviridae)(例如HIV及HTLV)及肝DNA病毒科(hepadnaviridae)(例如B型肝炎)之成員； (3) 來自造成獸醫學疾病之病原體，諸如病毒病原體、呼腸孤病毒(例如非洲馬病或藍舌病毒)及疱疹病毒(例如馬疱疹)、***病毒、蜱媒腦炎病毒、登革熱病毒、SARS、西尼羅河病毒及漢坦病毒； (4) 來自免疫缺陷病毒，諸如SIV或貓免疫缺陷病毒； (5) 係新抗原(例如由癌症/腫瘤中之突變基因編碼)、腫瘤抗原，諸如睪丸抗原(例如MAGE家族之成員(MAGE 1、2、3等)、NY-ESO-1及SSX-2)、分化抗原(例如酪胺酸酶、gp100、PSA、Her-2及CEA)、突變的自身抗原及病毒性腫瘤抗原(例如來自致癌HPV類型之E6及/或E7)、MART-1、Melan-A、p97、β-HCG、GaINAc、MAGE-1、MAGE-2、MAGE-4、MAGE-12、MUC1、MUC2、MUC3、MUC4、MUC18、CEA、DDC、P1A、EpCam、黑素瘤抗原gp75、Hker 8、高分子量黑素瘤抗原、K19、Tyr1、Tyr2、pMel 17基因家族之成員、c-Met、PSM (***黏蛋白抗原)、PSMA (***特異性膜抗原)、***分泌蛋白、α-胎蛋白、CA 125、CA 19.9、TAG-72、BRCA-1及BRCA-2抗原。The DNA construct of claim 6, wherein the coding sequence of interest comprises a DNA vaccine encoding the following antigen: (1) For the treatment or prevention of conditions such as cancer, allergies, toxicity and pathogen infections (eg fungi, viruses, such as human papillomavirus (HPV), HIV, HSV2/HSV1, influenza viruses (A, B and C) Type), poliovirus, RSV virus, rhinovirus, rotavirus, hepatitis A virus, Norwalk Virus Group, enterovirus, astrovirus, measles virus, parainfluenza virus, mumps virus , varicella-zoster virus, giant cell virus, Epstein-Barr virus, adenovirus, rubella virus, human T-cell lymphoma type I virus (HTLV-I), hepatitis B virus ( HBV), hepatitis C virus (HCV), hepatitis D virus, poxvirus, Marburg and Ebola; bacteria (such as Mycobacterium tuberculosis, Chlamydia) Chlamydia), Neisseria gonorrhoeae, Shigella, Salmonella, Vibrio cholerae, Treponema pallidum, Pseudomonas, Bordetella pertussis Ella pertussis), Brucella, Francisella tularensis, Helicobacter pylori, Leptospira interrogans, Legionella pneumophila, plague Yersinia pestis, Streptococcus (types A and B), Pneumococcus, Meningococcus, Haemophilus influenza (b) Type), Toxoplasma gondii, Campylobacteriosis, Moraxella catarrhalis, Donovanosis and Actinomycosis; fungal pathogens such as rosary Candidiasis and Aspergillosis; parasitic pathogens (such as Taenia, trematode, aphids, Amoebiasis, Giardiasis, Cryptosporidium) Genus (Cryptosporidium), Schistosoma, Pneumocystis carinii, Trichomoniasis, and Trichinosis; (2) From the adenoviridae (eg human adenovirus), herpesviridae (eg HSV-1, HSV-2, EBV, CMV and VZV), papovaviridae (eg HPV), poxyiridae (eg, smallpox and vaccinia), parvoviridae (eg, small virus B19), reoviridae (eg, rotavirus), coronaviridae (coronaviridae) (eg SARS), flaviviridae (eg yellow fever, West Nile virus, dengue fever, hepatitis C and tick-borne encephalitis), picoraviridae (eg polio, rhinovirus and A) Hepatitis), togaviridae (eg rubella virus), filoviridae (eg Marburg and Ebola), paramyxoviridae (eg parainfluenza, respiratory syncytial) Virus, mumps and measles), rhabdoviridae (eg rabies virus), bunyaviridae (eg Hantaan virus), orthomyxoviridae (eg A) Type, B and C influenza viruses), reversal Virus family (retroviridae) (such as HIV and HTLV) and liver DNA virus family (hepadnaviridae) (such as hepatitis B) of the members; (3) From pathogens causing veterinary diseases, such as viral pathogens, reoviruses (such as African horse disease or bluetongue virus) and herpes viruses (such as horse herpes), foot-and-mouth disease virus, tick-borne encephalitis virus, dengue virus, SARS, West Nile virus and Hantavirus; (4) from an immunodeficiency virus, such as SIV or feline immunodeficiency virus; (5) A new antigen (eg, encoded by a mutated gene in a cancer/tumor), a tumor antigen, such as a testis antigen (eg, members of the MAGE family (MAGE 1, 2, 3, etc.), NY-ESO-1, and SSX-2 ), differentiation antigens (eg tyrosinase, gp100, PSA, Her-2 and CEA), mutated autoantigens and viral tumor antigens (eg E6 and/or E7 from oncogenic HPV types), MART-1, Melan -A, p97, β-HCG, GaINAc, MAGE-1, MAGE-2, MAGE-4, MAGE-12, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, P1A, EpCam, melanoma antigen gp75 , Hker 8, high molecular weight melanoma antigen, K19, Tyr1, Tyr2, members of the pMel 17 gene family, c-Met, PSM (prostate mucin antigen), PSMA (prostate specific membrane antigen), prostate secretory protein, alpha - Fetal protein, CA 125, CA 19.9, TAG-72, BRCA-1 and BRCA-2 antigens. 如請求項6之DNA構築體，其中該所關注之編碼序列包含用於基因療法之治療性DNA分子，其中該治療性DNA分子： (1) 在患有由該功能基因之功能異常形式(例如杜興氏肌肉萎縮症(Duchenne muscular dystrophy)、囊腫性纖維化、高歇氏病(Gaucher's Disease)及腺苷脫胺酶(ADA)缺乏症、發炎疾病、自體免疫、慢性及感染性疾病、AIDS、癌症、神經疾病、心血管疾病、高膽固醇血症、各種血液病症(包括各種貧血症、地中海貧血症及血友病，及肺氣腫)及實體腫瘤之基因)引起之基因病症的個體中表現該功能基因； (2) 編碼毒性肽(亦即化學治療劑，諸如蓖麻毒素、白喉毒素及眼鏡蛇毒因子)、腫瘤抑制基因(諸如p53)、編碼轉化致癌基因之反義mRNA序列的基因、諸如腫瘤壞死因子(TNF)及其他細胞介素之抗腫瘤肽、或轉化致癌基因之反式顯性陰性突變體； (3) 編碼活性RNA形式(例如小干擾RNA (siRNA、miRNA、shRNA)或小活化RNA (saRNA)；或 (4) 編碼CRISPR/Cas組分(諸如Cas9酶或sgRNA)。The DNA construct of claim 6, wherein the coding sequence of interest comprises a therapeutic DNA molecule for gene therapy, wherein the therapeutic DNA molecule: (1) Having a functionally abnormal form of the functional gene (eg, Duchenne muscular dystrophy, cystic fibrosis, Gaucher's Disease, and adenosine deaminase (ADA) Deficiency, inflammatory disease, autoimmune, chronic and infectious diseases, AIDS, cancer, neurological diseases, cardiovascular diseases, hypercholesterolemia, various blood disorders (including various anemia, thalassemia and hemophilia, and The functional gene is expressed in an individual with a genetic disorder caused by emphysema and a gene of a solid tumor; (2) encoding toxic peptides (ie, chemotherapeutic agents such as ricin, diphtheria toxin, and cobra venom factor), tumor suppressor genes (such as p53), genes encoding antisense mRNA sequences that convert oncogenes, such as tumor necrosis factor Anti-tumor peptides of (TNF) and other interleukins, or trans-dominant negative mutants that convert oncogenes; (3) encoding an active RNA form (eg, small interfering RNA (siRNA, miRNA, shRNA) or small activating RNA (saRNA); or (4) Encoding a CRISPR/Cas component (such as Cas9 enzyme or sgRNA). 一種封閉末端雙股DNA (cdsDNA)，其係藉由使如請求項1至8中任一項之DNA構築體與識別該一對原核端粒酶識別序列之原核端粒酶接觸而產生。A closed-end double-stranded DNA (cdsDNA) produced by contacting a DNA construct according to any one of claims 1 to 8 with a prokaryotic telomerase which recognizes the pair of prokaryotic telomerase recognition sequences. 一種封閉末端雙股DNA (cdsDNA)，其包含： (a) 所關注之DNA片段； (b) 側接該所關注之DNA片段的一對末端序列，其中該等末端序列為雙股DNA病毒之反向末端重複序列(ITR)、DNA病毒(諸如HSV)之長末端重複序列(LTR)或內部重複序列、或端粒序列；及 (c) 側接該一對末端序列之一對半原核端粒酶識別序列，其中該等半原核端粒酶識別序列中之每一者形成該cdsDNA之一個封閉末端。A closed-end double-stranded DNA (cdsDNA) comprising: (a) a DNA fragment of interest; (b) a pair of terminal sequences flanked by the DNA fragment of interest, wherein the terminal sequences are the inverted terminal repeats (ITR) of the double-stranded DNA virus, and the long terminal repeats of the DNA virus (such as HSV) (LTR) Or an internal repeat sequence, or a telomere sequence; (c) flanking one of the pair of end sequences to a half prokaryotel telomerase recognition sequence, wherein each of the semi-prokaryotic telomerase recognition sequences forms a blunt end of the cds DNA. 一種醫藥組合物，其包含如請求項9或10之cdsDNA。A pharmaceutical composition comprising the cdsDNA of claim 9 or 10. 如請求項11之醫藥組合物，其中該cdsDNA係由奈米粒子包圍。The pharmaceutical composition of claim 11, wherein the cdsDNA is surrounded by nanoparticles. 如請求項12之醫藥組合物，其中該奈米粒子為SNALP (穩定核酸-脂質粒子)、AtuPLEX、DACC、DBTC、RONDEL、DPC (Dynamic PolyConjugate)、SMARTICLE、DiLA2 或EnCore。The pharmaceutical composition according to claim 12, wherein the nanoparticle is SNALP (stabilized nucleic acid-lipid particle), AtuPLEX, DACC, DBTC, RONDEL, DPC (Dynamic PolyConjugate), SMARTCLE, DiLA 2 or EnCore. 一種產生無細胞封閉末端雙股DNA (cdsDNA)之方法，該方法包含： (1) 在該真核(例如哺乳動物)或原核細胞中擴增該DNA構築體後，分離如請求項1至8中任一項之DNA構築體； (2) 使用不會在該嵌段內消解之核酸內切酶使該DNA構築體線性化； (3) 使該DNA構築體與識別該對原核端粒酶識別序列之原核端粒酶接觸，以釋放該cdsDNA； (4) 在步驟(2)及(3)之後，用核酸外切酶移除不是cdsDNA之線性化DNA構築體或其片段； (5) 富集或純化該cdsDNA。A method of producing cell-free closed-end double-stranded DNA (cdsDNA), the method comprising: (1) separating the DNA construct of any one of claims 1 to 8 after amplifying the DNA construct in the eukaryotic (e.g., mammalian) or prokaryotic cell; (2) linearizing the DNA construct using an endonuclease that does not digest within the block; (3) contacting the DNA construct with a prokaryotic telomerase that recognizes the pair of prokaryotic telomerase recognition sequences to release the cds DNA; (4) after steps (2) and (3), removing the linearized DNA construct or fragment thereof that is not cdsDNA with an exonuclease; (5) Enriching or purifying the cds DNA. 如請求項14之方法，其中步驟(2)及(3)以任何順序或同時，均在步驟(1)之後進行。The method of claim 14, wherein the steps (2) and (3) are performed after the step (1) in any order or simultaneously. 一種產生封閉末端雙股DNA (cdsDNA)之方法，該方法包含： (1) 在該真核(例如哺乳動物)或原核細胞中擴增該DNA構築體後，分離如請求項1至8中任一項之DNA構築體； (2) 使該DNA構築體與識別該一對原核端粒酶識別序列之原核端粒酶接觸，以釋放該cdsDNA； (3) 富集或純化該cdsDNA。A method of producing a closed-end double-stranded DNA (cdsDNA), the method comprising: (1) separating the DNA construct of any one of claims 1 to 8 after amplifying the DNA construct in the eukaryotic (e.g., mammalian) or prokaryotic cell; (2) contacting the DNA construct with a prokaryotic telomerase that recognizes the pair of prokaryotic telomerase recognition sequences to release the cds DNA; (3) Enriching or purifying the cds DNA. 如請求項14至16中任一項之方法，其其進一步包含將cdsDNA囊封於奈米粒子(諸如SNALP、AtuPLEX、DACC、DBTC、RONDEL、DPC、SMARTICLE、DiLA2 或EnCore)中。The method of any one of claims 14 to 16, further comprising encapsulating the cdsDNA in a nanoparticle such as SNALP, AtuPLEX, DACC, DBTC, RONDEL, DPC, SMARTICLE, DiLA 2 or EnCore. 一種將所關注之靶基因(GOI)遞送至靶細胞中之活體外或離體方法，該方法包含使該靶細胞在活體外或離體，與包含如請求項10之cdsDNA的組合物接觸。An in vitro or ex vivo method of delivering a target gene of interest (GOI) to a target cell, the method comprising contacting the target cell in vitro or ex vivo with a composition comprising the cds DNA of claim 10. 一種包含如請求項10之cdsDNA之組合物或如請求項11至13中任一項之醫藥組合物的用途，其係用於製造將所關注之靶基因(GOI)活體內遞送至靶細胞中之藥物。Use of a composition comprising the cdsDNA of claim 10 or a pharmaceutical composition according to any one of claims 11 to 13 for the in vivo delivery of a target gene of interest (GOI) to a target cell The drug. 如請求項19之用途，其中該靶GOI為野生型迷你-或微型肌縮蛋白基因、DMD途徑中或與DMD之基因修飾物相關之基因，且其中該靶細胞為人類之肌肉(例如骨骼肌，諸如脛骨前肌細胞、心肌或平滑肌)細胞。The use of claim 19, wherein the target GOI is a wild-type mini- or micro-muscle protein gene, a gene in the DMD pathway or associated with a genetic modification of DMD, and wherein the target cell is a human muscle (eg, skeletal muscle) , such as tibialis anterior muscle cells, myocardium or smooth muscle cells.
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