基因转移和基因治疗

1、基因转移 与 基因治疗,基因：染色体上的DNA片段，是遗传信息结构和功能的基本单位。 基因 决定 生老病死 控制 高矮胖瘦 影响 喜怒哀乐 基因组：某一生物的细胞中所带有的全部遗传信息,基因与疾病 基因与人类的疾病密切相关： 遗传病是由于基因先天缺陷所致; 肿瘤的发生涉及多种基因改变, 包括癌基因激活或抑癌基因失活; 高血压, 糖尿病等多基因病也涉及到多种基因的改变; 由病原体所致的传染病也和人体基因密切相关, 存在易感人群和耐受人群,一、基因治疗概念 基因治疗(Gene Therapy) 指将正常基因或有治疗作用的基因通过一定方式导入靶细胞,纠正基因的缺陷或者发挥治疗作用，从而达到治疗疾病目

2、的的生物医学高技术,The application of genetic principles in the treatment of human disease By introduction of genetic material into target cells in order to counteract the effect of a disease gene or introduce a new function Somatic and Germline approaches possible,基因治疗的必要条件,Understanding of the disease proc

3、ess Structure/function of gene to be introduced Efficient delivery of gene control of gene expression Prevention/control of immune responses Animal model and assessment of function Clinical trial,研究基因治疗的三个基本步骤寻找适当的靶细胞导入目的基因基因表达,体细胞基因治疗始终需要考虑的问题基因转移的效率治疗的特异性基因表达的持久性及其调节治疗的毒副作用 什么疾病什么基因什么载体什么靶器官和靶细胞什么

4、基因导入方法,体细胞基因治疗 将基因作为一种特殊“药物”，通过体细胞基因转移治疗疾病 慢性治疗 急性治疗 预防 遗传性疾病 获得性疾病 功能丧失 功能获得,二、基因治疗和传统的基因工程的区别 二者都着眼于寻找可治病或有其他应用价值的“目的基因”。 基因工程： 目的基因载体导入大肠杆菌、酵母和哺乳动物细胞体外表达所需要的蛋白经过分离纯化获得能用于治疗或其他用途的蛋白纯品, 最终是制造出一种蛋白类的药物,基因治疗目的基因载体导入人体，目的基因在人体内的细胞中制造所需要的蛋白达到治病的目的。 基因治疗在技术上一旦成功, 其优势：制品为基因及其载体，非基因表达蛋白产物,不需复杂的蛋白产物分离和纯化工艺

5、生产成本远远低于基因工程产品从理论上讲,凡能治病的基因，都有可能开发成为“药物” 半衰期,但基因治疗难度高，技术要求极为苛刻。例如，针对各种疾病，必须具有能够达到治病目的的基因。在此基础上，还必须具有能有效地将基因导入人体的载体系统，这种系统要求高效，而且能定向地导入人体某种细胞。基因导入人体后，必须能够控制它的表达,因此，基因治疗是生物高技术的高度集成，是遗传学、分子生物学、细胞生物学、分子病毒学等多种学科知识和技术的高度综合,三、基因治疗的主要策略 Gene replacement Gene augmentation therapy (GAT) Gene correction (Chime

6、raplasty) Targeted killing of specific cells Targeted inhibition of gene expression (Gene ablation,Gene replacement: Deficient gene corrected by replacing the mutated allele with an intact allele; used for the treatment of autosomal dominant disorders. The strategies are as follows: a) kockout mutat

7、ion by single crossing-over (insertional inactivation) b) gene replacement by double (reciprocal) crossing-overc) target gene inhibition in dominant negative genetic disorders; usually mutated proteins which interact with normal cellular proteins to create altered properties; mutational inactivation

8、 of the one mutated copy will alleviate this mutant phenotype ; the mutation can be carried out by insertional inactivation, or antisense technology,Gene Augmentation Therapy (GAT) For diseases caused by loss of gene function more copies of normal gene raise levels of gene product restore normal phe

9、notype Apply to: monogenic recessive diseases cystic fibrosis, haemophilia, muscular dystrophy,Gene Correction - Chimeraplasty,tumour cell,tk,thymidine kinase gene,viral vector,ganciclovir,ganciclovir phosphate,Targeted Killing Genetic Pro-drug Activation Therapy,Targeted inhibition of gene expressi

10、on Ribozymes -can cleave (or repair) mRNA Triple helix oligonucleotides -block gene transcription Antisense oligos -block mRNA translation,All gene therapy strategies depend on getting the gene or genetic material into the target cells,四、基因转移的主要方法： 若干相关术语：Transfection 转染 ：过去：将病毒DNA或RNA 转入细胞。 现在：将外源D

11、NA转入动物细胞。DNA Transfer 转移：基因导入或转移广义的概念。Transduction 转导：噬菌体介导的细菌之间的基 因 转移。Transformation 转化：将裸DNA或质粒导入原核 和真核细胞,Two main routes of gene transfer: In vivo: i.v. or i.m. injectable; or non-invasive (eg “sniffable”) Ex vivo: hepatocytes, skin fibroblasts haematopoietic cells “bioreactors,a,a,a,a,a,a,Ex-vi

12、vo,In-vivo topical delivery,In-vivo systemic delivery,Examples: - bone marrow - liver cells - skin cells,Examples: - brain - muscle - eye - joints - tumors,Examples: - intravenous - intra-arterial - intra-peritoneal,THREE classes of anatomical gene delivery,基因治疗过程中的基因转移方法Viral vectorsNon-viral vecto

13、rsPhysical methods目前最有效的是病毒载体，但存在插入大小有限、免疫原性强和生产难等局限,1、病毒载体类型 反转录病毒（RV）载体 腺病毒（AV）载体 腺相关病毒（AAV）载体 单纯疱疹病毒（HSV）载体 慢病毒（lentivirus)载体等,对载体的基本要求： 特异并有效的基因转移 特异、高效、持续性表达，具有可控性 免疫原性低 易于生产,载体特征： reproducibility stable propagated purified to high titers mediated targeted delivery,Advantage and disadvantage of

14、 gene-transfer vector(1)Vector Advantage DisadvantageAV Very high transfection ex vivo repeat dosing ineffective owing to 未根本解决； 制备293/E2A, 293/E4细胞;相应载体; 扩大容量; 降低毒性和免疫性; 效果有限; 滴度下降. 293T细胞: 转染SV40大T 抗原,降低腺病毒免疫原性,腺病毒载体插入B7反义基因, 阻断共刺激途径; 腺病毒载体插入ICAM-1反义基因, 阻断粘附因子作用. 腺病毒载体插入CTLA4 基因, 胞内结合B7. 腺病毒载体插入MH

15、C-I,II基因的调控基因反义片段,增强腺病毒的感染效率,腺病毒与葡聚糖, 聚凝胺, 多聚赖氨酸,脂质体混合感染细胞,组织, 提高感染效率10-100倍, 降低炎症反应 . 重组腺病毒感染前, 进行腺病毒封闭 不同亚型腺病毒互换应用,a,a,a,a,a,a,Recombinant Adenoviruses,Approaches Generation I Generation III Hybrid adenos: Adeno-RV Adeno-AAV Adeno-Transposase,Advantages / Limitations 8 Kb capacity Generation I 30

16、Kb capacity Generation IIIAdeno can be grown at very high titers, However Do not integrate Can contain RCAs Are toxic /immunogenic,Examples OTC deficiency (clin, -) Cystic Fibrosis (clin, - ) Oncolytic viruses (clin,腺病毒载体研究进展与展望 进展 展望 Ad载体用于治疗性的angiogenesis 降低毒性的方法将继续获得进展, 取得进展 最终将提供安全的Ad载体系统. Ad引起的

17、毒性问题得到进一步认识 靶向方法的应用将进一步改进. 提高Ad载体的靶向性，改善疗效 缺陷型的Ad载体生产将不断进展. 新一代Ad载体能降低毒性，改善表达 将面临再次注射的挑战,反转录病毒载体基因转移系统 辅助细胞: 将含有病毒结构基因但缺失了顺式包装信号的缺陷型反转录病毒导入哺乳动物细胞（2 ，PA317，CRIP）。可产生病毒包装蛋白但不产生病毒颗粒； 反转录病毒载体:以外源目的基因替换病毒结构基因,含病毒包装信号 辅助细胞提供载体包装蛋白，使后者产生含外源目的基因的病毒颗粒,缺陷型的反转录病毒颗粒的RNA进入靶细胞后，变成前病毒并整合到宿主细胞的染色体上，可稳定表达外源基因。 反转录病毒

18、只能感染处于增殖期的细胞,a,a,a,a,a,a,Approaches Murine Retroviruses VSV-pseudotyped RV Lentiviruses Self-inactivating RV Combination viruses,Advantages / Limitations 9 Kb capacity + integration through transposition also in quiescent cells (HIV), permit in principle long-term treatments, however disturbed by: I

19、nsertional mutagenesis Gene silencing High mutation rate Low titer of production Examples SCID (IL2R defect, Paris) (clin, +) Adenosine Deaminase deficiency (clin, +!) Parkinson (preclin, +) Anti cancer (clin +/,Recombinant Retroviruses (includes HIV-based,反转录病毒安全性问题 病毒感染的可能性 病毒在靶细胞基因组整合可导致: 破坏细胞正常生

20、长必需的抑癌基因 LTR激活原癌基因 染色体重排激活原癌基因,腺相关病毒（AAV） 载体：4.7kb单链DNA，结构为 ITR-rep-cap-ITR ，病毒基因组简单，易于消除，可降低细胞毒性和T-淋巴细胞反应的危险性；病毒DNA可在人19号染色体上进行位点特异性整合，需要辅助因子出现才复制；其Rep蛋白可能介导这种定向整合宿主范围宽，易感染造血干细胞，能潜伏感染非分裂期细胞；在动物模型中表达可持续半年以上。 但AAV载体接纳的外源DNA小于4.5kb，载体整合效率较低，制备困难,a,a,a,a,a,a,Recombinant adeno-associated-virus (AAV,Appr

21、oaches Helper-dependent production Helper independent production Cis-complementing vectors Co-infection,Advantages / Limitations Persistence in the genome permits long-term expression, high titers are easily obtained, immunogenicity is very low, However Small capacity (4.5 kb) which does not allow t

22、o accommodate large genes or gene clusters. Examples Hemophilia B (clin, animal, +) Gaucher (clin, animal, +) Brain Ischemia (animal, +) Cystic fibrosis (animal, +/,Lentivirus（慢病毒载体） 优点: 感染非分裂细胞 ，有RV优势 结构: LTR- gag- pol - env-LTR TAR tat, rev, tev 应用: 宿主广泛; 艾滋病基因治疗 问题: 安全性,HSV病毒,双链DNA, 包括: HSV-1, 2,

23、 VZV, EB, CMV. 主要以HSV-1型为主.HSV载体含包装信号, 复制位点等顺序结构, HSV病毒启动子,目的基因表达框架. 由辅助病毒共转染辅助细胞M64A, 制备重组病毒. 辅助病毒的改造: IE3 温度敏感突变型; 缺失突变型: IE3缺失; 应用: 感染心肌细胞, 神经元, 神经胶质细胞. VEGF-血管形成; 脑肿瘤; 帕金森病,a,a,a,a,a,a,Recap: current limitations of popular vectors,Adenovirus - no persistence - limited packaging toxicity, immunog

24、enicity,Retrovirus (incl. HIV) - limited packaging - random insertion - unstable genome,General - antibody response - limited packaging - gene silencing,Solutions: - synthetic viruses (“Virosomes”,Biolistic bombardmentor local direct injection - limited area,Electroporation - limited organ access,Li

25、posomes, gene correction & Co. - very inefficient transfer,General - low transfer efficiency - no or little genomic integration,Solutions: - improved liposomes with viral properties (“Virosomes”,非病毒载体除了基因转移的效率比较低以外，比病毒载体具有更多的优越性，将来会有viral-like, but artificial vectors,Pharmacological considerations f

26、or DNA transfer Classical drug MW 50- 500 Daltons Synthetically prepared Rapid diffusion/action Oral delivery possible Cellular delivery: - act at cell surface - permeate cell membrane - imported through channels Can be delivered as soluble molecules ngstrom/nm size rapidly reversible treatment,Mw 2

27、0,000- 100,000 Da Biologically prepared Slower diffusion/action Oral delivery not possible Cellular delivery - act extracellularlyCan be delivered as soluble molecules nm size rapidly reversible treatment,Protein drug,Nucleic acid,Mw N x 1,000,000 Da Biologically prepared Slow diffusion Oral deliver

28、y inconceivable Cellular delivery: - no membrane translocation - no nuclear translocation - no biological import Must be delivered as complex carrier particles 50-200 nm size slowly or not reversible,Therapy with nucleic acids requires particulated formulation is much more complex than previous drug

29、 deliveries has a different degree of reversibility (dosage problem,2、非病毒及物理方法 脂质体介导法 磷酸钙转染法 机械法（显微注射、基因枪等） DEAE-葡聚糖和polybrene转染法 电穿孔法 超声波辅助法 纳米材料等,中性脂质体 由脂类形成的可高效包装DNA的人造单层膜，其结构和性质与细胞膜极为相似，二者易于融合，细胞的内吞作用使其进入细胞，操作简单 ，转染效率可高达50%，可用于体内试验，但目的基因表达不稳定,阳离子脂质体：阳离子脂质体在水中可形成大小约100-400nm单层脂质体。其带正电，带负电的DNA可自动结

30、合到带正电的脂质体上，形成DNA-阳离子脂质体复合物，与细胞膜作用使DNA进入细胞。在该系统体内基因导入效率低，且无靶向性。在体内应用，除肿瘤瘤内注射外，其前景仍存在问题,磷酸钙转染法：将氯化钙、目的DNA和磷酸缓冲液混合，沉淀形成含有DNA的极小的不溶性磷酸钙颗粒。磷酸钙-DNA复合物粘附到细胞膜，通过胞饮进入受体细胞的细胞质，转染过程简单有效 ，适用于贴壁、非贴壁细胞，转染效率可达20%左右，但目的基因表达不稳定,机械法如显微注射和基因枪（biolistic particle）。显微注射使用一根细针头将外源DNA直接转入受体细胞核。基因枪使用高压DNA分子导入细胞,基因枪工作原理,显微注射

31、,a,a,a,a,a,a,Approaches Antisense Ribozymes/DNAzymes Triple helix Decoy / competitors Gene-correcting oligos,Advantages / Limitations these procedures may be suitable for : handling dominant defects transient treatments (gene modulation) permanent treatments (gene correction,Examples Anti cancer (cl

32、in,preclin., +/-) Restenosis (clin, +) Muscular Distrophy (animal,Oligonucleotides,a,a,a,a,a,a,Approaches Naked DNA injection /biolistic Naked DNA + pressure Naked DNA + electroporation Liposomal formulations Combinations,Advantages / Limitations Unlimited size capacity + lower immunogenicity and lo

33、wer bio-risk of non viral formulations is disturbed by Low efficiency of gene transfer Even lower stable integration,Examples Critical limb Ischemia (clin, +) Cardiac Ischemia (clin, +/-) Vaccination (clin, +/-) Anti restenosis (preclin. +/,Naked / complexed DNA,a,a,a,a,a,a,Ideal properties of a sys

34、temically delivered non-viral formulation,Stability particle should resist serum inactivation particle should be inert to immune inactivationAddressability particle should possess a vascular addressing signature particle should bear a tissue-docking specificity DNA construct should include tissue-sp

35、ecific regulatory elementsEfficiency cargo should be protected from cytoplasmic inactivation (ex. lysosomes) cargo should contain nuclear-translocating signals DNA cargo should include genome-integration functions DNA element must be guaranteed to function after genomic integration (no silencing,Oth

36、er properties Particle should not include immunogenic/toxic surfaces cargo should not encode immunogenic/toxic products Cargo should include anti-apoptotic functions several independent problems must be solved for a nonviral formulation to be suitable for clinical treatment and for industrial produc

37、tion most viral vectors include many, if not all those properties,裸DNA基因转移和治疗 进展展望 裸DNA肌肉注射，活体转移非病毒基因转移越来越重要 电击促进质粒DNA进入肌肉皮肤未来几年遗传病临床试验将首选血友病 血管内导入质粒DNA，肝细胞基因转移Duchenne 肌营养不良，关节炎等 尾静脉导入pDNA，简单有效，转染大、 啮齿类的尾静脉注射作为快速检测表 小鼠肝细胞 达状况和基因治疗方法广泛应用 pDNA表达载体可高水平持续表达 血管内裸DNA导入为细胞有效涉入pDNARNAi将在基因治疗领域中成为重要手 裸DNA导入

38、法已经在临床研究（PAOD， 段 外周动脉闭锁症） SiRNA可通过血管内基因转移法有效导入 Knockout 小鼠,a,a,a,a,a,a,problems that must be solved to be suitable for clinical treatment and for industrial production are different between viral and non-viral vectors when ignoring their low efficiency, nonviral vectors appears largely superior,Most

39、 relevant issues in the two main vectorology sectors (viral versus nonviral,Viral vectors Packaging capacity from 4 to 30 kb problem for some large genes (ex. dystrophin gene or CFTR gene) important toxic load: ratio infectious/non-infectious particles from 1/10 to 1/100 strong immunogenicity: capsi

40、d and envelope proteins, residual viral genes contaminants: replication-competent viruses (ex. wild type revertant viruses) Viral amount (titre) obtainable with recombinants (ex. 10exp5 = poor, 10exp10=excellent) Complexity of production (existence or not of packaging cell systems) Emotional problem

41、s linked to pathogenicity of donor vectors (ex. lentiviruses,Nonviral vectors Packaging capacity not an issue, even very large constructs can be used (example entire loci up to 150 kb) minor toxic load: small percentage of non relevant adventitious materials moderate immunogenicity: methylation stat

42、us of DNA (example CpG motifs) contaminants: adventitious pathogens from poor DNA purification (ex endotoxins) Amount of DNA molecules is usually not a problem, the other components depends on chemical synthesis No particular complexity, except for specially formulated liposomes no particular emotional problems linked to the nature of the reagents,a,a,a,a,a,a,Random integrating vectors r-lentiviruses r-retroviruses r-AAV plasmids (low frequency) plasmids + transposase (eg sleeping beauty,Transient, non integrating vectors adenovirus plasmid RNA virus based oligonucleotides (SiRNA,