基因转移与

基因治疗

复旦大学 遗传学研究所薛京伦

2003 ， 10

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。

基因与疾病基因与疾病•基因与人类的疾病密切相关：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肿瘤的发生涉及多种基因改变 , , 包括癌基包括癌基因激活或抑癌基因失活因激活或抑癌基因失活 ; ; •高血压高血压 , , 糖尿病等多基因病也涉及到多种糖尿病等多基因病也涉及到多种基因的改变基因的改变 ; ; •由病原体所致的传染病也和人体基因密切由病原体所致的传染病也和人体基因密切相关相关 , , 存在易感人群和耐受人群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的的生物医学高技术。

•The application of genetic principles in the The application of genetic principles in the treatment of human diseasetreatment of human disease•By introduction of geneticBy introduction of genetic material into material into target cells in order to counteract the effect target cells in order to counteract the effect of a disease gene or introduce a new of a disease gene or introduce a new functionfunction• Somatic and Germline approaches Somatic and Germline approaches possiblepossible

基因治疗的必要条件基因治疗的必要条件•Understanding of the disease process

•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研究基因治疗的三个基本步骤寻找适当的靶细胞寻找适当的靶细胞导入目的基因导入目的基因基因表达基因表达

体细胞基因治疗始终需要考虑的问题体细胞基因治疗始终需要考虑的问题

基因转移的效率基因转移的效率治疗的特异性治疗的特异性基因表达的持久性及其调节基因表达的持久性及其调节治疗的毒副作用治疗的毒副作用 什么疾病什么疾病

什么基因什么基因什么载体什么载体什么靶器官和靶细胞什么靶器官和靶细胞什么基因导入方法什么基因导入方法

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：①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②生产成本远远低于基因工程产品③从理论上讲 ,凡能治病的基因，都有可能开发成为“药物” ④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三、基因治疗的主要策略

•Gene replacement•Gene augmentation therapy (GAT)•Gene correction (Chimeraplasty)•Targeted killing of specific cells•Targeted inhibition of gene expression (Gene ablation)

Gene replacement:Gene replacement:

Deficient gene corrected by replacing the mutated alleDeficient gene corrected by replacing the mutated allele with an intact allele; used for the treatment of autosle with an intact allele; used for the treatment of autosomal dominant disorders. omal dominant disorders.

The strategies are as follows: The strategies are as follows:

a) kockout mutation by single crossing-over (insertiona) kockout mutation by single crossing-over (insertional inactivation) al inactivation) b) gene replacement by double (reciprocal) crossing-ob) gene replacement by double (reciprocal) crossing-oververc) target gene inhibition in dominant negative genetic c) target gene inhibition in dominant negative genetic disorders; usually mutated proteins which interact witdisorders; usually mutated proteins which interact with normal cellular proteins to create altered properties; h normal cellular proteins to create altered properties; mutational inactivation of the one mutated copy will allmutational inactivation of the one mutated copy will alleviate this mutant phenotype ; the mutation can be careviate this mutant phenotype ; the mutation can be carried out by insertional inactivation, or antisense technried out by insertional inactivation, or antisense technology. ology.

•Gene Augmentation Therapy (GAT)Gene Augmentation Therapy (GAT)

For diseases caused by loss of gene For diseases caused by loss of gene

functionfunction

more copies of normal gene more copies of normal gene

raise levels of gene productraise levels of gene product

restore normal phenotyperestore normal phenotype

Apply to: monogenic recessive diseases Apply to: monogenic recessive diseases

cystic fibrosis, haemophilia, muscular cystic fibrosis, haemophilia, muscular

dystrophydystrophy

•Gene Correction - Chimeraplasty

tumour cell

tkthymidine kinasegene

viral vector

ganciclovir

ganciclovir phosphate

•Targeted Killing –Genetic Pro-drug Activation Therapy

•Targeted inhibition of gene expression Targeted inhibition of gene expression Ribozymes Ribozymes

-can cleave (or repair) mRNA-can cleave (or repair) mRNA Triple helix oligonucleotidesTriple helix oligonucleotides

-block gene transcription-block gene transcription Antisense oligosAntisense oligos

-block mRNA translation-block mRNA translation

All gene therapy strategies All gene therapy strategies dependdepend

on getting the gene or genetic on getting the gene or genetic

material into the target cells!!!material into the target cells!!!

四、基因转移的主要方法： 若干相关术语：若干相关术语：Transfection Transfection 转染转染 ：：过去：将病毒过去：将病毒 DNADNA 或或 RNA RNA 转入细胞。 转入细胞。 现在：将外源 现在：将外源 DNADNA 转入动物细胞。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 transferTwo main routes of gene transfer::

In vivo:In vivo: i.v. or i.m. injectable; or i.v. or i.m. injectable; or non-invasive (eg “sniffable”)non-invasive (eg “sniffable”)

Ex vivo: Ex vivo: hepatocytes, skin fibroblastshepatocytes, skin fibroblastshaematopoietic haematopoietic cellscells““bioreactors”bioreactors”

Ex-vivo In-vivotopical delivery

In-vivosystemic delivery

V

Examples:- bone marrow- liver cells- skin cells

Examples:- brain- muscle- eye- joints- tumors

Examples:- intravenous- intra-arterial- intra-peritoneal

THREE classes of anatomical gene deliveryTHREE classes of anatomical gene delivery

基因治疗过程中的基因转移方法基因治疗过程中的基因转移方法

Viral vectorsViral vectorsNon-viral vectorsNon-viral vectorsPhysical methodsPhysical methods目前最有效的是病毒载体，但存在目前最有效的是病毒载体，但存在插入大小有限、免疫原性强和生产插入大小有限、免疫原性强和生产难等局限难等局限

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：对载体的基本要求：

•特异并有效的基因转移特异并有效的基因转移•特异、高效、持续性表达，具有可控性特异、高效、持续性表达，具有可控性•免疫原性低免疫原性低•易于生产易于生产

载体特征： reproducibility

stable propagated

purified to high titers

mediated targeted delivery

Advantage and disadvantage of gene-transfer vector(1)Advantage and disadvantage of gene-transfer vector(1)VectorVector Advantage Disadvantage Advantage Disadvantage

AV Very high transfection ex vivo repeat dosing ineffective owing to AV Very high transfection ex vivo repeat dosing ineffective owing to & in vivo strong immune response & in vivo strong immune response Transfects proliferating & non- Insert-size limit 0f 7.5kb Transfects proliferating & non- Insert-size limit 0f 7.5kb proliferating cells , substantial manufacture, storage, QC are proliferating cells , substantial manufacture, storage, QC are clinical experience acquired moderately difficult clinical experience acquired moderately difficult efficient retargeted transfction shout duration of expression efficient retargeted transfction shout duration of expression RV fairly prolonged expression lower transfection efficiency in vivo RV fairly prolonged expression lower transfection efficiency in vivo high transfection efficiency ex vivo insert-size limit of 8kb high transfection efficiency ex vivo insert-size limit of 8kb substantial clinical experience ex vivo transfect only proliferating cells,safty substantial clinical experience ex vivo transfect only proliferating cells,safty low immunogenicity concern of insertional mutagenesis low immunogenicity concern of insertional mutagenesisLentivirus transfects proliferating & non- safety concern from immunodeficiencyLentivirus transfects proliferating & non- safety concern from immunodeficiency proliferating , haematopoietic virus origins, manufacturing, storage, proliferating , haematopoietic virus origins, manufacturing, storage, stem cells QC are extremely difficult, insert-size stem cells QC are extremely difficult, insert-size limit of 8kb, no clinical experience limit of 8kb, no clinical experienceAAV efficiently transfects a wide variety insert-size limits 4.5kbAAV efficiently transfects a wide variety insert-size limits 4.5kb 0f cells in vivo, very prolonged manufacture, QC are very difficult 0f cells in vivo, very prolonged manufacture, QC are very difficult expression in vivo little clinical experience, safety concern expression in vivo little clinical experience, safety concern low immunogenicity of insertional mutagenesis, repeat low immunogenicity of insertional mutagenesis, repeat dosing affectedly by neutralizing dosing affectedly by neutralizing antibody responses antibody responses

基因治疗病毒载体比较 RV AV AAV HSV LV基因组成 ssRNA dsDNA ssDNA dsDNA dsRNA基因组长度 10kb 36kb 5kb 152kb 10kb装载容量 <8kb 8kb 4.5kb 30kb 9kb病毒滴度 107 1011 108 108 108感染细胞谱 窄 宽 宽 窄 宽感染能力 中 很强 强 强 中整合能力 有 无 有 无 强毒性作用 遗传毒 细胞毒 遗传毒 细胞毒 遗传毒免疫原性 弱 中等 弱 强 弱

Advantage and disadvantage of gene-transfer vector(2)Advantage and disadvantage of gene-transfer vector(2)

VectorVector Advantage Disadvantage Advantage DisadvantageNaked DNA manufacturing, storage, QC very short duration of expression inNaked DNA manufacturing, storage, QC very short duration of expression in

are simple and cheap, very most tissues, very ineffecient are simple and cheap, very most tissues, very ineffecient

low immunogenicity, clinical transfection ex vivo and in vivo low immunogenicity, clinical transfection ex vivo and in vivo

limb ischaemia, good safety retargeting transfection very limb ischaemia, good safety retargeting transfection very

profile difficult profile difficult

Cationic lipids relative simple manufacturing , inefficient transfection in vivoCationic lipids relative simple manufacturing , inefficient transfection in vivo

storage ,QC efficient transfection very short duration of expression storage ,QC efficient transfection very short duration of expression

ex vivo , low immunogenicity little clinical experience ex vivo , low immunogenicity little clinical experience

condensed relative simple manufacturing , inefficient transfection in vivocondensed relative simple manufacturing , inefficient transfection in vivo

DNA particles storage, QC . Efficient trancfection very short duration of expressionDNA particles storage, QC . Efficient trancfection very short duration of expression

ex vivo, low immunogenicity now clinic experience ex vivo, low immunogenicity now clinic experience

good safety profile good safety profile

retargeted transfection demostraded retargeted transfection demostraded

Transfection

Infection

exposed to106 particles/cell12 hours

exposed to 1 particle/cell30 min

• virally mediated gene transfer is millions of times more efficent than nonviral transfer (when calculated in terms of transfer/particle)

Transfection versus InfectionTransfection versus Infection

Vector used in gene therapyVector used in gene therapyType approximate integration duration ofType approximate integration duration of insert-size capacity persistenceinsert-size capacity persistenceNucleic-acid basedNucleic-acid based Oligonucleotides: Decoys, Oligonucleotides: Decoys, antisense, Ribozomes, siRNA 10-100 kb no hours-daysantisense, Ribozomes, siRNA 10-100 kb no hours-daysExpression plasmids 2-10kb extremely rare daysExpression plasmids 2-10kb extremely rare daysTransposons 2-10kb efficient stableTransposons 2-10kb efficient stable relative randomrelative randomBacteriohage integrase 2-10kb efficient ,site restricted stableBacteriohage integrase 2-10kb efficient ,site restricted stableArtificial chromosome 50-300kb episomal +/- stableArtificial chromosome 50-300kb episomal +/- stableVirus basedVirus basedRV 2-6 kb efficient stableRV 2-6 kb efficient stable relative randomrelative randomLentiviral 2-10kb efficient stableLentiviral 2-10kb efficient stable relative randomrelative randomAAV 2-5kb rare months-yearsAAV 2-5kb rare months-yearsAV 2-30kb NO weeks-monthsAV 2-30kb NO weeks-monthsHerpes virus 2-40kb episomal moths-yearsHerpes virus 2-40kb episomal moths-yearsEB virus 2-40kb episomal moths-yearsEB virus 2-40kb episomal moths-yearsSV40 1-5kb moderate unprovenSV40 1-5kb moderate unproven

腺病毒腺病毒载体载体：双链：双链 DNADNA ，在基因转，在基因转移中的应用相当广泛。其优点是易制移中的应用相当广泛。其优点是易制备、高滴度、宿主广、能感染非增殖备、高滴度、宿主广、能感染非增殖细胞，低毒，不整合入机体细胞染色细胞，低毒，不整合入机体细胞染色体，容量达体，容量达 36kb36kb ，但免疫原性强，，但免疫原性强，表达时间短。表达时间短。

改进的腺病毒载体改进的腺病毒载体1. 1. 早期早期 AV E1+E3AV E1+E3 区 缺失的有限性区 缺失的有限性2. E2, E42. E2, E4 区的置换区的置换 ::

Wilson Wilson 报道报道 : : 温度敏感温度敏感 E2E2突变突变 AAVV降低免疫性降低免疫性 , , 表达延长表达延长 ; ; 未根本解决；未根本解决； 制备 制备 293/E2A, 293/E4293/E2A, 293/E4 细胞细胞 ;; 相应载相应载体体 ; ; 扩大容量扩大容量 ; ; 降低毒性和免疫性降低毒性和免疫性 ; ; 效果有限效果有限 ; ; 滴度下降滴度下降 . . •293T293T 细胞细胞 : : 转染转染 SV40SV40 大大 T T 抗原抗原

降低腺病毒免疫原性降低腺病毒免疫原性•腺病毒载体插入腺病毒载体插入 B7B7反义基因反义基因 , , 阻断共刺阻断共刺激途径激途径 ;;•腺病毒载体插入腺病毒载体插入 ICAM-1ICAM-1 反义基因反义基因 , , 阻断阻断粘附因子作用粘附因子作用 ..•腺病毒载体插入腺病毒载体插入 CTLA4 CTLA4 基因基因 , , 胞内结合胞内结合B7.B7.• 腺病毒载体插入腺病毒载体插入 MHC-I,IIMHC-I,II 基因的调控基基因的调控基因反义片段因反义片段 ..

增强腺病毒的感染效率增强腺病毒的感染效率

•腺病毒与葡聚糖腺病毒与葡聚糖 , , 聚凝胺聚凝胺 , , 多聚赖氨酸多聚赖氨酸 ,,

脂质体混合感染细胞脂质体混合感染细胞 ,, 组织组织 , , 提高感染效提高感染效

率率 10-10010-100 倍倍 , , 降低炎症反应 降低炎症反应 ..

•重组腺病毒感染前重组腺病毒感染前 , , 进行腺病毒封闭进行腺病毒封闭

不同亚型腺病毒互换应用不同亚型腺病毒互换应用 ..

Recombinant AdenovirusesRecombinant Adenoviruses

ApproachesGeneration IGeneration IIIHybrid adenos:Adeno-RVAdeno-AAVAdeno-Transposase

Advantages / Limitations

8 Kb capacity Generation I >30 Kb capacity Generation IIIAdeno can be grown at very high titers, However Do not integrate Can contain RCAs Are toxic /immunogenic

ExamplesOTC 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反转录病毒载体基因转移系统辅助细胞辅助细胞 :: 将含有病毒结构基因但缺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（毒导入哺乳动物细胞（ψ—2 ， PA317，ψCRIP）。可产生病毒包装蛋。可产生病毒包装蛋白但不产生病毒颗粒；白但不产生病毒颗粒；反转录病毒载体反转录病毒载体 :: 以外源目的基因替换以外源目的基因替换病毒结构基因病毒结构基因 ,,含病毒包装信号含病毒包装信号辅助细胞提供载体包装蛋白，使后者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。

反转录病毒只能感染处于增殖期的细胞。

ApproachesMurine RetrovirusesVSV-pseudotyped RVLentiviruses Self-inactivating RVCombination viruses

Advantages / Limitations9 Kb capacity + integration throughtransposition also in quiescent cells(HIV), permit in principle long-termtreatments, however disturbed by:Insertional mutagenesisGene silencingHigh mutation rateLow titer of productionExamplesSCID (IL2R defect, Paris) (clin, +++)Adenosine Deaminase deficiency (clin, +++!!!)Parkinson (preclin, +++)Anti cancer (clin +/-)

Recombinant Retroviruses (includes HIV-basedRecombinant Retroviruses (includes HIV-based))

反转录病毒安全性问题反转录病毒安全性问题•病毒感染的可能性病毒感染的可能性•病毒在靶细胞基因组整合可导致病毒在靶细胞基因组整合可导致 ::

破坏细胞正常生长必需的抑癌基因破坏细胞正常生长必需的抑癌基因 LTRLTR 激活原癌基因激活原癌基因 染色体重排激活原癌基因染色体重排激活原癌基因

腺相关病毒（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

Recombinant adeno-associated-virus (AAV)Recombinant adeno-associated-virus (AAV)

ApproachesHelper-dependent productionHelper independent productionCis-complementing vectorsCo-infection

Advantages / LimitationsPersistence in the genome permits long-term expression, high titers are easily obtained, immunogenicity is very low,HoweverSmall capacity (<4.5 kb) which does not allow to accommodate large genes or gene clusters.ExamplesHemophilia 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

HSVHSV 病毒病毒•双链双链 DNA, DNA, 包括包括 : HSV-1, 2, VZV, EB, CMV.: HSV-1, 2, VZV, EB, CMV.

主要以主要以 HSV-1HSV-1 型为主型为主 .HSV.HSV 载体含包装信号载体含包装信号 , ,

复制位点等顺序结构复制位点等顺序结构 , HSV, HSV 病毒启动子病毒启动子 ,, 目的基目的基因表达框架因表达框架 . . 由辅助病毒共转染辅助细胞由辅助病毒共转染辅助细胞 M64A, M64A,

制备重组病毒制备重组病毒 ..• 辅助病毒的改造辅助病毒的改造 : IE3 : IE3 温度敏感突变型温度敏感突变型 ; ;

缺失突变型缺失突变型 : IE3: IE3 缺失缺失 ; ; • 应用应用 : : 感染心肌细胞感染心肌细胞 , , 神经元神经元 , , 神经胶质细胞神经胶质细胞 ..

VEGF---VEGF--- 血管形成血管形成 ; ; 脑肿瘤脑肿瘤 ; ; 帕金森病 帕金森病

Recap: current limitations of popular vectorsRecap: current limitations of popular vectorsAdenovirus- no persistence- limited packaging-toxicity, -immunogenicity

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

Liposomes, 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比较低以外，比病毒载体具有更多

的优越性，将来会有的优越性，将来会有 viral-like, buviral-like, bu

t artificial vectors.t artificial vectors.

Pharmacological considerationsPharmacological considerations for DNA transferfor DNA transfer Classical drugClassical 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•Ångstrom/nm size•rapidly reversible treatment

•Mw 20,000- 100,000 Da•Biologically prepared•Slower diffusion/action•Oral delivery not possible•Cellular delivery

- act extracellularly

Can be delivered as soluble molecules•nm size•rapidly reversible treatment

Protein drugProtein drug

Nucleic acidNucleic acid•Mw N x 1,000,000 Da•Biologically prepared•Slow diffusion•Oral delivery 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

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 DN

A 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

•机械法机械法如显微注射和基因枪（如显微注射和基因枪（ biolistic particlebiolistic particle ）。）。显微注射使用一根细针头将外源显微注射使用一根细针头将外源 DNADNA 直接转直接转入受体细胞核。基因枪使用高压入受体细胞核。基因枪使用高压 DNADNA 分子导分子导入细胞。入细胞。

基因枪工作原理

显微注射

ApproachesAntisenseRibozymes/DNAzymesTriple helixDecoy / competitorsGene-correcting oligos

Advantages / Limitationsthese procedures may be suitable for :handling dominant defectstransient treatments (gene modulation)permanent treatments (gene correction)

ExamplesAnti cancer (clin,preclin., +/-)Restenosis (clin, +++)Muscular Distrophy (animal, +++)

OligonucleotidesOligonucleotides

•电穿孔法通过将细胞暴露在短暂的高场强电脉冲中转导目的 DNA 。细胞悬浮液置于电场中会诱导沿细胞膜的电压差异，这种电压差异会导致细胞膜暂时穿孔。一般，成功的电穿孔过程都伴随高水平（ 50%

或更高）的毒性。

•DEAE-葡聚糖和 polybrene 转染法

带正电的 DEAE-葡聚糖或 polybrene 多聚体复合物和带负电的外源 DNA 分子作用，使得 DNA 可以结合在细胞表面。通过使用 DMSO 或甘油获得的渗透休克将外源 DNA 导入。两种试剂都已成功用于转染。 DEAE-葡聚糖仅限于瞬时转染。

ApproachesNaked DNA injection /biolisticNaked DNA + pressureNaked DNA + electroporationLiposomal formulationsCombinations

Advantages / LimitationsUnlimited size capacity + lowerimmunogenicity and lower bio-riskof non viral formulations isdisturbed by

Low efficiency of gene transferEven lower stable integration

ExamplesCritical limb Ischemia (clin, +++)Cardiac Ischemia (clin, +/-)Vaccination (clin, +/-)Anti restenosis (preclin. +/-)

Naked / complexed DNANaked / complexed DNA

Ideal properties of a systemically delivered Ideal properties of a systemically delivered non-viral formulationnon-viral formulationStabilityparticle should resist serum inactivationparticle should be inert to immune inactivationAddressabilityparticle should possess a vascular addressing signatureparticle should bear a tissue-docking specificityDNA construct should include tissue-specific regulatory elementsEfficiencycargo should be protected from cytoplasmic inactivation (ex. lysosomes)cargo should contain nuclear-translocating signalsDNA cargo should include genome-integration functionsDNA element must be guaranteed to function after genomic integration (no silencing)

Other 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 production•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 pDNA RNAi 将在基因治疗领域中成为重要手

裸 DNA 导入法已经在临床研究（ 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

∴ problems that must be solved to be suitable for clinical treatment and for industrial production are different between viral and non-viral vectorswhen ignoring their low efficiency, nonviral vectors appears largely superior

Most relevant issues in the two main Most relevant issues in the two main 'vectorology' sectors (viral versus nonviral)'vectorology' sectors (viral versus nonviral)

Viral vectorsPackaging 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 1/100strong immunogenicity: capsid and envelope proteins, residual viral genescontaminants: 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 problems linked to pathogenicity of donor vectors (ex. lentiviruses)

Nonviral vectorsPackaging 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 materialsmoderate immunogenicity: methylation status 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 synthesisNo particular complexity, except for specially formulated liposomes no particular emotional problems linked to the nature of the reagents

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, antisense,

ribozymes) artificial chromosomes

Gene correction vectors chimeroplasts (RNA-DNA chimeric

oligos) single stranded DNA (homologous

recom)

• genotoxic non-genotoxic

Specifically integrating vectors hybrid vectors (HSV-AAV) Phage 31 integrase-based designer integrase

Not all gene therapy approaches are 'random shooting'Not all gene therapy approaches are 'random shooting'

Rapid and transient action required

Adeno II, Plasmid, modulatory oligonucleotides

Trauma or infection(Ischemia, fracture, burn, wound, acute infection, anaphyllaxis)

rapid & transient expression of cytotoxic or immunomodulators

Adeno II, Plasmid, oncolytic recombinant viruses

Solid tumors +/- metastat.(cervical, breast, brain, skin)

No rapid expression necessary, persistence required, low toxicity

AAV, nonviral, LentiLocal chronic or progressive (ex. CNS,

joints, eyes)

Justifications /IssuesMost suitable vector

persistence of expression of the transferred gene, minimize readministration

Chronic Metabolic (ex. OTC, Gaucher, Haemophilia, hematopoietic)

AAV, Lenti, Adeno III, r-retroviruses, repair oligo

Disease Type

Which vector for which disease categoryWhich vector for which disease category ？？

基因治疗载体的发展趋势基因治疗载体的发展趋势

•综合病毒载体与非病毒载体的优势综合病毒载体与非病毒载体的优势 ,, 构建构建杂种病毒载体杂种病毒载体•其需要达到的境界：其需要达到的境界：　　　　靶向性进入特异细胞　　　　靶向性进入特异细胞　　　　表达持久、可控、安全、高效　　　　表达持久、可控、安全、高效•商业化定做（商业化定做（ tailortailor 　　 made)made)　　　　依据特异的治疗　　　　依据特异的治疗

　　 依据不同个体依据不同个体

五、基因治疗的靶细胞五、基因治疗的靶细胞• 体细胞：体细胞：造血干细胞，皮肤成纤维细胞，

肝细胞，血管内皮细胞，淋巴细胞，肌肉细胞，肿瘤细胞

• 生殖细胞生殖细胞 在实际应用中需根据具体目的和条件进行选择，但一般最好为组织特异性细胞；较易获得、生命周期较长；在离体情况下较易被外源基因转化，离体细胞经转染和培养后再植回体内，仍较易成活

Target cells for gene transfer in ongoing Target cells for gene transfer in ongoing clinical studiesclinical studies

Disease target cellsDisease target cells Cancer tumor cells, antigen-presenting cells,Cancer tumor cells, antigen-presenting cells,

blood progenitor cellsblood progenitor cells

T cells, fibroblasts, muscle cellsT cells, fibroblasts, muscle cells

Inherited monogenic Inherited monogenic Lung epithelial cells, macrophages, skinLung epithelial cells, macrophages, skin

disorders disorders fibroblast, T cells, blood progenitor cells,fibroblast, T cells, blood progenitor cells,

antigen-presenting cells, muscle cellsantigen-presenting cells, muscle cells

Infectious diseases Infectious diseases T cells, blood progenitor cells, antigen- T cells, blood progenitor cells, antigen-

presenting cells, muscle cellspresenting cells, muscle cells

Cardiovascular diseases Cardiovascular diseases endothelial cells, muscle cellsendothelial cells, muscle cells

Rheumatoid arthritis Rheumatoid arthritis sinovial lining cellssinovial lining cells

Cubital tunnel syndrome Cubital tunnel syndrome Nerve cellsNerve cells

ApplicationApplication

•Haematopoietic stem cells – easiestHaematopoietic stem cells – easiest

•Muscle, Liver - good targetsMuscle, Liver - good targets

•Eye retina – accessible but fragileEye retina – accessible but fragile

•Brain (neurons) – v. difficultBrain (neurons) – v. difficult

•Tumours – access to interior may be Tumours – access to interior may be

difficultdifficult