Therapy of the Hereditary Disease Medical Genetics Therapy

Therapy of the Hereditary Disease 遗传病的治疗

Medical Genetics Therapy of the Hereditary Disease 遗传病的治疗

Strategy of hereditary disease Therapy 遗传病治疗的策略 • Treatment on gene level 基因水平的治疗 • Treatment on enzyme and protein level 酶和蛋白质水平的治疗 • Treatment on metaboilic level • Treatment on clinical level 代谢水平的治疗 临床水平治疗

Principles of therapy 治疗原则 • Operative therapy • Pharmacotherapy • Dietetic therapy • Gene Therapy

Section 1 Operative Therapy 手术治疗 v Operation correction 矫正畸形 v Tissue and organ transplant 器官和组织移植

Section 2 Pharmacotherapy 药物治疗 v prenatal treatment v Pre-symptomatic treatment v symptomatic treatment

Prenatal treatment 出生前治疗 drug treatment can carry out in prior to birth. for example, methylmalonic aciduria, 甲基丙二酸症 give vitamin B 12 for pregnant women

Pro-symptomatic treatment 症状前治疗 For example: • thyroid hypofunction in newborn Give thyroxine preparation in presymptom • phenylketonuria; PKU in newborn screening Give Dietetic therapy

symptomatic treatment 现症患者治疗 Rules: vto remove Remnant v. Supply deficiency

Supply deficiency 补其所缺 Suppy estrogenic hormone for turner syndrome • Suppy growth hormone for hypophyseal dwarf • Suppy steroid hormone for congenital adrenal hyperplasia • Suppy Insulin for diabetic •

Methods of remove Remnant 去其所余 • chelating agent 应用螯合剂 • promote eccritic 应用促排泄剂 • metabolic inhibitor 利用代谢抑制剂 • Plasmapheresis or plasmafilter 血浆置换或血浆过滤 • equilibrium depletion 平衡清除法

Enzyme therapy 酶疗法 • enzyme induction 酶诱导治疗 • Enzyme replacement therapy 酶补充疗法

Section 3 Dietetic Treatment 饮食疗法 Rules: • Prenatal treatment • Symptomatic treatment

Section 4 Gene Therapy 基因治疗 Gene therapy is a technique for correcting defective gene responsible by transferring of a functional normal copy of the gene into cells.

Strategy of Gene Therapy 基因治疗的策略 v Gene correction 基因修正 specific correction of mutant gene without other changes in target genome. v Gene replacement 基因替代 remove whole variation gene, to substitute with normal gene.

v Gene augmentation 基因添加(增强) modifying content or expression of defective cells by introducing foreign normal gene sequences. v genic suppression or gene inactivation 基因抑制和基因失活 Interfereing and inhibiting gene expression of defective cells by introducting foreign gene.

Pathway of Gene Therapy 基因治疗的途径 O Somatic gene therapy 体细胞基因治疗 O Germ cell gene therapy 生殖细胞基因治疗

Qualification of Gene Therapy 基因治疗须具备条件 v Understanding of the disease process v Structure/function of gene to be introduced v Efficient delivery of gene v Control of gene expression v Prevention/control of immune responses v Animal model and assessment of function v Clinical trial

Techniques of gene therapy 基因治疗的技术方法 • To obtain the purpose gene • Gene delivery methods • Selection of target cell

To obtain the purpose gene 目的基因的获得 • Cloning gene 基因的克隆化 • Segregation of genomic 基因组中分离 • Artificial synthesis 人 合成 • PCR amplification PCR扩增

Gene Delivery Method 基因的转移方法 v In vivo 在体转移 v Ex vivo 回体转移

v In vivo O The 在体转移 genetic material is transferred directly into the body of the patient

v In vivo O More or less random process; small ability to control; less manipulations O Only available option for tissues that can not be grown in vitro; or if grown cells can not be transferred back

v Ex vivo 回体转移 Cells removed from body O The genetic material is transferred into the cells O O Cells cultured in vitro O Cells returned to the body

v Ex vivo O Controlled process; transfected cells are selected and expanded; more manipulations O Cells are usually autologous; they are then returned back to the patient

Gene Delivery Method 基因的转移方法 • Physical Methods • Chemical Methods • Membrane Fusion • Homologous Recombination • Virus Vectors

Physical Methods 物理方法 v Direct injections v Electroporation v Microparticle bombardment (gene gun)

Chemical Methods 化学方法 v Coprecipitation with calcium phosphate O Transfer efficiency: 1% ~0. 1%

Membrane Fusion 膜融合法 v Artificial liposomes v Erythrocyte ghosts v Microcell v Spheroplast

Membrane Fusion J Advantages Stable complex v Can carry large sized DNA v Can target to specific cells v Does not induce immunological reactions v L Disadvantages Low transfection efficiency v Transient expression v Inhibited by serum v

Homologous Recombination 同源重组法 Xba I Bst I Xba I 11. 1 Kb Xba I Bst I neo Xba I 16. 5 Kb neo 13. 1 Kb Bst I 7. 7 Kb Xba I

Virus mediated gene transfer 病毒介导转移法 O More efficient than other methods O Can infect every cell in a target population

Retrovirus, RV 逆转录病毒 J Advantages High transfection efficiency: 100% v Integration and persistent expression v Wide host cells v L Disadvantages Can only accomodate 7 kb maximum v Random insertion, influence host gene v Potential for carcinogenesis v Only infecting dividing cells v

Retrovirus, RV v Moloney murine leukemia virus (Mo-MLV) ψ U 3 R U 5 5’ LTR U 3 R U 5 gag pol env LTR 3’

Retrovirus, RV v Moloney murine leukemia virus (Mo-MLV) ψ U 3 R U 5 5’ LTR Exogenous gene U 3 R U 5 LTR 3’

Retrovirus, RV v Packaging cell line ψ- Helper virus Packaging cell RNA ψ+ Packaging protein 病毒颗粒 RNA 重组病毒颗粒

Adenovirus, AV 腺病毒 J Advantages v v Can carry large sized DNA No insertional mutagenesis Infecting dividing and nondividing cells Wide host cells L Disadvantages Transient expression v Highly immunogenic v

Adenovirus, AV 腺病毒 v Packaging cell line DNA Cell 293 E 1 E 3

Adenovirus, AV v Packaging cell line DNA E 3 Cell 293

Adeno-Associated Virus, AAV 腺病毒相关病毒 J Advantages v v Integration (chr 19) and persistent expression No insertional mutagenesis Infecting dividing and nondividing cells Safe L Disadvantages Size limitation, 4. 9 kb v Immunogenic v Dependent on a helper virus for replication v

Herpes Simplex Virus, HSV 单纯疱疹病毒 J Advantages v ds. DNA viruses that infect a neurons v Wide host cells v Infecting dividing and nondividing cells L Disadvantages v Cell toxicity

Vaccinia Virus, VV 痘苗病毒 J Advantages v Easy v Safe L Disadvantages v Highly immunogenic

Selection of target cell 靶细胞的选择 Qualification of target cell 靶细胞须具备条件 靶细胞 v easy draw the materials from body, no danger in autoplastic transplantation v Easy cultivation and delivery of gene v No Influence gene expression due to cell senescence in cell transplantation v high speed vascularize after embedding cell

commonly used cell • bone marrow stem cell 骨髓干细胞 • skin flbroblast 皮肤成纤维细胞 • peripheral blood lymphocyte 外周血淋巴细胞 • hepatic cell 肝细胞 • vascular endothelial cell 血管内皮细胞 • muscle cell 肌细胞

clinical application of gene therapy • gene therapy of hereditary diseases • gene therapy of tumors

gene therapy of hereditary diseases v ADA gene therapy (MIM 102700) 腺苷脱氨酶缺乏症 O Severe combined immunodeficiency (SCID) caused by mutation in adenosine deaminase gene in T cells.

v ADA gene therapy O French Anderson (NIH) O September 14, 1990 O Retrovirus (Mo-MLV) LTR ADA SV 40 neo LTR

Gene Therapy Trials Ashanti was the first patient to be treated with gene therapy. Ashanti de Silva Injections repeated 7 times in first 10. 5 months v ADA: 1% 25% v

Gene Therapy Trials v Hemophilia B O Xue Jinglun (Fudan university) O 1991, Retrovirus O Skin fibroblast O Factor IX: O 2003, r. AAV 2 -h. F. IX 5%

Antisense Nucleic Acid 反义寡核苷酸技术 A nucleic acid molecular with a nucleotide sequence complementary to a specified m. RNA. v Antisense RNA expression vector v Direct injection of antisense RNA/DNA v Antisense RNA delivered by liposome v Others

Suicide Gene “自杀基因”疗法 A gene that transforms a nontoxic form of a drug (pro-drug) into a toxic substance. v herpes simplex virus thymidine kinase gene (HSV 1 TK)

Suicide Gene v By-stander effect The toxic substance (transform from pro-drug) diffuses out into wide surrounding area and kills other tumour cells. Ø 1992, HSV-TK/GCV (brain tumor)

Suicide Gene v By-stander effect

Suicide Gene v By-stander effect

Suicide Gene v By-stander effect

Suicide Gene v By-stander effect

MDR Gene 多药耐药基因疗法 v Multidrug-Resistance Gene

Tumor Suppressor Gene 抑癌基因疗法 v Anti-oncogene v Gene replacement therapy p. RB, p 53, p 16, p 21, p 27 A method for replacing a mutated or missing gene (usually a tumor suppressor gene) that serves to keep cell growth and division under control with a normal copy of that gene.

Gene Replacement Therapy

Gene Replacement Therapy Apoptosis

Cytokine 细胞因子疗法 Ø 1984 -1987, lymphokine activated killer cell (LAK) Ø 1986, tumor infiltrate lymphocytes (TIL)/IL-2 Ø 1991, tumor necrosis factor (TNF) Ø Vascular endothelial cell growth factor (VEGF)

Problem of Gene Therapy v Persistent expression v High-efficiency expression v Safe

Gelsinger Case v 1999: Death of Jesse Gelsinger in Penn OTCD trial O OTCD: orthinithin transcarbamylase (a urea cycle enzyme) deficiency. O OTCD: X-linked, 1/40 000 birth v Jesse Gelsinger: 18 yr-old from Arizona, died on 9/17/1999, 4 day after gene transfer.