最新昆医分生基因治疗1不考试精品ppt课件教案资料

1、昆医分生基因治疗1不考试v 恶恶 性性 肿肿 瘤瘤v 心心 血血 管管 疾疾 病病v 遗遗 传传 病病v 感感 染染 性性 疾疾 病病威胁人类健康的重大疾病威胁人类健康的重大疾病基因治疗对象基因治疗对象基因治疗概况基因治疗概况基因治疗药与基因工程药基因治疗药与基因工程药DNAPROTEINRNA基因工程药基因工程药基因治疗药基因治疗药基因治疗概况基因治疗概况治疗基因治疗基因载体载体靶细胞靶细胞基因治疗制品基因治疗制品基因治疗要素基因治疗要素基因治疗概况基因治疗概况最理想的方式最理想的方式目前的主要方式目前的主要方式基因治疗策略基因治疗策略基因治疗概况基因治疗概况l 基因替代（基因替代（Gene

2、 Replacement）l 基因修复（基因修复（Gene Modification）l 基因添加（基因添加（Gene Addition）l 基因失活（基因失活（Gene block）v 体细胞基因治疗体细胞基因治疗 v 生殖细胞基因治疗生殖细胞基因治疗基因治疗种类基因治疗种类基因治疗概况基因治疗概况体外基因治疗体外基因治疗(Ex vivo)：从病人体内获取细胞，体：从病人体内获取细胞，体外培养并进行基因转移后，重新输入患者体内外培养并进行基因转移后，重新输入患者体内前列腺癌细胞转前列腺癌细胞转染细胞因子基因染细胞因子基因后回输到患者体后回输到患者体内，肿瘤转移灶内，肿瘤转移灶被被T T细胞及

3、细胞及NKNK细细胞附着。胞附着。基因治疗途径基因治疗途径基因治疗概述基因治疗概述体内基因治疗体内基因治疗(In vivo)：直接将基因或改造后的：直接将基因或改造后的病 毒病 毒 (插 入 目 的 基 因插 入 目 的 基 因 )导 入 人 体 患 病 组 织 中导 入 人 体 患 病 组 织 中携携p53p53基因的重组腺病毒导入人基因的重组腺病毒导入人体肿瘤组织引起肿瘤细胞裂解体肿瘤组织引起肿瘤细胞裂解基因治疗途径基因治疗途径基因治疗概况基因治疗概况生物传导的方法Adenoviruses enter cells by receptor-mediated endocytosis: Bind

4、ing of viral coat protein to a specific receptor on the plasma membrane of cells is followed by endocytosis, a process in which the plasma membrane invaginates and then pinches off to form an intracellular vesicle (endosome). Subsequent vesicle disruption by adenovirus proteins allows virions to esc

5、ape and migrate towards the nucleus where viral DNA enters through pores in the nuclear envelope. Adapted from Curiel (1994) with permission from the New York Academy of SciencesGene transfer via the receptor-mediated endocytosis pathway. The negatively charged plasmid DNA can bind reversibly to the

6、 positively charged polylysine attached to the transferrin molecule. During this process, the DNA is condensed into a compact circular toroid with the transferrin molecules located externally and free to bind to cell surface transferrin receptors. Following initial endosome formation, a portion of t

7、he endocytosed conjugates can migrate to the nucleus, although a very significant fraction is alternatively transferred to lysosomes where the DNA is degraded. The efficiency of transfer can be increased by the further refinement of coupling an inactivated adenovirus to the DNA transferrin complex:

8、following endocytosis and transport to lysosomes, the added adenovirus causes vesicle disruption , allowing the DNA to avoid degradation and to survive in the cytoplasm. Adapted from Curiel (1994) with permission from The New York Academy of Sciences. In vivo liposome gene delivery. (A) and (B) Stru

9、cture of liposomes. Liposomes are synthetic vesicles which can form spontaneously in aqueous solution following artificial mixing of lipid molecules. In some cases, a phospholipid bilayer is formed, with hydrophilic phosphate groups located on the external surfaces and hydrophobic lipids located int

10、ernally (left). In other cases there is a multilamellar lipid envelope. Anionic liposomes have a negative surface charge and when the lipid constitutents are mixed with negatively charged DNA molecules (see panel C below), the DNA is internalized. Cationic liposomes have a surface positive charge an

11、d DNA molecules bind to the surface of liposomes. (C) Use of liposomes to transfer genes into cells. This figure illustrates the use of anionic liposomes to transfer internally located DNA into cells. The plasma membranes of cells are fluid structures whose principal components are phospholipids, an

12、d so mixing of cells and liposomes can result in occasional fusion between the lipid bilayer of the liposome and the plasma membrane. When this happens the cloned genes can be transferred into the cytoplasm of a cell, and can thence migrate to the nucleus by passive diffusion through the pores of th

13、e nuclear envelope. Note that, in practice, cationic liposomes have been more widely used for transferring DNA into cells. 基因的整合途径Exogenous genes that integrate into chromosomes can be stably transmitted to all daughter cells, unlike episomal (extrachromosomal) genes. The figure illustrates two poss

14、ible fates of genes that have been transferred into nucleated cells. Gene therapy involving chromosomal integration of exogenous genes offers the possibility of continued stable expression of the inserted gene and a permanent cure, but carries certain risks, notably the possibility that one of the i

15、ntegration events may result in cancer . By contrast, episomal genes which do not integrate but replicate extrachromosomally (under the control of a vector origin of replication) may not segregate to all daughter cells during subsequent mitoses. As a result, this type of approach has been particular

16、ly applied in gene therapies where the target tissue consists of nondividing cells 。 Ex vivo gene augmentation therapy for adenosine deaminase (ADA) deficiency. Note that identification of suitably transformed cells is helped by having an appropriate selectable marker in the retrovirus vector, such

17、as a neoR gene which confers resistance to the neomycin analog G418 . Following infection, the target cells can be cultured in a medium containing G418 to select for the presence of retroviral sequences, and then assayed by PCR for the presence of the inserted ADA gene. Suitable ADA+ cells can then

18、be expanded in culture before being reintroduced into the patient. Examples of gene therapy trials for inherited disordersDisorder Cells altered Gene therapy strategyADA deficiencyT cells and hemopoietic stem cellsEx vivo GAT using recombinant retroviruses containing an ADA geneCystic fibrosisRespir

19、atory epitheliumIn vivo GAT using recombinant adenoviruses or liposomes to deliver the CFTR geneFamilial hypercholesterolemiaLiver cellsEx vivo GAT using retrovirus to deliver the LDL receptor gene (LDLR)Gauchers disease glucocerebrosidaseHemopoietic stem cellsEx vivo GAT using retroviruses to deliv

20、er the gene (GBA)GAT, gene augmentation therapy.v 直接基因水平治疗；直接基因水平治疗；v 副作用小；副作用小；v 技术含量高，生产成本低；技术含量高，生产成本低；v 病毒液性质稳定；病毒液性质稳定；v 生产简单可靠，无环境污染生产简单可靠，无环境污染。基因治疗的优势基因治疗的优势基因治疗概况基因治疗概况v 1990年年,美国美国W.F.Anderson v 严重复合免疫缺陷病严重复合免疫缺陷病SCID v 4岁女孩，岁女孩， Ashanti De silva v 13岁，正常生活岁，正常生活第一项体细胞基因治疗临床试验第一项体细胞基因治疗临床试

21、验基因治疗概况基因治疗概况两项新措施：两项新措施：（1 1）制定了基因治疗临床试验监）制定了基因治疗临床试验监查计划；（查计划；（2 2）定期开办基因治疗）定期开办基因治疗安全性专题研讨会。安全性专题研讨会。并否决了一项由少数激进分子提并否决了一项由少数激进分子提出的出的“停止基因治疗临床试验停止基因治疗临床试验”议案。议案。19991999年年9 9月月1717日，日，1818岁的岁的Jesse Gelsinger参加宾夕法尼亚大学治参加宾夕法尼亚大学治疗鸟氨酸甲酰氨基转移酶（疗鸟氨酸甲酰氨基转移酶（OTCOTC）缺乏症临床试验后死亡。）缺乏症临床试验后死亡。美国美国Gelsinger事件事

22、件调查结果：调查结果：应只包括女性；血氨值已偏高；应只包括女性；血氨值已偏高；门静脉注射大剂量的重组腺病毒门静脉注射大剂量的重组腺病毒（1X101X101414VPVP）调查结论：调查结论：临床试验存在违规行为，而与进临床试验存在违规行为，而与进行的基因治疗的制品本身无直接行的基因治疗的制品本身无直接关系。关系。基因治疗概况基因治疗概况巴黎巴黎Necker Enfants Malade Necker Enfants Malade 医院采用逆转录病毒载体携带医院采用逆转录病毒载体携带IL2RG/cIL2RG/c基因治疗重症联合免疫缺陷病（基因治疗重症联合免疫缺陷病（SCID-X1SCID-X1）

23、的临床试验；）的临床试验；1999.101999.10起，起，9/109/10名儿童的免疫力得到了恢复；名儿童的免疫力得到了恢复； 2002.10-2005.12002.10-2005.1， 3/93/9例分别在近例分别在近3 3年及年及5 5年后相继表现出年后相继表现出T T细胞白血病的症状细胞白血病的症状法国法国“气泡儿童气泡儿童”事件事件法国政府立即终止了该项试验。美国法国政府立即终止了该项试验。美国FDAFDA暂停了暂停了2727项同类以逆转录项同类以逆转录病毒为载体基因治疗临床试验。英国则表示不会停止这类基因治疗病毒为载体基因治疗临床试验。英国则表示不会停止这类基因治疗临床试验。意大利的米兰，类似的临床试验仍在进行。临床试验。意大利的米兰，类似的临床试验仍在进行。 研究表明研究表明IL2