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1 Gene Therapy ( 基因治療 ) 徐松錕 (Song-Kun Shyue ) Institute of Biomedical Science, Academia Sinica 中央研究院生物醫學科學研究所

2 References 1. Understanding gene therapy / N.R. Lemoine, New York, N.Y. : BIOS Scientific Publishers, 生技公司網站 :Clontech, Bio-Rad, Stratagene. 3. Annu. Rev. Genomics Hum. Genet : Adenovirus DNA : the viral genome and its expression. Edited by Walter Doerfler, Boston : M. Nijhoff, 1986

3 Gene Therapy and Transfer 基因治療與基因傳遞 傳遞一個具有治療效果的基因到特定的位置或細胞 體細胞基因治療 (Somatic gene therapy) 取代有問題的基因 增加保護功能的基因, 做短期或長期的治療 僅限於嚴重的疾病 ( 未知的副作用 ) 生殖細胞的基因治療的危險性

4 基因治療的理想與現實 理想 : 利用基因來治療疾病 取代損壞的基因 ( 遺傳性疾病 ) 利用基因產物來保護身體 打一針就 OK! 可以到特定器官做治療 無副作用 ( 理想 : 還做不到的事物 )

5 理想的基因治療載體 感染效果好, 容易製造成高濃度 : 濃度越高感染效果越好 安全性的問題 : 毒性 免疫反應越低越好 基因能表現的時間長短 (duration), 與位置或器官 (tissue specific expression) 可以控制 只感染特定的細胞或器官 可以感染分裂中與不分裂的細胞 在特定的染色體的特定位置插入 ; 避免 插入性突變 (integration mutation); 可以長時間表達, 隨細胞分裂而複製

6 現實 進入動物體, 全都不一樣! 細胞實驗與動物實驗的不同 感染效率 接受體表達量不同 ( 肝細胞 ) 生理上的差異 : 不同蛋白表達 改善中, 仍須努力 ( 創造就業機會 ) 還有很長的路要走!

7 Methods (Vectors) for Gene Transfer Non-viral vectors ( 非病毒類載體 ) Naked DNA Liposome( 微脂體 ): Lipid/DNA complex Viral vectors ( 病毒類載體 ) RNA virus ( 反轉錄病毒 ): Retrovirus, Lentivirus (HIV) DNA virus: Adenovirus ( 腺病毒 ), Adenoassociated virus (AAV) ( 腺相關病毒 ), HSV-1 ( 泡疹病毒 ) Other viral vectors

8 Ex Vivo( 體外 ) In Vivo( 體內 ) Patient

9 Naked DNA Purified DNA (Unlimited size) hydro-gel (very low efficiency): Report to improve blood supply the ischaemic limb with pvegf (Isner et al., 1996) VEGF: 血管內皮細胞生長因子 Stopped blood flow (high efficient in specific area, need to be further studied) Gene gun ( 基因槍 ) DNA vaccine, with electric pulse( 電擊 ) Limited delivery area (e.g., 血管內皮細胞 肌肉細胞 )

10 Understanding gene therapy N.R. Lemoine 1999

11 Gene Gun Helios Gene Gun DNA or RNA and gold carriers Require small amount of DNA Gene therapy DNA vaccination Bio-Rad

12 Liposomes Cationic liposome/dna complex; 帶正電脂質內包 DNA Controlled toxicity; 毒性可以控制 Unlimited DNA size;dna 大小沒有限制 Very low integration; 插入染色體機率低 Very low transfected efficient, below 1%; 基因傳遞效率低

13 帶正電油脂, 微脂體材料 Understanding gene therapy N.R. Lemoine 1999

14 Understanding gene therapy N.R. Lemoine 1999

15 Liposomes Various properties Thermo-sensitive ph-sensitive, Immunoliposome Cancer gene therapy human trails ( 癌症的基因治療臨床實驗 )

16 聯合報 5/8/2002

17 動物病毒載體 數千萬年來與動物同時演化 ; 病毒演化去感染動物 動物演化來避免被病毒感染 可以有效的感染動物細胞, 將基因物質傳遞到細胞內以表達蛋白 ; 小而精美 ; 寄生 共生的基因片段 ( 自私的基因 )

18 如何使用病毒載體? 知己知彼, 百戰百勝 了解越多越能有效控制 各蛋白的功能 : 對細胞的作用 對病毒複製的重要性 病毒基因如何調控, 去除有毒的基因 如何感染 進入細胞 表達蛋白 複製 無法自我複製的病毒 (replication deficient)

19 Good and Bad of Viral Vectors Advantages High efficiency( 高感染效率 ) Replication deficient( 無法自我複製 ) viral vectors available High titer preparation is possible for many vectors( 有些已可以高濃度製備 ) Longer expression period with DNA integration, 2 nd generation Ad and helper dependent Ad. ( 長時間的表達,6 月到 2 年 )

20 Disadvantages May exert immune response against viral vector or viral transfected cells, second administration limited ( 病毒蛋白的免疫反應限制第二次的病毒注射治療 ) Possible of integration mutation (AAV and retrovirus), risk of cancer or other genetic diseases( 插入性突變的可能危險 ). 直接注射時會到處擴散

21 Viral Vectors Retrovirus (~ 10 kb): 反轉錄病毒 Lentivirus (HIV base, ~ 10 kb) Adenovirus (5, 7, or 35 kb): 腺病毒 Adeno-Associated Virus (~ 5kb): 腺相關病毒 Herpes Simplex Virus-1 (human): 泡疹病毒 SV40, Sandi Virus, and other viruses

22 AAV

23 Retrovirus Moloney murine leukemia virus (MMLV) Clontech

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25 Retrovirus transduction system RNA virus Infect primary, dividing cells 5 LTR as promoter, 3 LTR as terminator 病毒釋放到溶液裡, 收集後再純化 ; 濃度低, 較難純化成高濃度

26 Retrovirus transduction system Integration into chromosome: provirus( 潛伏病毒 ) Stable transduction, 穩定的感染 High efficiency Non-specific integration, 插入染色體無專一位置 Requires mitosis: dividing cells only, 只能感染分裂細胞 Replication-incompetent vectors Cis elements( 必須包含的 DNA)(trans elements) LTR, RNA primer binding site and polypuring tract (PPT) Packaging signal: Ψ: for encapsulation 改進結構增加病毒產量 :Replacement of U3 in the 5 LTR: 100-fold increase in viral titer and high transcriptional activity, 許多不同的修飾

27 *no poly A signal at 3 end: replacement of U3/R *mutation in the U3 region of 3 LTR: increase biosafety

28 Replication-incompetent( 無法自我複製 )retrovirus gag and pol expressing cell Co-transfect with pvsv-g

29 Retrovirus transduction system 將病毒 DNA 分成兩部分以減少重組回原病毒的機率 (Minimize recombination to produce virus: split genome to two parts) VSV-G: G protein of vescular stomatitis virus: pseudotyped viruses: replace env protein Extreme broad host range of VSV (Bind nonspecifically to phospholipid) More stable: purified to higher titer (1000 x) Titer: > 10 9 IU/ml; 難感染幹細胞 (stem cells, 不一定在分裂中 ).

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32 Lentiviral vector Complex retrovirus (lente: slow) Infect dividing and non-dividing cells (MLV retrovirus can only infect dividing cells) Nuclear targeting without nuclear membrane disassembly Integrated into host genome HIV( 愛滋病毒 )

33 Lentiviral vector modification Replacement of HIV envelop glycoprotein with VSV-G: first useful lentiviral vector Viral particle can be concentrated (~1000-fold) by ultracentrifugation Titers: 1 x x IU/ml 293T packaging cells Production by cotransfection of several recombinant lentiviral plasmids Other lentivirus packaging systems: Feline IV ( 貓 ), Simian IV ( 猴 ), HIV-2, Equine IVA ( 馬 ), and maedi/visna virus (sheep disease; maedi which effects the lungs; visna which effects the central nervous system)

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35 Stable transduction of quiescent DC34 + CD38 - human hematopietic cells by HIV-1-based lentiviral vector Compared with MLV GFP as reporter Non-dividing DC34 + cells: 45.5% DC34 + CD38 - cells in G0: 12.4% Stable transfection of DC34 + CD38 - cells for over 15 weeks Both MLV and lentiviral vectors efficiently transduced cytokine-stimulated CD34 + cells PNAS 96:2988, 1999.

36 Adenovirus (Ad, 腺病毒 ) Well studied Double stranded DNA ~36 kb, 70~100 nm 47 characterized serotypes Ad5 and Ad2 are less oncogenic; less safety concerns Infect both dividing and nondividing cells

37 Adenovirus ~100% seropositive of Ad5 in Taiwan, 台灣幾乎每人都被感染過 : 治療時免疫反應 High transfer efficiency in a wide variety of cell types and tissues; 可感染許多不同種類的細胞及器官 Very high titer: up to 10E12 PFU/ml Large scale production available Low integration frequency,10e-6

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41 ptp ψ POL ITR ssdbp E1 Late genes E2 E3 E4 ITR E1 Late gene expression Packaging

42 Recombinant Ad construction (First-generation, E1 and/or E3 deleted) 100/0 ϕ pbrx E1 pjm17 (0~1.3%) ITR ϕ p cdna pa pad-pgk-cdna pbr322 Ad5 (9.25%-16%) pbr cells (0% ~ 12% of Ad5); Homologous recombination ITR ϕ p cdna pa pa rad-cdna [E1 and E3 (partial) deleted] ITR

43 PNAS 95: (He,.., Vogelstein)

44 ptp ψ POL ITR E1 ssdbp Late genes E2 E3 E4 ITR E1-like activity from infected cell Late gene expression Immune response

45 Second-Generation Ad 第二代腺病毒載體 Remove E2a, E2b, or one of the ORF of E4 plus the E1/E3 Less immune response 增長基因表現時間從 2 到 6 週到 2 到 6 月以上

46 ptp ψ POL ITR ssdbp E1 Late genes E2 E3 E4 ITR E1-like activity? 延長基因表現時間降低免疫反應 Late gene expression Immune response

47 Helper Dependent Ad Gutless Ad or Gutted Ad( 無腸腺病毒 ) Recombinant Ad without viral gene: 沒有病毒基因 Only cis elements included, ITR and packaging signal Helper Ad needed to produce HD virus Helper Ad contamination (0.01%: ) Problems with large-scale production: 大量製造有問題需解決

48 Cre/loxP: bacteriophage P1 Proc. Natl. Acad. Sci. USA Vol. 93, pp , 1996

49 E2B

50 First generation Ad E1 plus E3 deletion: 5 ~ 7 kb Stronger immune response of E3 deleted Ad 些許的腺病毒基因表現會造成免疫反應, 縮短基因表現期限 Second generation Ad Part of E2 and/or E4 deleted 需要一個互補的裝備細胞 幾乎沒有或非常低的病毒蛋白產生, 延長基因表現期限至 6 月以上, Helper dependent Ad (Gutless or Gutted) Containing only cis elements, ITR and Y, needs helper virus Very large capacity, up to 35 kb 沒有病毒蛋白表現, 非常低的免疫反應 ( 幾乎沒有 ) 不同態別的病毒可以做重複感染, 而免除免疫的排斥性 可以表現一年以上, 低濃度可以維持將近兩年 ( 老鼠實驗 )

51 Gene Therapy (1999) 6, 1565:573 Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration RJ Parks 1,2,3, CM Evelegh 1 and FL Graham 1,2 Departments of 1 Biology and 2 Pathology, McMaster University, 1280 Main Streeet West, Hamilton, Ontario, L8S 4K1, Canada

52 Hybrid vectors 組合型 ( 子母彈 ) 病毒 Nat. Biotechinol. 15,

53 Ad Gene Therapy on Trial 17 September 1999: Dr. J. Wilson, Univ. Penn. in Phil. Jesse Gelsinger (OTC deficiency) died with 38 trillion Ad particles, directly liver injection E1 and E4 deleted Ad( 第二代病毒 ) Died from a massive immune response to Ad vector ( 死於強烈的免疫反應 ) High and sustained levels of interleukin-6; Dose related IL-6 increase 2 to 4 hours after injection Precursor of red blood cells had been wiped out: maybe due to a preexisting parvovirus infection

54 Ad Gene Therapy on Trial Very low CAR in human liver compared to mouse liver, misleading of rodent model? Much higher dose needed for human liver gene transfer, low gene transfer rate (<1%) 25 cancer patients (R. Warren) with as high dose with no such problems Empty capsids appear to be immunogenic like intact virus Patient was very sick and not qualified for gene therapy

55 Clinical trials Over 400 clinical trials have been conducted or are underway More than 6000 patients > 70% are cancer related Immuno-modulation, tumor associated antigen, tumor vaccine: 免疫調節與癌症疫苗 Suicide genes and prodrugs: 自殺基因 Tumor suppressor genes: 抑癌基因 Anti-angiogenesis genes: 抑制血管生成 Conditional replicative adenovirus: 有條件的複製病毒

56 Adenovirus E1b-55kD cancer gene therapy E1b: form complex with p53 protein, inactivation Essential for efficient viral replication: transforming and antiapoptosis activities Suggested to replicate preferentially in p53- and some p53+ tumor cell lines but to be attenuated in primary cultured cells (Heise et al., Nat. Med. 1997) Clinical trial: 2x10 12 particles, 24 times [Gene Therapy (2001) 8, ] Combined with chemotherapy Fever 13 % effective rate [Gene Therapy (2001) 8,.89-98]

57 ptp ψ POL ITR ssdbp E1b Late genes E2 E3 E4 ITR E1 Specific promoter: only replicate in targeted cells Kill cells Late gene Packaging expression 只能在缺乏 p53 活性的細胞複製

58 CV706 with PSA promoter Phase I clinical trial [CANCER RESEARCH 61, , 2001] Prostate-specific antigen (PSA): a widely used marker for the diagnosis and management of prostate cancer. Minimal enhancer/promoter of PSA derives the E1A gene, CV706. Intraprostatic injection: 1x10 11 ~1x10 13 particles Side effects: Fever, Amnesia, Hematuria Lower PSA levels

59 Enzymes for cancer gene therapy Bystander-killing effect: A percentage of the tumor cells needs to be transduced for eradication of a tumor Can not treat secondary, distal tumors that are not directly accessible by gene transfer

60 Prodrug: GCV/TK system Suicide genes: HSV-thymidine kinase (tk) Treat with ganciclovir; phosphorylated to ganciclovir triphosphate then blocks the DNA synthesis machinery and kills the cells Phase III trail: Mouse producer cells making vector articles carrying the HSV-tk gene are inoculated into residual tumor and peritumor areas following tumor resection After 7 days, the patient is treat with ganciclovir

61 GCV/TK Tumor cells can be killed by: Direct effect on the transduced tumor cells Bystander effect through cell gap junctions and kill neighboring cells Local inflammatory effect caused by the injected mouse cells Systemic immune response Mutated HSV-tk to generate more sensitive tk Random oligonucleotide mutagenesis Nanomolar IC50 (30 µm for WT) 10 fold lower dose of GCV 35 fold increase in the Km for thymidine Liver injury causes by systemic delivery of GCV

62 Promoters ( 控制基因表現的地方 ) Midkine promoter-based Ad vector for pediatric solid tumors MK is a heparin-binding GF, highly expressed in many human malignant tumors Not expressed in liver (Ad accumulated in liver) Wilms tumor treatment: Ad-TK/GCV did not induce liver toxicity - Cancer Res. 60:4305, 2000 VEGF promoter against hypoxic tumor cells( 缺氧的癌細胞 ) Hypoxia response element of the mouse VEGF Up-regulated expression in poorly vascularized regions

63 Heat-induced gene expression: Targeted gene therapy Hsp70 promoter, 400 bp 500 to 1000 fold induction of GFP (in vitro) at 42 C for 30 min. TNFαand mil12: Reduced tumor volume, 42.5C 40 min each, 7 days apart. [Cancer Res. 60:3435, 2000]

64 VEGF inhibition 抑制血管生成 Over express extracellular domain of VEGF receptor in a remote organ Ad-VEGF-ExR: flt-1 fused to the Fc portion of human IgG. Soluble receptor secreted from Ad-VEGF-ExR infected cells bound to VEGF and inhibited VEGF induction in EC. Injected into skeletal muscle Receptor can be detected in blood for 3 weeks, tumor growth ceased after 10 days and size declined thereafter.

65 Cardiovascular gene therapy 心血管疾病 ( 保護 增進血管生成 ) Angiogenesis gene therapy VEGF, FGF-1, FGF-2, Endothelial progenitor cells (EPCs) Bone marrow derived EPCs Stimulate hematopoietic progenitor cells with GM- CSF

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67 Vasoprotective molecule gene therapy Restenosis( 血管再狹窄 )prevention 粥狀動脈硬化 gene therapy 胸腔性高血壓 (PGI 2 ) VEGF, EGF,, growth factors enos COX-1 and/or PGIS Antioxidants: SOD, catalase, GPX,

68 Prostacyclin, PGI 2 ( 攝護腺環素 ) Vasoprotective molecule Powerful vasodilator( 血管放鬆 ) Potent naturally occurring inhibitor of platelet aggregation( 抑制血小板凝固 ) Disperse existing aggregates Inhibition of monocyte and endothelium interaction ( 抑制單核球與內皮細胞接觸 ) Physiological antagonist of thromboxane A 2 (TXA 2 )( 凝血因子的拮抗劑 )

69 Biosynthetic pathway of PGI 2 Phospholipid Phospholipases A 2 Arachidonic Acid (AA) PGH 2 COX (PGG 2 ) PGIS TXAS PGI 2 TXA 2 PGE 2, PGD 2, PGF 2α 6-keto-PGF 1α

70 Figure 3. Movat's pentachrome stain of porcine carotid arteries removed 10 days after balloon-catheter injury. In the Ad-COX-1 treated artery, the lumen is patent and free of thrombus (A), and there is minimal neointimal formation (B). In the vehicle-treated artery, the lumen is nearly filled with thrombus (C), and there is neointimal formation (D). Arrow indicates disrupted internal elastic lamina. L denotes lumen; I, intimal and neointimal formation; M, media; and T, thrombus. Bar=0.25 mm for A and C; mm for B and D. (Circulation, 1996;93:10-17)

71 The Future Can be injected Target to specific cells; 鎖住特定器官 Safe and efficient gene transfer: high percentage of transfection; 安全 高效率 Insert themselves into appropriate regions of the genome ( or persist as stable episomes); 在特定位置插入染色體 Regulated either by administered agents or by the body s own physiological signals; 由病人的身體情況或藥物來控制基因的表達 Cost-effective to manufacture; 經濟上的考量