1、基于病毒進入機制的抗HIV造血干/祖細胞基因治療與AIDS病人免疫重建01INTRODUCTIONAIDS/HIV1981年第一次被診斷破壞免疫系統機會性感染及癌癥不經治療平均存活期9-10年感染免疫系統細胞降低CD4+T細胞的數量到一個臨界水平后，人體的細胞免疫將會喪失Picture: https:/wiki/HIV/AIDS#/media/File:HI-virion-structure_en.svg01INTRODUCTION免疫重建外周血液CD4+細胞計數的恢復顯示患者艾滋進程的一項重要指標不等價于病毒控制Cenderello G, De M A. Discordant respons

2、es to cART in HIV-1 patients in the era of high potency antiretroviral drugs: clinical evaluation, classification, management prospects.J. Expert Review of Anti-infective Therapy, 2015, 14(1):1-12.01INTRODUCTIONHAART高效抗逆轉錄病毒治療顯著提升患者的生存率和生存質量病毒載量能被抑制到無法檢測的水平缺點需要終生保持，很難堅持副作用治療的花費1Cenderello G, De M A.

3、 Discordant responses to cART in HIV-1 patients in the era of high potency antiretroviral drugs: clinical evaluation, classification, management prospects.J. Expert Review of Anti-infective Therapy, 2015, 14(1):1-12.2Yoshimura K. Current status of HIV/AIDS in the ART era.J. Journal of Infection & Ch

4、emotherapy Official Journal of the Japan Society of Chemotherapy, 2016.01INTRODUCTION造血干/祖細胞（HSPC）移植自我更新、增殖并分化為成熟免疫細胞T淋巴細胞，巨噬細胞和樹突細胞終生持續提供抗HIV免疫細胞柏林病人CCR5缺陷的異源CD34+外周血干細胞移植超過8年沒有被檢測到HIV1Pernet O, Yadav S S, Dong S A. Stem cell-based therapies for HIV/AIDS J. Advanced Drug Delivery Reviews, 2016, 103

5、:187-201.2Htter G, Nowak D, Mossner M, et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation.J. New England Journal of Medicine, 2009, 360(7):692-698.01INTRODUCTIONHIV進入機制病毒和宿主細胞表面結合，Env與CD4結合CD4的結合造成了Env的構象改變，引起了輔助受體的參與gp41亞基使病毒和宿主細胞膜融合Wilen C B, Tilton J C, Doms R W. Mo

6、lecular Mechanisms of HIV EntryJ. Advances in Experimental Medicine & Biology, 2012, 726(1):223-242.Pernet O, Yadav S S, Dong S A. Stem cell-based therapies for HIV/AIDS J. Advanced Drug Delivery Reviews, 2016, 103:187-20101INTRODUCTION病毒嗜性病毒感染的細胞類型R5嗜性的HIVX4嗜性的HIV02CCR5HIV感染的主要輔助受體保護細胞免受R5型HIV感染修飾不

7、會對人類造血系統和免疫系統造成明顯的不良反應02CCR5AAV6（腺病毒相關病毒）+ZFN（鋅指核酸酶） mRNAHSPC（造血干/祖細胞）高水平的基因組編輯被移植到免疫缺陷小鼠后出現了長期的多譜系分化Wang J, Exline C M, Declercq J J, et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using zinc finger nuclease mRNA and AAV6 donorsJ. Nature Biotechnology, 2015, 33(12)

8、:1256-1263.02CCR5In vitroGFP表達盒插入CCR5轉入外周血來源的CD34+細胞ZFN mRNA進行轉染20%基因修飾Wang J, Exline C M, Declercq J J, et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using zinc finger nuclease mRNA and AAV6 donorsJ. Nature Biotechnology, 2015, 33(12):1256-1263.In vivo胎肝CD34+細胞進行基

9、因組編輯移植進新生免疫缺陷小鼠第8周,第12周,第16周取外周血，第16周取骨髓和脾臟02CCR5Wang J, Exline C M, Declercq J J, et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using zinc finger nuclease mRNA and AAV6 donorsJ. Nature Biotechnology, 2015, 33(12):1256-1263.Fig. 1 Lineage analysis of human cells in

10、 NSG mice engrafted with genome edited HSPCs02CCR5基于miRNA的載體在T細胞中有效抑制CCR5在體外環境中阻止HIV感染移植經過預選的轉染后CD34+細胞能保持顯著降低的HIV病毒載量Myburgh R, Ivic S, Pepper M S, et al. Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice.J.

11、 Journal of Virology, 2015, 89(13):6761-72.02CCR5mirGE直接移植mirGE轉導的CD34+細胞R5嗜性的HIV感染GFP陽性CD4+T細胞比例和絕對數量增加整體CD4+T細胞數量未增加HIV復制的比率無明顯區別Myburgh R, Ivic S, Pepper M S, et al. Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Huma

12、nized Mice.J. Journal of Virology, 2015, 89(13):6761-72.02CCR5Myburgh R, Ivic S, Pepper M S, et al. Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice.J. Journal of Virology, 2015, 89(13):6761-72.Fig. 2 Homeos

13、tatic expansion of GFP-positive CD4 T cells in R5 knockdown mice despite sustained R5-tropic (YU-2) HIV infection02CCR5Myburgh R, Ivic S, Pepper M S, et al. Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice.J

14、. Journal of Virology, 2015, 89(13):6761-72.Fig. 3 Sustained HIV load inhibition in FACS-sorted R5 knockdown mice02CCR5Myburgh R, Ivic S, Pepper M S, et al. Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice.J

15、. Journal of Virology, 2015, 89(13):6761-72.Fig. 4 Increased engraftment and central memory T cells in blood and spleens of FACS-sorted R5 knockdown mice.02CCR5ZFN臨床研究永久失活自體CD4+T細胞中的CCR5基因對12名接受HAART治療的慢性HIV患者進行自體CD4+T細胞輸注Tebas P, Stein D, Tang W W, et al. Gene Editing of CCR5 in Autologous CD4 T Ce

16、lls of Persons Infected with HIVJ. New England Journal of Medicine, 2014, 370(10):901-10.02CCR5Tebas P, Stein D, Tang W W, et al. Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIVJ. New England Journal of Medicine, 2014, 370(10):901-10.Fig. 5 Lymphocyte Values02CCR5Tebas P,

17、 Stein D, Tang W W, et al. Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIVJ. New England Journal of Medicine, 2014, 370(10):901-10.Fig. 6 CCR5-Modified CD4 T Cells in the Circulation02CCR5Tebas P, Stein D, Tang W W, et al. Gene Editing of CCR5 in Autologous CD4 T Cells of

18、 Persons Infected with HIVJ. New England Journal of Medicine, 2014, 370(10):901-10.基因修飾的T細胞在所有時間點都能被檢測到CCR5修飾的CD4+T細胞在所有直腸活檢標本中被檢測到02CCR5Tebas P, Stein D, Tang W W, et al. Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIVJ. New England Journal of Medicine, 2014, 370(10):901

19、-10.Fig. 7 Changes in Viremia during Treatment Interruption03CXCR4CXCR4HIV有可能從CCR5抑制中逃逸CCR5修飾不能抑制X4嗜性的HIV03CXCR4Didigu C A, Wilen C B, Wang J, et al. Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection.J. Blood, 2014, 123(1):61-9

20、.ZFN同時修飾ccr5和cxcr4CCR5和CXCR4共修飾細胞能夠實現對R5嗜性HIV和X4嗜性HIV感染的抑制分組第一組第二組第三組處理空白對照只轉導R5-ZFN同時進行R5和X4-ZFN轉導03CXCR4Didigu C A, Wilen C B, Wang J, et al. Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection.J. Blood, 2014, 123(1):61-9.Fig.

21、8 Dual R5- and X4-ZFN treatment confers protection in vivo.03CXCR4Didigu C A, Wilen C B, Wang J, et al. Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection.J. Blood, 2014, 123(1):61-9.Fig. 9 Cells lacking both ccr5 and cxcr4 foll

22、owing R5/X4-ZFN treatment have a survival advantage in vivo in the presence of HIV.03CXCR4Limitation參與體內多種生理機制HPSC上CXCR4的敲除可能有不利影響一般只使用于成熟CD4+T細胞04C46HIV膜融合抑制劑抑制R5嗜性和X4嗜性的HIV感染對HIV包膜gp41具有特異性阻止病毒包膜和細胞膜融合所需的構象改變04C46LVsh5/C46同時表達抑制CCR5表達的sh5和C46（via RNAi）提供兩種獨立的防止HIV入侵的機制Burke B P, Levin B R, Zhang J

23、, et al. Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral VectorJ. Molecular Therapy - Nucleic Acids, 2015, 4(4).04C46Burke B P, Levin B R, Zhang J, et al. Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vec

24、torJ. Molecular Therapy - Nucleic Acids, 2015, 4(4).Fig. 10 Protection of CD4+ T-cells and reduced HIV-1 viral load within peripheral blood04C46Burke B P, Levin B R, Zhang J, et al. Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral VectorJ. Molecular Ther

25、apy - Nucleic Acids, 2015, 4(4).Fig. 11 Protection of CD4+ cells and reduced HIV-1 proviral load within lymphoid tissues04C46Pernet O, Yadav S S, Dong S A. Stem cell-based therapies for HIV/AIDS J. Advanced Drug Delivery Reviews, 2016, 103:187-201.Table 1 A summary of anti-HIV HSPC gene therapy rese

26、arch05LIMITATION臨床經驗不足HSPC移植成活率不高不能達到完全的免疫重建06REFERENCE1 Cenderello G, De M A. Discordant responses to cART in HIV-1 patients in the era of high potency antiretroviral drugs: clinical evaluation, classification, management prospects.J. Expert Review of Anti-infective Therapy, 2015, 14(1):1-12.2 Yosh

27、imura K. Current status of HIV/AIDS in the ART era.J. Journal of Infection & Chemotherapy Official Journal of the Japan Society of Chemotherapy, 2016.3 Pernet O, Yadav S S, Dong S A. Stem cell-based therapies for HIV/AIDS J. Advanced Drug Delivery Reviews, 2016, 103:187-201.4 Htter G, Nowak D, Mossn

28、er M, et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation.J. New England Journal of Medicine, 2009, 360(7):692-698.5 Wilen C B, Tilton J C, Doms R W. Molecular Mechanisms of HIV EntryJ. Advances in Experimental Medicine & Biology, 2012, 726(1):223-242.6 Wang J, Exline C M, Declercq J J, et al. Homology-d