醫(yī)學(xué)免疫學(xué)課件：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

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(tǒng)機會性感染及癌癥不經(jīng)治療平均存活期9-10年感染免疫系統(tǒng)細胞降低CD4+T細胞的數(shù)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(shù)的恢復(fù)顯示患者艾滋進程的一項重要指標(biāo)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(zhuǎn)錄病毒治療顯著提升患者的生存率和生存質(zhì)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(xù)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(jié)合，Env與CD4結(jié)合CD4的結(jié)合造成了Env的構(gòu)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(tǒng)和免疫系統(tǒng)造成明顯的不良反應(yīng)02CCR5AAV6（腺病毒相關(guān)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(xiàn)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(zhuǎn)入外周血來源的CD34+細胞ZFN mRNA進行轉(zhuǎn)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(huán)境中阻止HIV感染移植經(jīng)過預(yù)選的轉(zhuǎn)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(zhuǎn)導(dǎo)的CD34+細胞R5嗜性的HIV感染GFP陽性CD4+T細胞比例和絕對數(shù)量增加整體CD4+T細胞數(shù)量未增加HIV復(fù)制的比率無明顯區(qū)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?qū)?2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(biāo)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?qū)崿F(xiàn)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(zhuǎn)導(dǎo)R5-ZFN同時進行R5和X4-ZFN轉(zhuǎn)導(dǎo)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(nèi)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(gòu)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(jīng)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