Novel Vaginal Microbicides Based On Stable AAV-Neutralizing Antibody Gene Transfe

描述（由申请人提供）：在全球艾滋病大流行中，一半以上的新艾滋病毒-1感染是妇女通过阴道内接触艾滋病毒而获得的。尽管宫颈阴道上皮细胞排列在女性下生殖道的粘膜表面，为HIV-1感染提供了最初的防御系统，但这种保护往往是不完整的。HIV-1通过粘膜屏障的运输对于HIV-1定植和随后的病毒传播至关重要，因此，通过局部表达抗HIV-1中和抗体（nab）来增强粘膜细胞表面的抗HIV-1体液免疫，从而阻断上皮细胞附着和病毒进入，可能提供一种重要的新干预措施，可以减缓HIV/AIDS的传播。这个R21/R33项目代表了Marasco（抗体工程，基因治疗），Anderson（粘膜免疫）和Mansfield（艾滋病毒/艾滋病猕猴模型）实验室的共同努力，研究稳定的腺相关病毒(AAV)-nAb基因转移到宫颈阴道上皮干细胞是否可以提供一种策略，从而导致持久的抗HIV-1保护。在R21期，我们将首先确定9种AAV血清型中哪一种提供了最佳的GFP基因转移，对人（Hu）和恒河猴（Rh）的原代生殖上皮细胞（PGECs）（包括宫颈内、宫颈外和阴道上皮细胞）无毒，并特别关注将基因稳定转移到能够更新层状上皮的p63+CK17+上皮干细胞中。AAV-GFP转导的持久性、潜在的毒性和促炎细胞因子、激素条件、精液和阴道分泌物对转导效率和转基因持久性的影响将被检查。我们将构建IgG1和二聚体IgA2格式的广泛中和的人抗gp120单抗b12的小型化版本（minibody），并通过抗体治疗研究和AAV基因递送到器官型人阴道和宫颈内模型以及Hu和Rh pges来评估b12对HIV-1/SHIV的中和活性。在成功证明体外保护后，R33期将开始，我们将首先在Rh （n=12）中进行aav转导剂量递增研究，以评估p63+，CK17+干细胞转导的深度，均匀性和程度，b12scFv-FcG1和b12scFv-FcA2分泌的PK和毒性。随后将进行第二次阴道内转导研究，分别使用两种AAV-b12scFv载体的最佳剂量，然后用SHIV阴道攻击（Rh=15-16）。最后，我们将通过凝胶形成聚合物和AAV的混合物来评估增强SHIV保护，以增加体内AAV转导和b12scFv-Fc分泌（Rh=9）。总共将测试13个假设。这些重要的研究实现了R21/R33计划的一个主要目标，即支持可能具有高风险/影响并有可能推进艾滋病杀微生物剂战略的研究。鉴于基于AAV的基因治疗在众多临床试验中的安全性、低免疫原性和快速进展，这种新方法的成功很可能很快转化为人类研究。艾滋病毒-1感染通常是通过性接触获得的，一半以上的新感染是妇女通过阴道内接触艾滋病毒而获得的。我们建议开发一种基因杀菌剂，当它被递送到宫颈和阴道的粘膜表面时，将允许衬里细胞稳定地产生一种中和的人类抗hiv抗体，阻止HIV-1的附着和感染。一种保护性的基因杀微生物剂被注射到女性的下生殖道，可以显著减缓艾滋病的传播。

DESCRIPTION (provided by applicant): In the global AIDS pandemic, more than half of new HIV-1 infections are acquired by women through intravaginal HIV exposure. Although cervico-vaginal epithelial cells lining the mucosal surfaces of the female lower genital track provide the initial defense system against HIV-1 infection, the protection is often incomplete. Transport of HIV-1 across this mucosal barrier is absolutely critical for HIV-1 colonization and subsequent virus dissemination and thus enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by the local expression of anti-HIV-1 neutralizing antibodies (nAbs) that block epithelial cell attachment and virus entry may provide an important new intervention that could slow the spread of HIV/AIDS. This R21/R33 Project represents the combined efforts of the Marasco (Antibody Engineering, Gene Therapy), Anderson (Mucosal Immunity) and Mansfield (HIV/AIDS Macaque Model) laboratories to investigate whether stable adeno-associated virus (AAV)-nAb gene transfer to the cervico-vaginal epithelial stem cells can provide a strategy that will lead to durable protection against HIV-1. In the R21 phase, we will first determine which of 9 AAV serotypes provides optimal gene transfer of GFP without toxicity to primary human (Hu) and rhesus macaque (Rh) primary genital epithelial cells (PGECs) comprising endocervical, ectocervical and vaginal epithelial cells with special focus on stable gene transfer into p63+CK17+epithelial stem cells which are capable of renewing stratified epithelium. Persistence of AAV-GFP transduction, potential toxicities and effects of proinflammatory cytokines, hormonal conditions, semen and vaginal secretions on transduction efficiency and transgene persistence will be examined. We will construct a miniaturized version (minibody) of broadly neutralized human anti-gp120 Mab b12 in both the IgG1 and dimeric IgA2 format and assess b12 neutralizing activity against HIV-1/SHIV by both Ab treatment studies and AAV gene delivery to organotypic human vaginal and endocervical models and Hu & Rh PGECs. Upon successful demonstration of in vitro protection, the R33 phase will begin where we will first conduct an AAV-transduction dose escalation study in Rh (n=12) to evaluate depth, uniformity and extent of p63+,CK17+ stem cell transduction, the PK of b12scFv-FcG1 and b12scFv-FcA2 secretion, and toxicity. This will be followed by a second intravaginal transduction study with the optimal dose of the two AAV-b12scFv vectors each alone and together followed by vaginal challenge with SHIV (Rh=15-16). Finally, we will evaluate enhanced SHIV protection through mixtures of gel-forming polymers and AAV to increase in vivo AAV transduction and b12scFv-Fc secretion (Rh=9). Overall, 13 hypotheses will be tested. These important studies fulfill a major objective of the R21/R33 program to support research that may be high risk/impact and have the potential to advance AIDS microbicide strategies. Given the safety profile, low immunogenicity and rapid advancement of AAV based gene therapy in numerous clinical trials, it is likely that success of this novel approach could be quickly translated to human studies. HIV-1 infections are acquired most often through sexual contact and more than half of new infections are acquired by women through intravaginal HIV exposure. We propose to develop a genetic microbicide that when delivered to the mucosal surface of the cervix and vagina will allow the lining cells to stably produce a neutralizing human anti-HIV antibody that blocks HIV-1 attachment and infection. A protective genetic microbicide delivered to the female lower genital track could dramatically slow the spread of HIV/AIDS.