Novel AAV vector generation methods to prevent immunogenic unmethylated CpGs that trigger efficacy-limiting CTLs in human gene therapy

总结 基于腺相关病毒（AAV）的基因转移载体已被证明是安全的和具有变革性的。 对几种遗传性疾病的治疗效果，包括RPE 65-/-视网膜病变（FDA批准2017年），脊髓 肌萎缩（FDA批准，2019年）和血友病A和B（关键试验正在进行中），验证其 巨大的潜力然而，宿主免疫应答仍然是成功的基于AAV的产品的主要障碍。 发展特别值得关注的是，本申请的重点是产生captain特异性CD 8+细胞， 细胞毒性T淋巴细胞（CTL），可导致炎性毒性和损失 通过破坏载体转导的细胞进行治疗性转基因表达。失去表达能力是一种主要的 问题，因为暴露于AAV研究产品的受试者产生高滴度和广泛交叉 反应性AAV抗体，其妨碍将来施用基于AAV的治疗剂。新方法， 防止CTL应答的初始引发是迫切需要的。先前的报道支持AAV载体 CpG二核苷酸胞嘧啶（MenegCpG）的基因组低甲基化是导致形成 衣壳特异性CTL。这些未甲基化的CpG结合并二聚化Toll样受体9（TLR 9）病原体。 相关分子模式（PAMP）传感器蛋白存在于核内体/溶酶体区室， 浆细胞样树突状细胞（pDC）激活MyD 88先天途径，导致产生 炎性细胞因子触发适应性细胞免疫反应。我们将测试增加CpG是否 AAV载体基因组中的甲基化是可行的，并且可以通过消除MenegCpG-1来纠正这个问题。 相关的PAMP，从而防止逆转初始治疗的有害免疫应答。 这是从AAV介导的治疗性基因转移获得的益处。我们将在体外使用人和鼠， 体内模型系统，以评估通过添加或不添加CpG来增加AAV载体中的MeposCpG的免疫效果。 在载体基因组生物合成和包装成AAV期间对HEK 293细胞的甲基转移酶活性 颗粒（Aim 1）或通过在转染和基因组包装到AAV中之前甲基化载体质粒DNA HEK 293细胞中的颗粒（Aim 2）。我们的目标是制定强有力的战略，提高持久性和有效性， 的AAV载体介导的转基因表达，从而导致临床试验中的改善的结果。积极的 我们的方法的结果将促进产生AAV载体的方法的重要改进， 他们在人类基因治疗中的表现。最关键的是，先天免疫刺激会减少 通过这些去免疫化的AAV载体，在人类中实现持久的治疗水平的基因表达， 这将直接导致更大的概念验证研究（R 01或类似），以及随后的人体临床试验。

SUMMARY Gene transfer vectors based on adeno-associated virus (AAV) have demonstrated safety and transformative therapeutic effects for several genetic diseases including RPE65-/- retinopathy (FDA-approved 2017), spinal muscular atrophy (FDA-approved, 2019), and hemophilia A and B (pivotal trials ongoing), validating their enormous potential. However, host immune responses remain a major barrier to successful AAV-based product development. Of particular concern, and the focus of this application, is the generation of capsid-specific, CD8+ cytotoxic T-lymphocytes (CTLs) following vector administration that can lead to inflammatory toxicities and loss of therapeutic transgene expression by destruction of vector-transduced cells. Loss of expression is a major problem because subjects exposed to an AAV investigational product develop high titer and broadly cross reactive AAV antibodies that preclude future administration of AAV-based therapeutics. New approaches, that prevent initial priming of the CTL response, are urgently needed. Previous reports support that AAV vector genome hypomethylation at the cytosine of CpG dinucleotides (MenegCpG) is a key trigger leading to formation of capsid specific CTLs. These unmethylated CpGs bind and dimerize Toll-like receptor 9 (TLR9) pathogen- associated molecular pattern (PAMP) sensor proteins present in the endosome / lysosome compartments of plasmacytoid dendritic cells (pDCs) to activate the MyD88 innate pathway, leading to the generation of inflammatory cytokines that trigger adaptive cellular immune responses. We will test whether increasing CpG methylation in AAV vector genomes is feasible and can correct this problem by eliminating the MenegCpG- associated PAMPs and thereby preventing the deleterious immune responses that reverse the initial therapeutic benefit achieved from AAV-mediated therapeutic gene transfer. We will use both human in vitro and murine in vivo model systems to evaluate the immunological effect of increasing MeposCpG in AAV vectors by either adding methyltransferase activity to HEK293 cells during the biosynthesis and packaging of vector genomes into AAV particles (Aim 1) or by methylating vector plasmid DNA prior to transfection and genome packaging into AAV particles in HEK293 cells (Aim 2). Our goal is to develop robust strategies that improve the durability and efficacy of AAV vector mediated transgene expression, thus leading to improved outcomes in clinical trials. A positive outcome of our approach will facilitate important improvements in methods to generate AAV vectors leading to their improved performance in human gene therapy. Most critically, will be reduced innate immune stimulation by these de-immunized AAV vectors to achieve durable therapeutic levels of gene expression in humans, which will lead directly into a larger proof of concept study (R01 or similar), and subsequent human clinical trials.