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

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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.