Characterizing the immune response to AAV9-CRISPR/Cas9 in a humanized mouse model of Duchenne muscular dystrophy using single cell RNA-sequencing


Scientific Other

Poster Number: 184


Michael Emami, BS, Courtney Young, Feiyang Ma, Matteo Pellegrini, Melissa Spencer, PhD


1. UCLA, 5. Molecular Biology Institute, Department of Neurology, Center for Duchenne Muscular Dystrophy at UCLA

Duchenne muscular dystrophy (DMD) is an x-linked disease caused by out-of-frame mutations in the DMD gene, which encodes dystrophin. Due to the nature of these mutations, DMD is amenable to gene-editing and gene replacement therapies. Ongoing phase I/II clinical trials for DMD rely on a gene replacement strategy using adeno-associated virus (AAV) to achieve systemic delivery. However, immune responses against AAV vectors have resulted in serious adverse events (SAEs) in current and past studies. Yet, it is poorly understood why certain patients develop SAEs following AAV gene therapy and what specific immune responses arise. To study immune responses in vivo following AAV administration, we dosed a humanized dystrophic mouse model (hDMD del45) with AAV serotype 9 carrying CRISPR/Cas9 and performed 10x Genomics single cell RNA-sequencing (scRNA-seq). We tested three different AAV9-CRISPR/Cas9 doses: 1.2E12vg, 6E12vg, and 1E13vg and isolated PBMCs pre-AAV administration and 4 weeks post-AAV administration. Four main immune cell populations were observed: monocytes, NK cells, B cells, and T cells, all of which demonstrated a clear shift in phenotype between pre- and post-AAV treatment. Within the T cell population, we detected shifts in the phenotypes of all subpopulations including CD4+, CD8+, regulatory T cells (Tregs), and gamma delta (γδ)T cells between pre- and post-AAV treatment, regardless of dose. Assessment of gene expression within T cell subpopulations revealed upregulation of Dusp1 and Dusp2 in CD8+, CD4+ and γδT cells. These two genes are downstream of Toll-Like Receptor signaling and may represent a potential target to suppress SAEs with AAV administration. Additional analysis and validation studies are needed identify critical immune cell populations and genes that elicit responses to AAV, and to separate capsid vs transgene-specific immune reactions. These studies will enable the identification of new target genes involved in immune responses to AAV.