Exploring genomic and cellular determinants of gene therapy durability

PROJECT SUMMARY Since the identification of disease-causing genes, the promise of gene correction seemed out of reach for decades. New delivery vehicles and gene-editing tools have brought the field to an inflection point in our ability to address the molecular basis of disease. Remarkable results in spinal muscular atrophy, inherited blindness,

and hemoglobinopathies are just the beginning of a revolution in precision medicine. However, many unknowns remain including the durability of gene replacement therapy, the adaptive immune response to gene therapies, and unintentional genomic changes including vector integrations. In the clinic, these concerns are reminiscent

of gene therapy failures of the past, and recent clinical trial deaths in high-dose adeno-associated virus (AAV) clinical trials illustrate the significance of improving our understanding of the host response to gene therapy. We have previously shown that, in the absence of an immune response, CRISPR-mediated genome editing effects

are maintained for the lifetime of an animal. However, the adaptive immune response can reverse gene correction. Additionally, we observed a high level of vector integration leading to chimeric transcripts. The biological consequences of these genomic and transcriptomic alterations are unknown. The goal of this MIRA

research program is to develop a better understanding of genomic, epigenomic, and transcriptomic outcomes of gene therapy. We aim to answer long-standing questions including: (1) What are the epigenetic and transcriptional consequences of vector integration on long-term outcomes? (2) Is there a cell, tissue, or promoter

dependency on the behavior of the vector genome? (3) Is there a differential host response to integrated vectors, episomal vectors, or alternate delivery vehicles? To answer these questions, we will use a convergence of tools and techniques including CRISPR-mediated targeted integration, PCR-free long-read DNA sequencing, and

spatial biology. These approaches are supported by robust preliminary data in the PI’s lab. Using nanopore long- read sequencing, we have developed methods for characterizing full-length AAV insertions and epigenetic alterations to AAV genomes and resulting transcriptional changes. We are adapting spatial biology techniques

to characterize host responses to divergent gene therapy vehicles in skeletal muscle, cardiac tissue, liver, and others. Answers to these questions will enable improved design of gene therapy approaches with the aim of life- long correction in a range of diseases.