THERAPEUTIC GENOME EDITING IN RETINA AND LIVER

In vivo gene therapy with adeno-associated viral (AAV) vectors has been successful at treating several inherited diseases, specifically those caused by loss of function mutations which require transfer of a correct copy of a gene. This would not benefit dominant diseases due to gain of function mutations which produce toxic protein products. In addition, since AAV genomes persist as episomes in target cells, AAV mediated transgene expression might be short lived in tissues where cell proliferation occurs when newborn or after damage, like for example the liver. To overcome these challenges, I have developed AAV-based therapeutic approaches which use genome editing to introduce stable modifications at specific genomic loci. First, an allele-specific approach which targets the Rhodopsin P347S dominant mutation was developed and tested both in vitro and in vivo. I achieved allele-specific targeting of human P347S rhodopsin, which reduced mRNA levels and improved retinal electrical function in a mouse model of autosomal dominant retinitis pigmentosa. Second, I developed a mutation- and homology-independent targeted integration (HITI) approach for gene correction in photoreceptors. I demonstrated feasibility of this approach in mouse and pig photoreceptors using a reporter gene and characterized on-target precision of HITI in the murine rhodopsin locus. I then tested the therapeutic potential of this approach in a mouse model of autosomal dominant retinitis pigmentosa and observed mild and transient improvement of retinal function in treated eyes, which suggests that the levels of editing obtained need optimization. Third, I developed a HITI approach for expressing therapeutic genes from the liver by targeting the albumin locus, which is highly transcribed in hepatocytes. I demonstrated feasibility and efficiency of this approach using a reporter gene, and characterized on-target precision of HITI, as well as off-target integration due to Cas9 cleavage. I then tested the therapeutic potential of the integration of a copy of the human arylsulfatase B (ARSB) gene, which is mutated in a rare lysosomal storage disease, mucopolysaccharidosis type VI (MPS VI), in the albumin locus in the liver of newborn MPSVI mice. I demonstrated that this approach achieves stable expression of ARSB at levels that reduce glucosaminoglycan (GAG) urinary secretion, one of the main readouts of MPSVI phenotype. This stable expression of ARSB is contrary to the decrease of transgene expression observed in neonatal MPSVI mice injected with the same dose of a conventional gene therapy vector, thus overcoming the potential loss of transgene expression caused by hepatocyte proliferation. Overall, I have developed different genome editing approaches for conditions that are inherited as either dominant or recessive. I have tested these approaches in two relevant tissues for gene therapy like retina and liver and shown the potential to provide AAV with persistent transgene expression in proliferating tissues like the newborn liver.