Genetic disorders of children are individually rare but collectively frequent, affecting the lives of approximately 500,000 children in CanadaFORGE. They often are serious, life threatening or fatal, but because each rare disease affects a relatively small population few treatments have been developed. Efforts by national and international groups such as the Canadian Pediatric Genetic Disorders Sequencing Consortium (FORGE) and the International Rare Disease Research Consortium (IRDiRC) are uncovering disease- causing genes that could be corrected through genetic therapies. In vivo gene transfer strategies have coalesced around the use of recombinant Adeno-Associated Virus (AAV), a vector that has been used successfully in the clinic for a variety of indications, including haemophilia. However, despite the achievement of safe and efficient gene transfer, therapeutic effects in neonatal animals are only transient because the genome of recombinant vectors is in episomal form within the host cell and gets diluted in growing tissues, like the liver. Thus, for genetic diseases that manifest at a young age with irreversible consequences, early treatment is currently unlikely to succeed.
Targeted integration of the therapeutic transgenes using engineered nucleases, such as zinc-finger nucleases (ZFNs), could circumvent this technical problem. ZFNs are designed, sequence-specific endonucleases produced by linking an engineered zinc finger DNA-binding domain with the nuclease domain of the FokI restriction enzyme. We have previously shown that systemic delivery of ZFNs using liver-tropic AAV vectors can stimulate gene targeting of a corrective FIX transgene leading to the restoration of haemostasis in a mouse model of haemophilia B (Nature, 475(7355):217-21, 2011). Furthermore, this was achieved in neonates and the genetic and phenotypic correction remained persistent after induced liver regeneration suggesting that permanent genetic correction could be achieved in a single treatment.
Our goal is to develop liver-based enzyme replacement therapies for rare diseases via in vivo genome editing. We aim to couple systemic gene delivery using AAV to ZFN-driven genome editing in mice to deliver promoter-less therapeutic transgenes into the very highly expressed albumin locus, a potential “safe harbour”. Successful targeting would ensure adequate levels of expression, even if a relatively small percentage of alleles were targeted, in absence of deleterious effect on the host organism. Proof-of-concept experiments will be performed using genes affected in lysosomal storage diseases because they represent a paradigm for enzyme replacement therapies (ERTs).
This research may lead to significant improvements in quality of life for children affected by rare diseases and their families. This project aims to design, test, and apply a promising technology to develop novel therapeutics, a core research priority of the CIHR Institute of Genetics (IG).