Hemophilia A and B are monogenic disorders characterized by the loss of factor VIII (F8) or factor IX (F9) leading to delayed blood clotting and life-long treatment with recombinant clotting factors or blood products. Gene therapy has held promise for treating monogenetic diseases like hemophilia, however, has yet to deliver a curative treatment. Gene delivery barriers and the potential requirement of costly repetitive treatments characterize the drawbacks of technologies currently in the therapeutic pipeline. Gene editing has emerged as a method to make permanent modifications to the host genome. Specifically, RNA-guided nucleases (RGNs) have the potential to correct the genetic basis of a broad range of diseases including hemophilia. However, significant delivery barriers of RGNs or gene-encoded RGNs hinder in vivo gene editing. To date, the most reported in vivo gene editing approaches have used viral delivery vehicles due to robust expression and available tissue tropism. An exciting alternative is the generation of effective non-viral delivery vehicles for RGNs that address the safety concerns of viral delivery vehicles. Therefore, the overall goal of this work is to create a powerful biomacromolecule delivery vector for genome-editing technologies. The central hypothesis of this work is that non-viral delivery of gene-editing nucleases can precisely target replacement genes to safe-harbor loci in the human genome and restore protein expression in hemophilia A or B. The hypothesis will be evaluated with the following specific aims: 1) Characterize viral and non-viral gene replacement safety and efficacy in murine liver. 2) Apply viral and non-viral gene replacement in mouse models of hemophilia, and 3) Develop a screening platform for non-viral gene delivery vehicles.
In Aim 1, intravenous delivery of CRISPR/Cas9 will be evaluated for efficient gene replacement. Promising preliminary data for gene delivery to liver has been demonstrated by the PI for viral and non-viral delivery. Both viral and non-viral delivery strategies will be pursued in parallel. Quantitative readouts for single base pair changes, small insertions, and large insertions will be used to assess the efficacy of the delivery routes and determine immune response and off-target activity.
In Aim 2, mouse models of hemophilia will be evaluated for genomic modification and recovery of the absent clotting factor. Factor VIII is a model of a large insertion and FIX is a model of a small insertion.
In Aim 3, we propose to combine deep sequencing with gene editing to screen libraries of non-viral compounds in small numbers of animals. This method overcomes the limitations of high-throughput in vitro screens that cannot recapitulate complex in vivo delivery barriers. The end result of this work will be an efficient non-viral gene editing toolbox for targeted gene replacement. In addition, this work will generate new strategies for gene editing and gene therapy to improve clinical treatment of hemophilia which may be applied to other genetic diseases.
The proposed work will apply non-viral delivery to genome-editing technologies. This application of in vivo genetic correction can be applied to a wide range of genetic disorders and is therefore highly relevant to the NIH?s mission.
