The power of induced pluripotent stem cell (iPSC), xenograft and gene editing technologies will be combined to develop a comprehensive CRISPR-Cas9-based gene repair therapy to treat a broad spectrum of dystroglycanopathies caused by recessive inactivating mutations of Fukutin-Related Protein (FKRP). Loss of FKRP-mediated glycosylation of the cell surface glycoprotein ?-dystroglycan (?-DG) destabilizes muscle and CNS interactions with extracellular matrix (ECM) proteins, causing neurological and muscular deficiencies. As all reported mutations occur in the coding region exclusively localized to the terminal exon 4, we propose an all-in-one therapeutic strategy to use Cas9-based nucleases to introduce a gene repair cassette flanked by a strong splice acceptor and polyadenylation site into intron 3. Upon repair, transcription from the endogenous FKRP promoter will incorporate the wild-type exon 4 into the spliced mRNA, restoring gene function and achieving a permanent gene correction for all dystroglycanopathies caused by FKRP mutations. This FKRP gene repair strategy will be developed using ex vivo iPSC and in vivo muscle xenograft models of Walker Warburg Syndrome (WWS) that harbor mutations fully inactivating FKRP ribitol-5-phosphate (Rbo5P) transferase activity.
Aim 1 will focus on the development of SpCas9 nuclease/guide RNA combinations that efficiently and precisely insert the donor repair cassette into intron 3 of FKRP by homologous recombination (HR) or homology-independent targeted integration (HITI). Reconstitution of FKRP function will be assessed in ?repaired? WWS iPSCs and iPSC-derived myoblasts and myotubes based on ?- DG glycosylation levels and laminin-binding capacity. Once optimized, rAAV-based donor DNA, sgRNA and nuclease delivery systems for in vivo gene repair will be evaluated in vitro in WWS myotubes to define the most optimal constructs for in vivo studies.
Aim 2 will focus on adapting our FKRP gene repair technologies for delivery to immunodeficient mouse xenograft muscle engrafted with WWS iPSC-derived myoblasts. Initial studies to define conditions for efficient rAAV-based delivery of a sgRNA/donor repair cassette will be facilitated by creating WWS myoblast lines with constitutive expression of Cas9 and epitope-tagged ?-DG. The efficiency of in vivo gene repair will be evaluated in satellite cells and myofibers by FACS, IHC, and Western blotting assays. Once rAAV delivery has been optimized, co-delivery of Cas9 by rAAV will be investigated to complete the FKRP gene repair delivery system. This therapy will be broadly applicable to the full spectrum of muscle and neural FKRP dystroglycanopathies, including Limb Girdle Muscular Dystrophy 2i (LGMD2i), which is particularly amenable to the proposed intervention because of its lack of clinical complexity and early age of onset.
Protein coding mutations in the FKRP gene cause a broad spectrum of neurological and muscle diseases for which there are no therapies. This project combines iPSC, xenograft and CRISPR-Cas9 gene engineering technologies to develop a comprehensive gene repair therapy designed to treat all FKRP diseases. Importantly, the proposed all-in-one gene repair therapy has the potential to improve patient cognitive and/or muscle function, mobility and perhaps even reverse progression of FKRP-related diseases.
