MSCS ENGINEERED TO PRODUCE BDNF AND GENE EDITING CARGO FOR THE TREATMENT OF HUNTINGTON'S DISEASE

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Abstract Huntington's disease (HD) is a neurodegenerative disorder with no cure, and there is a critical unmet need for disease- modifying treatments. We are developing a novel therapy for HD: implantation of human Mesenchymal Stem/Stromal Cells (MSCs) engineered to secrete Brain-Derived Neurotrophic Factor (MSC/BDNF), a growth factor needed in the degenerating striatal regions of the brain, along with gene editing cargo to specifically reduce levels of production of the mutant RNA and protein. BDNF is low in humans and mice with HD, and up- regulation of BDNF in the brains of transgenic rodent models of HD has ameliorated the disease phenotype. Due to pro-survival effects in striatal neuropathology, BDNF is a strong candidate for neuroprotective therapies. Our labs have also developed targeted gene silencing tools using a novel variant of Cas9 (xCas9 3.7) that allows for broad protospacer adjacent motif (PAM) targeting. We have demonstrated this platform is effective in reducing huntingtin when fused to a powerful transcriptional repressor, KRAB. We have demonstrated that MSCs are an excellent delivery vehicle. We are testing injection via the cisterna magna as a surrogate for spinal cord/CSF fluid delivery in future patients who might receive this cell and gene therapy product developed by our team. Cellular Nanoengineered xCas9 Therapy (CellNeXT) combines the beneficial effects of MSC administration to the striata with the benefits of BDNF production while in combination reduction levels of mutant huntingtin. Unlike BDNF delivery via direct vector injection or protein administration into the brain, MSCs migrate into the areas of damage and have numerous beneficial effects. Although optimized MSCs will not persist longer than several months, we hypothesize that the neurorestorative effects of BDNF will outlast the survival of MSCs. This is supported by animal data from our laboratory and others. In our double-blinded efficacy studies, intrastriatal delivery of human MSC/BDNF significantly reduced anxiety and significantly increased neurogenesis in immune suppressed HD mice, with increased survival, in comparison to vehicle treated HD mice. We have demonstrated that treatment with MSC/BDNF decreased striatal atrophy as compared to vehicle treated HD mice (PMID:26765769). This recovery may be due to the stimulation of endogenous neurogenesis promoted by BDNF and enhanced by the secretion of various complementary therapeutic factors by the MSCs. In the planned studies, we will perform the following studies in support of an investigational new drug filing to the FDA: in Aim 1 we will evaluate the selectivity and durability of xCas9 in patient iPSC-derived NSC and neurons. In Aim 2 we will assess dose and route of administration of CellNeXT in our novel HD mouse model. Our studies will define reproducible techniques and methods, at the level of Good Laboratory Practice, for evaluation of cell and gene therapy candidates to be used in neurodegenerative disorders. We will be positioned to move the CellNeXT candidate into clinical trials for HD initially, and to assist others in using the product for additional disorders in the future. We will better define mechanism of action of CellNeXT and will define potency assays, using reduction of mutant huntingtin and preservation of striatal atrophy as key readouts.