Biodegradable nanoparticles, a genome editing platform to treat hemophilia

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PROJECT SUMMARY Humans suffer from more than 6,500 rare diseases, of which hemophilia A (HA) affects an estimated 20,000 patients in the US and over 400,000 patients worldwide. These patients have a high risk of life- threatening bleeding and serious complications, including joint and muscle diseases. HA is caused by gene mutations of factor VIII (FVIII), resulting in impaired coagulation. Currently, no effective therapy has been shown capable of curing HA, because the half-life of the FVIII protein (the standard treatment) or new FVIII products is less than twenty hours in patients. The goal of this project is to develop biodegradable nanoparticles (BNPs), a genome editing platform to achieve a cure for hemophilia A. Previously, we reported that the efficient delivery of CRISPR/Cas9 and repair template DNA can induce gene-editing and correction of genetic disease in adult mammals by combining viral and non-viral delivery systems. This treatment fully restored weight loss, alleviated liver damage and generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes by correcting a FAH splicing mutation in FAH-mutated mice. Moreover, we developed lipid-like nanoparticles via an orthogonal array design for efficient delivery of mRNA. Our optimized TT3 LLNs was able to restore functional protein to normal physiological values in a hemophilia B mouse model. In this proposal, we aim to develop biodegradable nanoparticles for delivery of CRISPR/Cpf1 and hFVIII cDNA. CRISPR/Cpf1 is a new series of CRISPR effectors with single guide RNA. We anticipate maximizing the function of CRISPR/Cpf1 and enhancing genome editing efficiency for HA therapy. In this study, we will synthesize and characterize BNPs, study their cutting efficiency of the albumin (mAlb) locus, a designed safe-harbor gene- insertion site in vivo, thereby advancing this novel platform toward future clinical trials for treating HA. This approach has several advantages over other strategies currently used for HA treatment. (i) If successful, BNPs offer a curable therapy for HA. (ii) Cpf1 mRNA can be translated for short-term expression in order to induce gene-cutting, avoiding potential off-target effects and toxicity due to long term expression of Cpf1 protein. (iii) mRNA does not integrate into the genes of host cells, avoiding potential genotoxicity. (iv) BNPs are biodegradable, thus minimizing accumulation in the liver and relevant side effects. The following specific aims will be carried out to accomplish our goals: 1). To synthesize and characterize novel biodegradable nanoparticles (BNPs). 2). To optimize chemically modified Cpf1 mRNA and sgRNA for mAlb gene-cutting in vitro; and 3). To evaluate genome editing efficiency and safety profiles of BNPs in a hemophilia A mouse model. Based on current lead material as well as new BNPs to be identified from the proposed study, this research program will be able to successfully discover and develop new drug candidates for treating hemophilia A and potentially other rare diseases.