SBIR Phase I: A block copolymer delivery system for the Cpf1 ribonucleoprotein

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project is to bring curative gene editing therapies using CRISPR to patients with genetic diseases. CRISPR-based therapeutics have the potential to be next generation therapeutics, particularly for genetic diseases due to their ability to cut DNA with sequence specificity. However, this type of targeted genome editing has not yet been successfully demonstrated for human therapeutics with existing methods. The translation of CRISPR-based gene editing to the clinic remains a significant unmet need and the most difficult aspect of translating CRISPR-based gene editing into therapeutics is the lack of safe and effective delivery methods to the target the tissues. Existing viral-based delivery systems have limitations that include immunogenicity, pre-existing antibodies against them, broad tropism, off-target effects, restricted DNA cargo packaging capacity, and manufacturing challenges. As a result, non-viral methods that employ synthetic materials are being widely investigated as potential alternatives. Developing non-viral delivery vehicles that can effectively deliver CRISPR components to target tissues will improve the ability to broadly use CRISPR-based therapeutics for many genetic diseases. The intellectual merit of this SBIR Phase I project is to develop a non-viral delivery system for the CRISPR enzyme Cas 12a (Cpf1) for delivery to target muscle tissue, and, potentially, enable the development of a novel gene editing treatment for Duchenne muscular dystrophy (DMD). The approach involves proprietary CRISPR-nanoparticles that are composed of peptide PEG-PAsp(DET) complexed to Cpf1 RNP, which possess good biocompatibility and high gene editing efficiency with the potential to manufacture and scale up under GMP for use in clinical trials. Preliminary data demonstrate that PEG-PAsp(DET) complexed to Cpf1 RNP can efficiently deliver Cpf1 RNP to the muscle tissue and can induce the expression of the dystrophin protein by deleting exon23 with a mutation. Under this proposal, the specific experiments will focus on improving the biocompatibility, stability, and muscle-targeting ability of the CRISPR-nanoparticles. The experimental plan is to synthesize various Peptide-PEG-PAsp(DET), followed by screening in primary myoblasts and reporter mouse system. This technology using CRISPR-nanoparticles will be the first example of a delivery vehicle that can simultaneously deliver Cpf1 protein and gRNA via intravenous injection and achieve gene editing. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.