CRISPR-based Modular Therapy for Precision Medicine 3 CTR 6 MCB Project Summary Efforts to treat genetic diseases have suffered from insufficiently transformative clinical technologies. The recent development of CRISPR genome editing tools is paving the way for new innovations in precision medicine. In 2013, we spearheaded the first successful correction of an inherited liver disease mutation in an adult mammal using the CRISPR-Cas9 system. However, in vivo delivery of CRISPR components for disease treatment remains a challenge as we strive to reduce off target effects, provide transient editing systems and increase the rates of gene correction. In this application, we propose to develop an innovative modular delivery strategy to help address these issues. Our programmable modular approach will allow us target a large number of genetic mutations in mouse models of human disease. The modules comprise: (1) Cas9, (2) single-guide RNA (sgRNA), and (3) a DNA repair template. By breaking CRISPR into smaller components, we can use both adeno-associated virus (AAV) and non-viral vehicles to achieve transient therapeutic delivery to various organs. This new method will advance precision medicine research for an array of genetic diseases. Specifically, we will develop a Cas9 module using lipid nanoparticle delivery of Cas9 mRNA and AAV delivery of self-targeting Cas9 in order to improve the precision of genome editing. We will fine-tune DNA repair pathways in vivo to increase the rate of gene correction and explore whether inhibiting non-homologous end joining (NHEJ) can promote homology-directed repair (HDR) in non- dividing somatic cells such as pancreatic cells and neurons. We will also develop platforms for CRISPR-based modular therapy for a panel of genetic disease, especially those which require a high rate of gene correction such as alpha-1 antitrypsin (AAT) deficiency. These investigations will form the basis of a clinically-relevant platform capable of precisely targeting a wide range of disease gene mutations in somatic organs.
Efforts to treat genetic diseases have suffered from insufficiently transformative clinical technologies. We propose to develop an innovative modular delivery strategy of CRISPR-based therapy for a wide spectrum of genetic diseases. First, we will develop a transient Cas9 module using lipid nanoparticle delivery of Cas9 mRNA and AAV delivery of self-targeting Cas9 in order to improve the precision of genome editing. Second, we will fine-tune DNA repair pathways in vivo to increase the rate of gene correction and explore whether inhibiting non-homologous end joining (NHEJ) can promote homology-directed repair (HDR) in non-dividing somatic cells. Finally, we will develop programmable platforms of CRISPR-based modular therapy for a panel of genetic disease. These investigations will form the basis of a clinically-relevant platform capable of precisely targeting a wide range of disease gene mutations in somatic organs.
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