Wen Xue, Ph.D. - US grants

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.

Project Summary/Abstract ? Project 3: The ability to correct disease gene mutations in vivo has broad potential utility for both therapy and basic research. CRISPR/Cas9 is a powerful RNA-guided tool for genome editing. Our recent discovery that CRISPR/Cas9 delivery can cure genetic disease in adult mouse liver provided proof-of-concept of gene correction therapy. This subproject will interact synergistically with all other Projects and Cores of this tPPG to develop new rAAV CRISPR tools to treat alpha- 1 antitrypsin deficiency (AATD). The main goal of this proposal is to establish a pre-clinical rAAV paradigm for CRISPR-mediated correction of AAT deficiency in mouse models carrying the mutant human AAT gene. The impact of this project is to develop somatic gene correction using rAAV systems to (1) maximize efficiency and safety of CRISPR delivery, (2) maximize the rate of homologous recombination for gene correction, and (3) efficiently correct AAT mutation in the liver to treat lung disease in mice. The development of safe and effective delivery vehicles and genome editing tools to correct AAT deficiency will guide future clinical trials for CRISPR- mediated gene therapy for AAT lung disease. Because AAV serotypes can target a wide range of tissues, our approach has broad basic research and clinical applications beyond AATD. Project 3 has three Aims that focus on different aspects of liver-directed somatic AAT correction: Aim 1: Develop liver-directed rAAV vehicles to maximize efficiency and precision of Z-AAT genome-editing in mice. Aim 2: Investigate rAAV HDR templates to correct Z-AAT mutation in mouse liver. Aim 3: Explore Z-AAT correction in mouse models in vivo to treat the lung disease.

Project Summary The ability to correct disease gene mutations in vivo has broad potential utility for both therapy and basic research. CRISPR/Cas9 is a powerful RNA-guided tool for genome editing. Our recent discovery that CRISPR/Cas9 delivery can cure genetic disease in adult mouse liver provided proof-of-concept of gene correction therapy in vivo. The main goal of this proposal is to establish innovative delivery technologies to maximize the efficiency of CRISPR delivery and gene correction in disease-relevant lung cell types. The impact of this project is a novel paradigm of lung-targeted delivery tools for CRISPR-mediated gene correction. The development of safe and effective delivery vehicles and genome editing tools will guide future studies for CRISPR-mediated gene therapy. This project has three aims that focus on different aspects of lung-directed somatic genome editing: Aim 1: Optimize NP+AAV combination for delivery and gene correction in mouse lung; Aim 2: Identify the best AAV capsid for lung delivery and optimize AAV genome as donor template for HDR; Aim 3: Characterize NP+AAV developed in the UG3 phase in macaque models. This project will develop innovative designs of adeno-associated virus and nanoparticles to significantly improve the efficiency, cell specificity, and safety of CRISPR delivery in vivo.

PROJECT 2 - Project Summary/Abstract Gene editing has the potential to correct mutations and provide long-term therapeutic benefit for patients with rare monogenic diseases like alpha-1 antitrypsin deficiency (AATD). AATD is caused by mutations in the AAT (or PI) gene, which encodes a serine protease inhibitor that is made in hepatocytes and delivered to lung to neutralize neutrophil elastase. The PI*Z mutation encodes mutant Z-AAT protein that aggregates in hepatocytes, which can cause liver disease and reduces serum AAT. Reduced serum AAT causes progressive airway disease and emphysema. Gene correction would address both aspects of AATD. CRISPR-mediated homology directed repair (HDR) can be used to partially correct mutations in mouse liver. Yet, HDR is limited by the need to deliver a long DNA repair template, its inefficiency in non-dividing or slow- dividing cell types, and its generation of genotoxic double-strand breaks. To address these limitations, this proposal will develop two CRISPR-based gene correction strategies that do not require a double-strand break: prime editing and adenine base editing. Prime editor (PE) is comprised of Cas9 nickase fused to reverse transcriptase and an extended guide RNA that doubles as a template for reverse transcriptase to copy editing information into the genomic target. Adenine base editor (ABE), comprised of a Cas9 nickase fused to an adenosine deaminase, can correct G-to-A point mutations in mouse liver. The PI*Z allele results from a G-to-A mutation; and thus, is a good candidate for gene correction via ABE and PE. The goal of this project is to optimize PE and ABE tools for AAT gene correction in vivo by developing ABE and PE vectors that can be accommodated by adeno-associated virus (AAV) capsids; maximizing on-target editing and minimizing off-target editing; and determining how immune responses affect editing. Aim 1 will develop novel PE tools for in vivo AAT gene correction. A split AAV PE platform will be developed to maximize prime editing efficiency in vivo, then PE gene correction and lung phenotype will be measured in a PI*Z transgenic mouse model and a clinically-relevant AAT null/PI*Z mouse model. Aim 2 will enhance the specificity of ABE for in vivo AAT gene correction. Long-term ABE expression can induce off-target editing. Therefore, new ABE variants will be optimized to increase activity and reduce RNA editing effects, and split AAV delivery of ABE will be investigated in a PI*Z model. This Aim will also develop self-inactivating ABE to reduce off-target effects. Aim 3 will characterize and mitigate immune responses to PE and ABE, which harbor viral reverse transcriptase and bacterial TadA protein, respectively, and Cas9, a known antigen. This Aim will investigate antibody and T cell response to PE and ABE in mice, how immune response regulates editing, and whether CAR-Treg can mitigate immune response. Project 2 will benefit from extensive interactions with the other projects and cores in this P01. Completing this project will improve the efficiency and safety of PE and ABE in vivo, providing an HDR-independent gene editing blueprint for AATD, and other monogenic diseases.