The revolution in gene editing technology promises to transform the development of therapeutics to treat human disease. As part of the Somatic Cell Genome Editing consortium, the goal of this project is to build mouse resources and provide an animal model testing platform to support the optimization of novel genome editing technologies for future translational applications.

The difficulty of delivering genome editing agents into many types of cells in animals and patients is a major challenge that must be overcome to realize their full potential to cure genetic diseases. We propose to develop two new strategies for the delivery of genome editing agents into animals and patients that will increase editing efficiency, target cell selectivity, and DNA specificity, as well as a new tool to rapidly and sensitively evaluate the delivery of these agents in mice with minimal effort and expense. These developments will advance the safety and efficacy of genome editing methods for clinical development.

This project aims to advance the NIH Somatic Cell Genome Editing Program’s objective to identify novel delivery technologies that enable genome editing in therapeutically relevant somatic cell populations. We will use proven virus engineering methods to develop new vehicles that can deliver genome editing machinery throughout the adult mammalian central nervous system. Accomplishing this objective would pave the road for applying gene editing, and gene therapy more broadly, to the study and treatment of neurological and psychiatric disorders.

RNA-guided CRISPR genome editing systems promise to revolutionize the treatment of inherited disease. Safe, effective, and target-tissue-specific delivery of the guide RNA that directs editing is a critical hurdle in the development of clinical applications for engineered CRISPR systems. Using strategies validated for the delivery of other categories of nucleic acid therapeutics, we have established a framework for complete chemical modification of CRISPR guides, thereby conferring in vivo stability and effective biodistribution properties. The proposed research will optimize these guides, as well as other editing components, for clinical use.

The proposal is designed to screen lipid nanoparticles as novel materials for RNP (ribonucleoprotein) delivery of editing machinery into the mammalian sensory organ inner ear, to expand the cell types that can be edited to treat genetic hearing loss and to establish a method to perform the study in wildtype large animals. This study has direct relevance to bringing editing based therapy to clinic.

Genome editing systems have the potential to cure some of the most severe human diseases. However, there are significant efficacy and safety issues that must be addressed before this technology can be applied in clinical trials. The BCM-Rice Resource Center for the Analysis of Somatic Gene Editing in Mice will create mouse reporter models for testing genome editing technologies, and to use these animal models to test genome editing delivery technologies and new genome editors developed by other Somatic Cell Genome Editing program members.

Genome editing technologies are poised to revolutionize the practice of modern medicine for the treatment of various types of genetic diseases. However, reliable testbed systems using human cells and tissues are needed to accurately predict both intended and unintended consequences of therapeutic interventions. The primary objective of this proposal is to develop and validate human tissue models capable of sensitively and accurately detecting adverse effects of genome editing on physiologic tissue function.

Genome editing technologies have extraordinary potential as the basis of new genomic medicines that address the underlying genetic causes of human disease; however, it is challenging to predict their long-term safety, because we do not know the consequences of potential unintended side effects of genome editing such as off-target mutations or immunogenicity. To define the biological effects of genome editing strategies, we will develop a human primary T-cell platform to sensitively detect functional effects coupled with an empirically-trained artificial intelligence models to predict them. Together, our platform will significantly improve confidence in safety assessments of promising genome editing therapeutics.

Recombinant adeno-associated viruses (AAV) have emerged as safe and effective vectors for clinical gene therapy applications including systemic treatment of neuromuscular diseases such as Spinal Muscular Atrophy (SMA), Duchenne Muscular Dystrophy (DMD), and Giant Axonal Neuropathy (GAN) amongst others. However, genome editing in neuromuscular tissue, in particular, is challenging. The current proposal is on a comprehensive and innovative approach to evolve high potency AAV variants for systemic neuromuscular genome editing.

The proposed research is relevant to the public health because genetic and acquired diseases affecting the airways pose major disease and economic burdens. By advancing the delivery of gene editing tools, it may be possible to therapeutically modify the cells lining the airways. This novel strategy has implications for the treatment of both monogenetic and acquired lungs disease, and may have applications for other somatic cell therapies.

In vivo genome editing using CRISPR/Cas9 is anticipated to be the next wave of therapeutics for various major health threats, including neurodegenerative diseases. However, to date, very few Cas9-gRNA ribonucleoprotein in vivo delivery methods have been reported, and delivery to the brain has been particularly challenging. The unique nanocapsules we plan to develop will ultimately enable high efficiency neuron-targeted genome editing in the brain, thereby offering new hope to treat devastating neurodegenerative diseases.

Efficacy and safety limitations in current gene editing technologies have hindered efforts to treat genetic lung diseases. This proposal seeks to develop and validate a combinatorial delivery approach that uses non-viral and viral vehicles to efficiently transport gene editing tools to disease-relevant cells in the lung. Completion of our work will establish safe and effective delivery vehicles that will guide the design of future gene therapies for genetic disorders.

Expanding genome editing tools through exploration of new CRISPR-Cas proteins and DNA repair enzymes NARRATIVE Fundamental research on bacterial adaptive immunity uncovered the genome editing properties of CRISPR-Cas systems, and it is clear that uncultivated microbes contain more pathways and enzymes that may be useful as genome editing tools. We will combine bioinformatics and biochemistry to identify new DNA- and RNA-associating proteins and will analyze their mechanisms of action. We will focus our investigation on newly described CRISPR-Cas systems and DNA-interacting proteins that occur in conserved genomic context.