In vivo precision genome editing to correct genetic disease

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PROJECT SUMMARY Genetic diseases impact over 1 in 50 newborns worldwide and yet there are no approved therapies capable of correcting the underlying genetic defects. As a result, most patients continue to suffer throughout their life and require frequent interventions to ameliorate symptoms. I aim to develop in vivo genome editing therapeutics that correct the underlying disease mutation in relevant tissues by a single injection into the patient. Base editors can efficiently correct transition point mutations, the most common form of disease- causing genetic mutation, without undesired editing outcomes such as indels. With one dose and no subsequent enrichment, over 95% of cells in tissue culture can be edited, and editing in over 60% of non- dividing cells in targeted adult mammalian tissues has been demonstrated in early in vivo work. Prime editors can correct any genetic perturbation of up to at least ~50 nt in length (encompassing ~89% of human disease mutations). I will develop and assess both precision genome editing technologies (base and prime editors) using suitable in vivo delivery tools in mouse models to develop therapeutics for genetic disease. Dilated cardiomyopathy (DCM) is a frequent form of genetic heart disease, affecting an estimated 300,000 people in the United States, and can lead to heart failure. Common causes of DCM are haploinsufficiency of important genes in cardiomyocytes including TTN and LMNA. I will employ screens to identify new editing strategies to treat haploinsufficiencies by enhancing transcription of the healthy allele. I will characterize the mechanism of identified edits to understand the associated changes in transcription factor occupancy and chromatin states. Simultaneously, I will use fluorescent reporter mice to characterize the in vivo delivery of base editor and prime editor tools in order to find the best method for editing cardiomyocytes. This work will include characterizations of tissue- and cell-specific editing following delivery via adeno-associated virus, lipid nanoparticles, or polymer nanoparticles. I will then combine the identified therapeutic editing strategy with the best vehicle for delivery to cardiomyocytes to treat a mouse model of TTN haploinsufficiency. I will measure on- and off-target editing as well as any improvement in the contractility defect that defines this model. Base editors and prime editors can be readily reprogrammed to correct one or even multiple simultaneous mutations by altering the co-delivered guide RNA. Future work will expand this screening methodology to additional haploinsufficiency disorders, and applying identified delivery methods to new models of genetic disease. The ultimate goal of this work is to develop in vivo genome editing therapeutics that can be readily adapted to treat even rare or one-of-a-kind disease variants.