Therapeutic gene editing strategies for neurological and renal disorders

Abstract

Driven by the desire to understand, manipulate, and potentially correct the genetic code that underlies life, scientists have long sought tools capable of predictably editing DNA. The discovery of CRISPR-Cas-based technologies has ushered in an era of highly precise, programmable genome editing tools suitable for the correction of genetic disease. In this thesis, I apply two such tools, base editing (BE) and prime editing (PE) to the correction of both neurological and renal disorders. In the first chapter, I provide an overview of CRISPR-Cas DNA cleavage, BE, and PE, highlighting some of the many discoveries that led to their development and comparing the capabilities of these technologies. I also describe recent improvements to each technology and their current clinical use. In the second chapter, I describe the proof-of-concept gene editing correction of Dravet syndrome (DS), a severe neurodevelopmental disorder characterized by drug-resistant epilepsy, temperature-sensitive seizures, cognitive impairment, and a high incidence of sudden unexpected death in epilepsy (SUDEP). DS is caused by loss-of-function variants in SCN1A, which encodes the voltage-gated sodium channel ⍺ subunit Nav1.1. Current clinically-approved DS treatments manage symptoms and do not address the root cause of the disease. In this chapter, I describe the use of adenine base editing (ABE) to directly correct SCN1AR613X, a variant found in DS patients. We identified ABE strategies to efficiently correct R613X in engineered homozygous SCN1AR613X HEK293T and Neuro-2a cells (72% and 92% correction efficiencies, respectively). We used a dual-AAV9 approach to deliver an optimized ABE system to Scn1aR613X/+ mice, which recapitulate key DS pathologies. AAV9-ABE treatment of Scn1aR613X/+ neonates resulted in efficient DNA and mRNA editing (59% and 97%, respectively, in bulk neocortex), restored parvalbumin-expressing inhibitory neuron excitability and function, rescued mice from hyperthermia-induced seizures, and led to a 7.0-fold improvement in 45-day survival over vehicle-treated mice. These findings represent the direct correction of the cause of DS, validate a strategy to correct SCN1A variants with ABE, and suggest the potential of precision genome editing treatments for DS and other neurodevelopmental disorders. In the third chapter, I describe efforts towards the development of precision gene editing treatments for three genetic causes of chronic kidney disease (CKD). The first target, MUC1 kidney disease (MKD) is an autosomal dominant proteinopathy affecting epithelial cells of the kidney nephron. MKD is caused by a heterozygous +1C frameshift mutation in a variable number tandem repeat region (VNTR) of MUC1 that is challenging to assay by high-throughput sequencing. To optimize prime editing (PE) conditions for this locus, we used a pooled, self-targeting lentiviral library to screen over 11,000 pegRNAs that precisely delete this +1C insertion. From this screen, we find multiple hit strategies that achieve >30% correction with interpretable characteristics. The second target, autosomal dominant tubulointerstitial kidney disease (ADTKD)-UMOD is a toxic proteinopathy caused by mutations in UMOD, which encodes uromodulin. We sought to develop an ABE treatment for a humanized UMOD-p.C77Y rat model, which mimics a variant found in a family with the disease. After in vitro optimization in a UMOD-p.C77Y HEK293T cell line, we find an ABE condition capable of 61.9% correction with 1.1% nonsilent bystander editing. We performed retrograde ureter injections of dual-AAVs packaging this ABE strategy and observed no renal transduction. The third target, X-linked Alport syndrome (AS), is caused by loss-of-function mutations in COL4A5, which encodes an extracellular collagen protein necessary for podocyte function in the kidney glomerulus. We developed a gene editing strategy to correct a Col4a5-p.G5X murine model which recapitulates a variant found in AS patients. Through systemic injection of a dual-AAV ABE editing strategy, we achieved 24.0% stop codon conversion in cDNA from isolated glomeruli, demonstrating glomerular dysfunction caused by AS pathology rendered affected podocytes accessible to AAV delivery. Collectively, these findings motivate further translational studies for precision gene editing of both DS and several genes causing CKD. I conclude this thesis with a brief discussion of future directions for these targets as well as forward-looking statements to the field of therapeutic genome editing at large.