UC Berkeley

Gene therapy, the delivery of therapeutic genetic material to diseased cells, has demonstrated clinical efficacy for numerous inherited disorders in the last decade. Recombinant adeno-associated viral (AAV) vectors, in particular, have enabled the development of breakthrough drugs for several rare genetic diseases, including biallelic RPE65-associated retinal dystrophy, spinal muscular atrophy Type I, and hemophilia B, due to their ability to safely mediate long-term transgene expression in human patients. While this clinical success has inspired widespread enthusiasm about the promise of AAV-mediated gene delivery to treat a variety of diseases, broad applicability of AAV vectors for gene therapy demands enhanced transduction efficiency and targeting of therapeutically relevant tissues. Innovative strategies that grant greater functional benefit, in conjunction with improved disease models, are also pivotal for realizing the full potential of AAV-based therapies in treating human disorders. Such challenges have driven my dissertation research in two major directions: engineering new AAV capsid variants to address unmet clinical needs, and combining AAV vectors and CRISPR/Cas9 technologies to efficiently generate preclinical models of human disorders and to correct disease-causing mutations through genome editing.

The AAV delivery problem has been tackled by engineering the viral capsid to evade immune detection, circumvent biological barriers, and exhibit high infectivity of clinically important tissues. In this dissertation, I describe the latest trends in AAV vector design that harness cell-type-specific selection schemes and our rapidly accumulating knowledge of AAV capsid structure and function to overcome longstanding obstacles in the gene therapy field. Of note, these techniques have yielded novel AAV variants with desirable biodistribution in the central nervous system, stimulating strong interest in adapting them for sparse neuronal labeling and physiological studies of the brain and spinal cord. I also discuss next-generation sequencing platforms that have powered unprecedented, high-throughput phenotypic profiling of AAV vectors and lent invaluable insight to AAV capsid engineering.

In addition to the development of more potent vectors, the efficient generation of preclinical animal models is key to continued progress in gene therapy. Specifically, genetically engineered mouse models of disease enable early assessment of safety and efficacy of new therapeutics. These mouse lines harbor large sequence insertions or modifications and thus require complex genomic manipulation; however, existing methods to produce such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP Electroporation and AAV Donor Infection) that couples AAV-mediated donor delivery with Cas9/sgRNA ribonucleoprotein (RNP) electroporation to induce large homology-directed, site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice, demonstrating that CRISPR-READI is applicable to most widely used knock-in schemes with potential applications in other mammalian species.

AAV vectors and CRISPR-based tools can also be leveraged for permanent correction of debilitating genetic diseases through genome editing in somatic tissues. Duchenne muscular dystrophy (DMD) is a particularly attractive disease target, as the massive DMD gene limits the feasibility of simple gene addition as a therapeutic approach. To date, most gene editing strategies for DMD employ reframing or exon skipping mediated by non-homologous end joining (NHEJ) to bypass mutations that disrupt the DMD open reading frame and produce a truncated but partially functional dystrophin protein. However, restoration of full-length dystrophin expression has not yet been achieved. To overcome this challenge, we devised a novel “excise-and-replace” approach that harnesses AAV-mediated co-delivery of CRISPR/Cas9 and a homology-independent targeted integration (HITI) donor to correct the DMD locus. Through dual gRNA directed deletion and repair of a target exon, our strategy yields wildtype dystrophin transcripts in an in vitro human cardiomyocyte model of DMD. To improve gene targeting efficiency, we also examine the ability of a third gRNA to preferentially drive desired transgene insertion over other genomic arrangements. Our gene correction method can potentially be applied to various exon deletions and small mutations in DMD to rescue full-length dystrophin expression and confer maximal clinical benefit in afflicted muscle cells.

In summary, my dissertation research focuses on the ongoing evolution of new AAV capsids and the creation of advanced genome editing strategies for generating preclinical animal models and treating human disease. Together, these technologies make important contributions to the development of improved AAV-mediated gene therapies.