There have been an increasing number of successful human gene therapy clinical trials, and in particular gene delivery vehicles or vectors based on the adeno-associated virus (AAV) have enabled success in trials for hemophilia B, spinal muscular atrophy, X-linked myotubular myopathy, and Leber’s congenital amaurosis type 2 (LCA2). The LCA2 trials led to the first FDA approval of a gene therapy for rare disease in the US in December, 2017.  That said, vectors based on natural versions of AAV face a number of delivery challenges that limit their efficacy and will thus preclude the extension of these successes to the majority of human diseases.  These delivery limitations arise since the parent viruses upon which these vectors are based were not evolved by nature for our convenience to use as human therapeutics.  We have been developing and implementing a high throughput approach termed directed evolution – involving the iterative genetic diversification of a viral genome and functional selection for desired properties – to engineer highly optimized variants of AAV for a broad range of cell and tissue targets.

In parallel, the advent of genome editing technologies such as the CRISPR/Cas9 system raise the possibility of using gene delivery not only for gene replacement but for repair or knockout of endogenous genes.  We have thus been combining engineered AAVs with CRISPR/Cas9 for a range of applications.  For example, delivery of a Cas9 targeting mutant superoxide dismutase (SOD1) in a murine model of amyotrophic lateral sclerosis delayed the onset of disease symptoms and significantly extended animal lifespan.  In addition, we engineered an AAV for enhanced transport along neuronal axons, and delivery of Cas9 enabled the knockout of specific genes in vivo within projection neurons from a distance, work with both basic and translational applications.  Finally, we have combined AAV delivery of donor constructs with Cas9 ribonucleoproteins to effect efficient homologous recombination within in genomic target loci.  The integration of these new technologies – Cas9 cargo with AAV delivery – can thus enable a broad range of basic and therapeutic applications.

Learning Objectives: 

1. Adeno-associated viral vectors have the capacity to mediate efficient delivery of CRISPR/Cas9, leading to effective genome editing in vivo.
2. As a result, CRISPR/Cas9 is an effective tool to knock out autosomal dominant alleles that underlie human disease.