CMV-SaCas9 AAV (Serotype 9)

AAV is the foundation of a multi-billion dollar industry, with hundreds of clinical trials using AAV delivery systems. Viral vector biotechs are the gene therapy platform of choice, with recombinant adeno-associated virus (rAAV) vectors often being the first choice due to their low toxicity, reliance on other viruses for replication, broad tropism, and ability to infect both dividing and non-dividing cells. Zolgensma uses rAAV9 to deliver a functional copy of the human SMN1 (survival of motor neuron 1) gene to patients with spinal muscular atrophy (SMA). SMA is the most common fatal monogenic disease in infants and is caused by autosomal recessive mutations in the survival motor neuron 1 gene (SMN1). Additional rAAV therapies are in development, with dozens of clinical trials currently underway. Commercial and research rAAV production typically involves a “triple transfection” approach. The triple transfection assembly consists of a plasmid encoding the transgene, a helper plasmid containing adenovirus type 5 (Ad5) helper genes (i.e., E1a/b, E2a, E4, and VA RNA) or their equivalents, and another plasmid encoding rAAV Rep and Cap proteins. Variants include combining helper and rAAV plasmid functions on a single plasmid or stable expression of helper functions preprogrammed into mammalian cell lines. Vector production improvements focus on particle metrics, such as the ratio of empty to non-empty vector particles. Increasing the number of full particles would reduce the number of particles required for gene therapy, thereby improving safety and efficacy.