Advances in Production of rAAV Through Mutations to the Cap Gene Sequence

• Balamurugan Saravanan, Chemical Engineering, University of Delaware

• Mark Blenner, Biomolecular and Chemical Engineering, University of Delaware

Gene Therapy is an emerging medical approach that treats and/or prevents diseases by correcting the underlying genetic problem. Recombinant Adeno-Associated Virus (AAV) is the ideal vector for gene therapy as it is non-Immunogenic, performs natural degradation, and has proven to be a safe and effective strategy in preclinical and clinical settings. AAV is administered as a drug delivery by replacing the viral DNA with modified DNA, which is the coded vector. However, AAV Production poses difficulties in production costs, scalability, and capsid ratio. Therefore, to correct the difficulties, a proposition to mutate the Cap Gene Sequence in the AAV is posed. The goal is to make a silent mutation to the cap sequence such that the three capsid proteins (VP1, VP2, and VP3) and MAAP/AAP will not be affected. The silent mutation is performed through a change of one of the bases in the DNA sequence of the cap gene to change the entirety of the nucleotide sequence. Although the nucleotide chain will change, the amino acids associated with it will remain the same. The purpose of the silent mutation is to prevent the BsmBI from cutting the sequence. The BsmBI restriction enzyme recognizes a sequence and cuts next to it, therefore a change to the bases in that sequence will prevent the cut, and corresponding amino acids will remain the same. A successfully mutated assembly plasmid gave way to perform an experiment on HEK293T cells (immortalized human embryonic kidney cells used for retroviral production) which evaluates cap mutations in the plasmid design to examine the AAV production levels. Data collected examines the concentration of successful AAV produced through triple transfection. These production levels are compared to the non-mutated AAV production levels to examine any improvements.