Bioengineered Hybrid Rep 2/6 Gene Improves Encapsulation of a Single-Stranded Expression Cassette into AAV6 Vectors.

Area of Science:

• Molecular Biology
• Gene Therapy
• Virology

Background:

• Clinical-grade adeno-associated viral (AAV) vector production is critical for gene therapy advancement.
• Current AAV research focuses on capsids, immunogenicity, and scale-up, with limited exploration of non-structural Rep genes.
• AAV serotype 2 Rep genes are traditionally used for packaging vector genomes.

Purpose of the Study:

• To investigate the potential of manipulating AAV Rep genes for improved vector production.
• To engineer a novel Rep2/6 hybrid gene by mutating AAV serotype 6 Rep.
• To evaluate the packaging ability and in vivo activity of the Rep2/6 hybrid.

Main Methods:

• Mutated four variable amino acids in the AAV serotype 6 Rep binding domain.
• Generated a Rep2/6 hybrid gene.
• Compared packaging ability, in vivo activity, and full/empty capsid ratios with wild-type Rep6 and standard AAV6 vectors.

Main Results:

• The Rep2/6 hybrid demonstrated improved packaging ability compared to wild-type Rep6.
• AAV vectors produced with Rep2/6 showed comparable in vivo activity to standard AAV6 vectors.
• The Rep2/6 hybrid enhanced the ratio of full to empty AAV capsids.

Conclusions:

• Rep gene bioengineering is a viable strategy to improve AAV vector manufacturing.
• The Rep2/6 hybrid offers a promising tool for increasing the efficiency of producing fully encapsulated AAV vectors.
• This advancement could significantly impact the upstream manufacturing processes for gene therapy trials.