AAV integration in human hepatocytes

Area of Science:

• Gene Therapy
• Molecular Biology
• Hepatology

Background:

• Recombinant adeno-associated viral (rAAV) vectors are promising for liver-directed gene therapy.
• rAAV is typically considered an episomal vector, but its genome can undergo recombination and integrate into host DNA.

Purpose of the Study:

• To determine the chromosomal integration frequency of rAAV in human hepatocytes.
• To analyze the integrity of integrated rAAV sequences and associated host genomic changes.

Main Methods:

• Human hepatocytes were transduced with rAAV ex vivo and in vivo.
• Cells were expanded in a mouse model of xenogeneic liver regeneration.
• Capture-PacBio sequencing, a long-read NGS method, was used to detect integration events and vector integrity.

Main Results:

• Chromosomal rAAV integration occurred at a high frequency of 1%-3% in both in vitro and in vivo settings.
• The majority of integrated rAAV sequences were significantly rearranged.
• Host genomic sequences at integration sites frequently exhibited deletions.

Conclusions:

• rAAV integration into the host genome is more frequent than previously assumed.
• Rearrangements and host DNA deletions at integration sites raise concerns for rAAV gene therapy safety and efficacy.