Adeno-Associated Vector-Delivered CRISPR/SaCas9 System Reduces Feline Leukemia Virus Production In Vitro

Area of Science:

• Veterinary Virology
• Gene Therapy
• Molecular Biology

Background:

• Feline leukemia virus (FeLV) causes progressive infections and death in cats.
• Regressive FeLV infections are characterized by low viral loads and effective immune responses.
• CRISPR/SaCas9 gene therapy offers a potential strategy to reduce viral loads and shift infections towards a regressive outcome.

Purpose of the Study:

• To evaluate adeno-associated virus (AAV) vectors for delivering CRISPR/SaCas9 into feline cells.
• To assess the efficiency of CRISPR/SaCas9 in targeting and disrupting the FeLV provirus.
• To investigate the potential of this gene therapy approach to reduce FeLV viral loads in vitro.

Main Methods:

• Screened nine natural AAV serotypes, two hybrid strains, and Anc80L65 for feline cell infection efficiency.
• Utilized AAV-DJ for transduction experiments due to its superior infection rates.
• Employed T7 endonuclease 1 (T7E1) and Tracking of Indels by Decomposition (TIDE) assays to confirm CRISPR/SaCas9 targeting and indel formation at 12 FeLV provirus sites.

Main Results:

• AAV-DJ demonstrated the highest efficiency in infecting various feline cell types.
• CRISPR/SaCas9 successfully introduced double-strand breaks in the FeLV provirus, with up to 80% nonhomologous end-joining (NHEJ) observed in conserved gag and pol regions.
• Transduction with AAV-DJ resulted in indel formation and a significant reduction in FeLV p27 antigen levels for specific targets in vitro.

Conclusions:

• CRISPR/SaCas9 gene therapy demonstrates efficient in vitro targeting of the FeLV provirus.
• AAV-DJ is a suitable vector for delivering gene editing systems to feline cells for FeLV therapy.
• Further in vivo studies are required to determine if this level of provirus targeting can help cats overcome FeLV infection.