Gene Editing of Primary Rhesus Macaque B Cells.

Area of Science:

• Immunology
• Gene Therapy
• Primate Models

Background:

• B cells are key for antibody production and attractive targets for gene editing therapies.
• Existing gene editing protocols are effective in mice and humans but lack scalability for larger animal models.
• Rhesus macaques are crucial for preclinical studies of novel therapeutics.

Purpose of the Study:

• To establish a feasible and scalable protocol for gene editing primary B cells in rhesus macaques.
• To enable in vitro studies of B cell-based therapeutics in a relevant non-human primate model.
• To facilitate the development of advanced gene therapies using B cells.

Main Methods:

• Developed CRISPR/Cas9 gene editing conditions for rhesus macaque B cells from peripheral blood and spleen.
• Optimized in vitro culture methods for primary B cells.
• Created a rapid protocol for producing recombinant adeno-associated virus serotype 6 (AAV6) for homology-directed repair, utilizing a novel adenoviral helper vector.

Main Results:

• Successfully demonstrated efficient in vitro gene editing of primary rhesus macaque B cells.
• Established conditions for culturing and genetically modifying these cells.
• Developed a robust method for generating AAV6 vectors for targeted integration of large DNA cassettes (<4.5 kb).

Conclusions:

• The developed protocol provides a critical tool for studying B cell therapeutics in rhesus macaques.
• This advancement overcomes previous scalability limitations for gene editing in larger animal models.
• Facilitates preclinical evaluation of gene-edited B cell therapies in a primate model relevant to human biology.