Identification of Novel Retinal Pericyte-Targeting rAAV Vectors Through Directed Evolution

Area of Science:

• Ophthalmology
• Gene Therapy
• Molecular Virology

Background:

• Retinal pericytes are crucial for retinal health and are implicated in diseases such as diabetic retinopathy and wet age-related macular degeneration.
• Targeting retinal pericytes with recombinant adeno-associated virus (rAAV) vectors is a promising gene therapy strategy but faces challenges in achieving efficient delivery.

Purpose of the Study:

• To develop novel rAAV vectors with enhanced tropism for retinal pericytes.
• To overcome current limitations in gene therapy delivery to retinal pericytes for treating vascular eye diseases.

Main Methods:

• A library of over 10 million rAAV2/2 capsid mutants was generated with modifications in surface-exposed variable regions.
• A reporter mouse model (Tg(Cspg4-DsRed.T1)1Akik/J) with fluorescently labeled pericytes was used for three rounds of screening.
• Flow cytometry and vector genome recovery were employed to identify and isolate pericyte-transducing mutants.

Main Results:

• Two specific rAAV mutants, Peri-E and Peri-G, demonstrated significantly enhanced transduction of retinal pericytes compared to unmodified rAAV2/2.
• Peri-E showed a 1.4-fold increase, and Peri-G showed a 2.8-fold increase in retinal pericyte transduction after intravitreal administration.
• Mutant vectors successfully delivered reporter genes to retinal pericytes, confirmed by postmortem flow cytometry.

Conclusions:

• Novel rAAV vectors (Peri-E, Peri-G) have been identified that effectively target retinal pericytes.
• Specific mutations likely contribute to improved transduction by modulating HSPG binding, antibody neutralization, antigenicity, and ubiquitination.
• These findings enable the development of advanced gene therapies for debilitating retinal vascular diseases.