Identification of Novel Retinal Pericyte-Targeting rAAV Vectors Through Directed Evolution. Transl Vis Sci Technol 2022 Aug 01;11(8):28
Date
08/27/2022Pubmed ID
36018583Pubmed Central ID
PMC9428359DOI
10.1167/tvst.11.8.28Scopus ID
2-s2.0-85136924759 (requires institutional sign-in at Scopus site) 3 CitationsAbstract
PURPOSE: Retinal pericytes play a vital role in maintaining retinal homeostasis, and their dysfunction underlies pathogenesis in such vascular eye diseases as diabetic retinopathy and wet age-related macular degeneration. Consequently, retinal pericytes are attractive therapeutic targets for gene therapy, but effectively targeting pericytes with recombinant adeno-associated virus (rAAV) vectors remains a challenge.
METHODS: We introduced genetic modifications into the surface-exposed variable regions of the rAAV2/2 capsid to generate a complex library (>1 × 107) of capsid mutants that were then screened for preferential tropism toward retinal pericytes. Using the Tg(Cspg4-DsRed.T1)1Akik/J reporter mouse model, which has red fluorescent pericytes that can be isolated via flow cytometry in order to recover vector genomes, we performed three rounds of screening and identified seven putative mutants capable of transducing retinal pericytes.
RESULTS: Following intravitreal administration of mutant vectors packaging ubiquitously expressing green fluorescent protein reporters and postmortem flow cytometry of enzymatically digested retinae, two mutants in particular, Peri-E and Peri-G, demonstrated significantly greater transduction of retinal pericytes than unmodified rAAV2/2 (1.4-fold and 2.8-fold, respectively).
CONCLUSIONS: Although difficult to characterize the effect of each point mutation in the context of multiple amino acid variations from the wild-type AAV2 sequence, we identified several point mutations that may play critical roles in limiting HSPG binding, evading neutralization by murine A20 monoclonal antibodies, modulating antigenicity, and evading ubiquitination to ultimately improve transduction efficiency of retinal pericytes.
TRANSLATIONAL RELEVANCE: Identification of novel retinal pericyte targeting rAAV vectors enables the development of new, long-lasting gene therapies for retinal diseases such as diabetic retinopathy and wet age-related macular degeneration.
Author List
Patel DD, Marsic D, Periasamy R, Zolotukhin S, Lipinski DMAuthor
Daniel M. Lipinski PhD Associate Professor in the Ophthalmology and Visual Sciences department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsDependovirus
Diabetic Retinopathy
Genetic Vectors
Macular Degeneration
Mice
Pericytes
Transduction, Genetic
