Validating editing efficiency of AAV-mediated gene therapy for Rett Syndrome (RTT)
Rett Syndrome (RTT) is a genetic neurodevelopmental disorder, observed almost exclusively in females, that severely impacts social, motor, and cognitive skills. It is caused by mutations in the X-linked MECP2 gene. A class of hematopoietic stem cell (HSC)-derived clade F adeno-associated virus (AAV) or HSC-derived AAV vector (AAVHSC) is a promising gene therapy candidate with the potential to rescue MECP2 function in RTS patients. AAVHSC replaces only the mutated section of MECP2 and avoids gene overexpression, unlike traditional gene therapies.
To validate AAVHSC gene editing efficiency with high accuracy, we developed a next-generation sequencing assay to quantify the editing efficiency of the MECP2 gene from an in vitro cell model system. We extracted and poly(A) purified RNA, then performed cDNA synthesis using biotinylated primers targeting the MECP2 3’ UTR region, which act outside the AAV vector used to target the MECP2 gene. MECP2 yield was quantified through TaqMan qPCR to identify optimal assay conditions and purification methods for primer efficiency and first strand cDNA synthesis. We also isolated pure clonal cell colonies from a female RTT patient through flow cytometry, and verified clonal purity through RNA sequencing to test gene editing efficiency in clonal cells expressing only the mutant MECP2 gene. We demonstrated three key conditions—cell type, RNA quantity, and primer annealing temperature—and a purifying streptavidin bead pulldown for assay development. Further optimization will fully establish proof-of-concept for an AAV-mediated gene replacement therapy efficiency assay using gene-specific primers targeted to the 3’UTR region.