Abstract
Purpose :
Gene therapy for choroideremia is developing rapidly, as an increasing number of sites recruit patients worldwide. The subretinal delivery of the therapeutic agent, AAV2-REP1, is an established procedure in vitreoretinal surgery. Concerns exist, however, that the contact of the vector solution with the standardised surgical device results in loss of physical titre and/or level of expression and activity. Here we assessed the biocompatibility and stability of AAV2-REP1 following passage through the injection device to mimic the clinical scenario.
Methods :
Three separate surgical devices (loading syringe and delivery syringe with tip needle) were screened using two doses of research-grade AAV2-REP1: high (1E+12 DRP/mL) and low (1E+11 DRP/mL). The low dose was prepared using either formulation buffer (with PF-68 as surfactant agent) or balanced salt solution (BSS). Vector preparations were analysed at baseline and over time after loaded into the systems (+4oC for 6 hours, followed by 90 and 180 min at +20oC). Samples were collected from all devices at all time-points and their titre determined by qPCR. The levels of REP1 expression and activity were determined by WB and in vitro prenylation assay, respectively, for some of the time-points.
Results :
The mean genomic titre (DRP/mL) analysis was run ensuring good precision between sample replicates. The high-dose samples did not show any significant losses at any of the time-points. The low-dose samples, however, showed significant losses in the genomic titre of samples diluted with BSS for all time-points: up to 75% drop when compared to baseline. These were therefore excluded from protein analysis. Low-dose samples prepared with formulation buffer showed no significant difference to baseline for any of the time-points. Analysis of REP1 expression corroborated the data obtained by qPCR: no significant differences were detected in REP1 protein levels. Similarly, the levels of biotinylated Rab substrate did not vary significantly from baseline.
Conclusions :
Neither the genomic titre nor the protein stability of an AAV2-REP1-containing solution was affected following passage through the surgical device when PF-68 was present as a surfactant. We provide evidence that inclusion of this surfactant in the formulation buffer ensures biocompatibility and stability of AAV2-REP1 even at a lower dilution, over a period up to 10 hours.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.