Choroideremia (CHM) is a rare, X-linked recessive retinal dystrophy caused by mutations in the CHM gene. CHM is ubiquitously expressed in human cells and encodes Rab escort protein 1 (REP1). REP1 plays a key role in intracellular trafficking through the prenylation of Rab GTPases, a reaction that can be reproduced in vitro. With recent advances in adeno-associated virus (AAV) gene therapy for CHM showing gene replacement to be a promising approach, an assay to assess the biological activity of the vectors is of the uttermost importance. Here we sought to compare the response of two Rab proteins, RAB27A and RAB6A, to the incorporation of a biotinylated lipid donor in a prenylation reaction in vitro. First, we found the expression of REP1 to be proportional to the amount of recombinant AAV (rAAV)2/2-REP1 used to transduce the cells. Second, prenylation of RAB6A appeared to be more sensitive to REP1 protein expression than prenylation of RAB27A. Moreover, the method was reproducible in other cell lines. These results support the further development of a prenylation reaction using a biotinylated lipid donor and RAB6A to assess the biological activity of AAV vectors for CHM gene therapy.

Recent advances in recombinant adeno-associated virus (rAAV) gene therapy for choroideremia show gene replacement to be a promising approach. It is, however, well known that contact of vector solution with plastic materials in the surgical device may result in non-specific adsorption with resulting loss of physical titer and/or level of protein expression and activity. Here we assessed the biocompatibility and stability of rAAV2-REP1 (Rab Escort Protein-1) before and following passage through the injection device over a period of time to mimic the clinical scenario. Three identical devices were screened using two concentrations of vector: high (1E+12 DNase-resistant particles [DRP]/mL) and low (1E+11 DRP/mL), to mimic high-and low-dose administrations of vector product. The low dose was prepared using either formulation buffer that contained 0.001% of a non-ionic surfactant (PF68) or balanced salt solution (BSS). We observed significant losses in the genomic titer of samples diluted with BSS for all time points. The addition of 0.001% PF68 did not, however, affect rAAV physical titer, or REP1 protein expression and biological activity. Hence we observed that neither the genomic titer nor the biological activity of a rAAV2-REP1-containing solution was affected following passage through the surgical device when PF68 was present as a surfactant and this was maintained over a period up to 10 h.

Epithelial cells were removed from bovine oviducts by enzyme digestion and either cultured on laminin-coated coverslips (for determination of [Ca2+]i) or on collagen filters (for determination of transepithelial potential difference [pd]). Cells on coverslips were loaded with Fura-2 to monitor [Ca2+]i. Application of extracellular ATP induced a transient increase in [Ca2+]i in a dose-dependent manner. This response was abolished by thapsigargin, indicating that the rise in [Ca2+]i was derived from intracellular stores. The order of potency of the nucleotide-induced rise in [Ca2+]i was uridine triphosphate (UTP)>ATP>ADP. Epithelial cells were grown on collagen filters, and when mounted in a modified Ussing chamber exhibited an electrical pd of 1.00 +/- 0.36 mV with the apical side negative with respect to the basal. Application of UTP, ATP, and ADP to the basal side induced transient increases in pd of 1.15 +/- 0.21, 0.77 +/- 0.16, and 0.26 +/- 0.06 mV, respectively. The order of potency of the nucleotides in eliciting transient increases in [Ca2+]i and pd suggests the presence of a P2u purinergic receptor in the bovine oviduct epithelium that could play a role in transepithelial ion movements and hence the control of oviductal fluid formation.

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