The Biological Activity of AAV Vectors for Choroideremia Gene Therapy Can Be Measured by In Vitro Prenylation of RAB6A

Abstract

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.

Beneficial effects on vision in patients undergoing retinal gene therapy for choroideremia

“…During the trial period, the clinical-grade gene therapy vector was tested annually to confirm REP1 protein expression and prenylation activity using in vitro assays. 33 Briefly, HEK293 cells were transduced with clinical-grade AAV2. Table S1).…”

Recombinant Adeno-Associated Viral Vectors (rAAV)-Vector Elements in Ocular Gene Therapy Clinical Trials and Transgene Expression and Bioactivity Assays

“…Then, 72 hours later, the cells are lysed by adding lysis buffer, and the lysate is incubated with all-trans-retinol and CRALBP for 2 hours in the dark to assess the enzymatic activity of RPE65 (an isomerohydrolase) to convert all-trans-retinol to 11-cis-retinol [ 260 ]. The rAAV- REP1 for the treatment of choroideremia is assessed on in vitro prenylation of RAB6A in HEK293 cells [ 261 ]. The directly injectable dose can also be assayed.…”

Adeno-Associated Viral Gene Therapy for Inherited Retinal Disease

“…The ability to restore functional REP1 expression by AAV2-mediated delivery of REP1 to affected cells was first investigated in in vitro models of choroideremia (83-85). Defective lymphocytes and fibroblasts devoid of REP1 were isolated from patients with choroideremia and used to test the ability of the AAV-REP1 vector to rescue the expression and function of the REP1 protein (84).…”

“…Results demonstrated the effective delivery of REP1 to the affected cells, as well as restoration of REP1 protein expression and function (84). In addition, in vitro reproduction of prenylation has been used to demonstrate the activity of REP1 delivered by an AAV2 vector (85). …”