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Aaron D. Black, Jean Bennett; Gene Replacement Therapy for Choroideremia. Invest. Ophthalmol. Vis. Sci. 2011;52(14):486.
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© ARVO (1962-2015); The Authors (2016-present)
Choroideremia is an inherited form of retinal degeneration caused by mutations in the X-linked CHM gene, which result in the lack of the gene’s protein product, REP-1. Our goal was to generate a recombinant adeno-associated virus (rAAV) vector which would allow intracellular production of exogenous functional REP-1 protein. This could ultimately be used as a potential treatment for choroideremia.
The coding sequence for the human CHM (hCHM) gene was inserted into an AAV2 construct under the control of a cytomegalovirus (CMV) enhancer and chicken beta actin (CBA) ubiquitous promoter. The resulting construct was then transfected into CHO-K1 cells and the cell lysates probed for human REP-1 by western blotting. The construct was then used to produce a rAAV vector, AAV2/8.CMV.CBA.hCHM. CHO-K1 cells were then transduced with AAV2/8.CMV.CBA.hCHM at multiple MOIs and cell lysates probed for human REP-1 via western blot.
Transfection of CHO-K1 with the hCHM construct resulted in robust human REP-1 expression observed by western blotting of cell lysates. Transduction of CHO-K1 cells with AAV2/8.CMV.CBA.hCHM at a range of multiplicities of infection resulted in a clear dose response in the production of REP-1 observed by western blotting of cell lysates.
Preliminary experiments with a rAAV vector expressing hCHM have demonstrated that high levels of REP-1 expression can be attained in vitro. Further experiments now underway will seek to ascertain whether REP-1 expression can also be induced in cells cultured from effected individuals and, in vivo, in mouse retinal tissue. Previous clinical trials have demonstrated that rAAV vectors can be an effective and safe means of gene replacement therapy in the eye, the development of a robust rAAV vector marks a significant step towards the potential treatment of choroideremia in humans.
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