Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-associated X-linked Retinitis Pigmentosa

Giacalone JC, Andorf JL, Zhang Q, Burnight E, Ochoa D, Reutzel AJ, Collins MM, Sheffield V, Mullins RF, Han IC, Stone E, Tucker BA

In a screen of 1000 consecutively ascertained families we recently found that mutations in the gene RPGR are the 3rd most common cause of all inherited retinal disease. As the two most frequent disease-causing genes, ABCA4 and USH2A, are far too large to fit into clinically relevant AAV vectors, RPGR is an obvious early target for AAV based ocular gene therapy. In generating plasmids for this application, we discovered that those containing wild-type RPGR sequence, which includes the highly repetitive low complexity region ORF15, were extremely unstable (i.e., they showed consistent accumulation of genomic changes during plasmid propagation). To develop a stable RPGR gene transfer vector we used a bioinformatics approach to identify predicted regions of genomic instability within ORF15 (i.e. potential non-B DNA conformations). Synonymous substitutions were made in these regions to reduce the repetitiveness and increase the molecular stability while leaving the encoded amino acid sequence unchanged. The resulting construct was subsequently packaged into AAV serotype 5 and the ability to drive transcript expression and functional protein production was demonstrated via subretinal injection in rat and pull down assays respectively. By making synonymous substitutions within the repetitive region of RPGR we were able to stabilize the plasmid and subsequently generate a clinical grade gene transfer vector (IA-RPGR). Following subretinal injection in rat, we demonstrated that the augmented transcript was expressed at levels similar to wild-type constructs. By performing in vitro pull down experiments we were able to show that IA-RPGR protein product retained normal protein binding properties (i.e. analysis revealed normal binding to PDE6D, INPP5E and RPGRIP1L). In summary, we have generated a stable RPGR gene transfer vector capable of producing functional RPGR protein, which will facilitate safety and toxicity studies required for progression to an IND application.

Human Gene Therapy
Publication Date: 
Mon, 05/20/2019
Pubmed ID: