Molecular Strategies for RPGR Gene Therapy

Abstract

Mutations affecting the Retinitis Pigmentosa GTPase Regulator (RPGR) gene are the commonest cause of X-linked and recessive retinitis pigmentosa (RP), accounting for 10%-20% of all cases of RP. The phenotype is one of the most severe amongst all causes of RP, characteristic for its early onset and rapid progression to blindness in young people. At present there is no cure for RPGR-related retinal disease. Recently, however, there have been important advances in RPGR research from bench to bedside that increased our understanding of RPGR function and led to the development of potential therapies, including the progress of adeno-associated viral (AAV)-mediated gene replacement therapy into clinical trials. This manuscript discusses the advances in molecular research, which have connected the RPGR protein with an important post-translational modification, known as glutamylation, that is essential for its optimal function as a key regulator of photoreceptor ciliary transport. In addition, we review key pre-clinical research that addressed challenges encountered during development of therapeutic vectors caused by high infidelity of the RPGR genomic sequence. Finally, we discuss the structure of three current phase I/II clinical trials based on three AAV vectors and RPGR sequences and link the rationale behind the use of the different vectors back to the bench research that led to their development.

Keywords: Retinitis Pigmentosa (RP); Retinitis Pigmentosa GTPase Regulator; adeno-associated viral; gene therapy.

Figure 1

Retinitis Pigmentosa GTPase Regulator (RPGR) gene structure and splicing variants. (A) Human RPGR gene exon-intron structure showing the combination of exons 1 to 19 to create the constitutive protein isoform, and alternative splicing of exon 15/intron 15 that creates the RPGR^ORF15 variant. (B) Mouse RPGR gene exon-intron structure showing the combination of exons 1 to 18 to create the constitutive protein isoform and alternative splicing of intron 14 creates the RPGR^OFR15 variant.

Figure 2

Clinical phenotypes associated with RPGR retinal degeneration—rod-cone phenotype (early stage (A–C) and a more advanced stage (D–F)) and cone-rod phenotype (G–I). The phenotypes are captured by Heidelberg fundus autofluorescence, (left column), MAIA microperimetry measuring central retinal sensitivity (central column; sensitivity is represented by a heat map: green/yellow—normal/mildly reduced; red/purple—reduced; black—not measurable) and Heidelberg optical coherence tomography showing retinal structures in cross-section (right column). In rod-cone phenotype there is extensive peripheral retinal atrophy with relative preservation of central retina as seen on autofluorescence associated with para-foveal hyper-autofluorescent ring (A). This is confirmed by near normal central retinal sensitivity (B) and preservation of ellipsoid zone (C). In more advanced stages of the disease there is reduction in size of the para-foveal hyper-autofluorescent ring (D) with corresponding reduction in retinal sensitivity (E) and length of ellipsoid zone (F). In contrast, in cone-rod phenotype there is early loss of para-foveal photoreceptors with associated hypo-fluorescent ring and marked reduction of retinal sensitivity with corresponding loss of the ellipsoid zone.

Figure 3

Clinical phenotype of RPGR female carriers. Fundus autofluorescence (Heidelberg) showing a typical macular radial pattern or ‘tapetal’ reflex in a female carrier of an RPGR mutation (A,B). Random X-chromosome inactivation generates clones of normal or affected photoreceptors giving rise to this mosaic pattern. Blue reflectance (C,D) and multicoloured (E,F) modes using Heidelberg scanning laser ophthalmoscope can be very helpful in showing the macular reflex.

Figure 4

AAV vector constructs used in current gene therapy trials: (A) the Nightstar Therapeutics (now Biogen Inc) trial, NCT03116113; (B) the Applied Genetic Technologies Corporation trial, NCT03316560; (C) the MeiraGTx trial, NCT03252847.