Letter to the editor: ‘Emerging gene therapy products for RPGR-associated X-linked retinitis pigmentosa’

KEYWORDS:

• Adeno-associated virus (AAV)
• gene therapy
• inherited retinal diseases
• retinitis pigmentosa
• retinitis pigmentosa GTPase regulator (RPGR)

This letter is to comment on the review article ‘Emerging gene therapy products on RPGR-associated retinitis pigmentosa’ by Martinez-Fernandez de la Camara and colleagues published in this issue of Expert Opinion On Emerging Drugs [1].

We would like to bring to the Editor’s notice our concerns regarding what we perceive as an unbalanced review of the existing scientific literature around RPGR biology and its relation to retinal gene therapy development, as well as the speculative, and in parts factually wrong conclusions that are drawn during the manuscript’s discussion of four clinical-stage experimental gene therapies.

We hereby provide clarifications on the structure and function of the RPGR gene, as well as the design and efficacy data of one of the gene therapy vectors discussed in the aforementioned manuscript. In addition, since the acceptance of the aforementioned manuscript for publication, the development program for the codon optimized RPGR vector developed by the University of Oxford/Nightstar Therapeutics Ltd. (sponsored by Biogen Inc.) has been suspended after Biogen announced in May 2021 that the primary endpoint was not met in its Phase 2/3 XIRIUS clinical trial.

1. The RPGR gene sequence and messenger RNA sequence

• The RPGR gene is transcribed into a full-length pre-mRNA, which then gives rise to multiple mature mRNA transcripts via alternative splicing. These different mRNA splice forms are then translated into different isoforms of the RPGR protein.

• In healthy human retinas, RPGR.ORF15 mRNA transcripts with in-frame deletions in the purine-rich repeat region (similar to the MeiraGTx vector see below) have been identified [2,3]. Indeed, the pleiotropy of RPGR.ORF15 and its glutamylated isoforms is also evident in healthy human retina immunoblots ([4] – Fig.1 H).

2. The RPGR protein in the retina

• The region of the RPGR protein corresponding to the purine-rich repeat sequence in ORF15 has >80% homology across a range of species, although the length of this region varies.

• The variability of the size and sequence of this region between species suggests that the precise length of the corresponding protein domain is not critical to endogenous RPGR function.

• Functional RPGR protein expressed in photoreceptors localizes to the cilia region of the cell, where it plays a role in the transport of proteins from the inner to the outer segment.

3. MeiraGTx viral vector construct AAV5-hRKp.RPGR

Vector structure and configuration; efficacy in murine model, human retinal organoids, and preliminary clinical benefit:

• The gene sequence used in the MeiraGTx construct contains a small in-frame deletion in the purine-rich repetitive region of ORF15.

• In-frame deletions in this region are present in the RPGR mRNA in retina of normally seeing individuals (see above and [2,3]) – while full length transcripts have never been isolated.

• The RPGR.ORF15 sequence in AAV5-hRKp.RPGR has not been codon-optimized and gives rise to a functional RPGR protein with a 126aa-shortened internal repeat domain. The AAV5-hRKp.RPGR sequence does not give rise to proteins prematurely truncated at the 3’ end.

• The MeiraGTx construct when delivered to the retinas of transgenic mice lacking RPGR [5], enabled restoration of RPGR gene function in terms of:

  • Restoration of correct localization of the RPGR protein encoded by AAV5-hRKp.RPGR to the cilia region of the photoreceptors, at endogenous levels normally found in healthy photoreceptors.

  • Correction of the mislocalization of cone opsin and rhodopsin in the outer segment of the photoreceptors observed in retinas lacking functional RPGR expression.

  • Amelioration of photoreceptor degeneration and the normalization of photoreceptor morphology.

  • Durable rescue of retinal function as measured by light-evoked electrophysiological responses up to 15 months following treatment.

• In addition to studying AAV5-hRKp.RPGR in mice, the MeiraGTxconstruct was also tested in human retinal organoids grown from iPSCs derived from patients with a disease-causing lack of function variant in the RPGR gene as well as CRIPSR-generated RPGR-deficient human retinal organoids ([6] and data presented at ARVO 2022). When human retinal organoids lacking RPGR were treated with the MeiraGTx construct:

  • RPGR protein was produced which localized to the correct position in the ciliary region of both rod and cone photoreceptors.

  • Strong levels of RPGR glutamylation, assessed by either immunoblotting or immunohistochemistry, occurred at the photoreceptor cilia margin, indicating correct glutamylation of the RPGR transgene-derived protein.

  • Significant reduction of GFAP (a known retinal cell stress marker) in RPGR-deficient human retinal organoids following treatment with the vector construct.

  • Significant reduction of rhodopsin mislocalization in human photoreceptors of treated RPGR-deficient human retinal organoids.

4. Preliminary clinical evidence of benefit

• Significant improvements on retinal function, functional vision, and visual function of AAV5-hRKp.RPGR has also been demonstrated in preliminary data from Phase I/II studies, out to 12 months following treatment.

• MeiraGTx and Janssen Pharmaceuticals, Inc., one of the Janssen Pharmaceutical Companies of Johnson & Johnson, are jointly developing AAV5-hRKp.RPGR as part of a broader collaboration to develop and commercialize gene therapies for the treatment of inherited retinal diseases.

5. Additional manuscript comments

With regard to the above functional data [5], Martinez-Fernandez de la Camara and colleagues speculated on the significance of these findings based on a factually wrong conclusion stemming from the fact that the mouse RPGR.ORF15 region is shorter than the human one (in nucleotide count). The authors link the reduced nucleotide count to the shortened RPGR.ORF15 gene sequence in AAV5-hRKp.RPGR and directly pointing to a potentially reduced glutamylation profile of the mouse RPGR.ORF15 or indeed glutamylation levels that mouse photoreceptors would require for normal function. The assumption that glutamylation levels are linked to the mere size of the repetitive RPGR.ORF15 region is not correct. Levels of glutamylation are more accurately predicted by the presence of glutamylation sites (GEEEG). These motifs exist in higher numbers in the mouse RPGR.ORF15 sequence compared to the human RPGR.ORF15 sequence: 10–11 in human vs 18–22 in mouse, depending on which partial sequences are used for full length sequence generation. This was also noted twice in a paper the authors reference in their manuscript [4]. As such, the argument that the mouse species does not adequately model the need for glutamylation to maintain the integrity and function of mouse RPGR-ORF15 is not a plausible one. Furthermore, it was the mouse model studies that demonstrated the importance of glutamylation and the functional significance of human TTLL5 mutations in the first place.

With regard to our reference of an unbalanced review of the current literature and in addition to the above points on drawn assumptions failing to take into account data of a referenced study [5] as well as not including the published work of West and colleagues showing human retinal organoid rescue data with the shortened RPGR.ORF15 construct [6], there is further incomplete referencing: The authors refer to a study from Hong et al., 2004 [7] suggesting that a shortened RPGR.ORF15 may cause a ‘severe phenotype due to a gain-of-function mechanism.’ In that study, a construct bearing a 654bp deletion in the RPGR.ORF15 was placed downstream of a strong, intronless CMV promoter for generation of transgenic lines. In that study, the phenotype observed was not a consequence of a shortened in-frame RPGR.ORF15 sequence, but rather because of promoter-driven aberrant mis-splicing that gave rise to nonsense mutations leading to premature stop codons. Hong and colleagues note this in the manuscript and changed the construct to drive expression for further work using a CBA promoter (also called CAG): ‘The CBA promoter contained an artificial intron and in our pilot studies CBA promoter-expression constructs appeared to reduce aberrant splicing compared with the conventional CMV promoter, allowing more faithful expression of the ORF15 sequence’ [7]. Hong and colleagues used the new construct for a follow-up study that was published a year later 8] where they showed the exact same shortened construct (654bp in-frame deletion) provided a structural and functional rescue in an RPGR-deficient background (in line with the AAV-mediated rescue seen by the MeiraGTx construct in Pawlyk et al., 2016) [4] Surprisingly, the second Hong et al.; 2005 study 8] is not referenced or discussed in the Martinez-Fernandez de la Camara and colleagues’ manuscript.

6. Summary

• Data from preclinical in vitro and in vivo studies and phase 1/2 clinical trials provide evidence supporting the expression of functional RPGR protein in photoreceptors following AAV5-hRKp.RPGR transduction.

• There is no evidence demonstrating that the gene sequence used in AAV5-hRKp.RPGR contains an ‘unstable’ DNA sequence that results in a higher rate of spontaneous mutation or that the RPGR protein resulting from AAV5-hRKp.RPGR transduction lacks function in murine or human photoreceptors.

• There is evidence to suggest that this particular sequence shows improved stability relative to wild-type full length RPGR.ORF15 one, which is key to both consistency in manufacturing of the product and reproducible functional activity of clinical supply materials. Reproducible functional activity is an important part of the release testing of the product.

• There is no evidence that the transcripts derived from AAV5-hRKp.RPGR are incompletely translated resulting in ‘truncated’ proteins.

• A number of gene therapy programs targeting RPGR are currently underway, each with differing designs for the vector used for RPGR gene replacement. While there are distinct design elements in each program, functional advantages of one design over another, while open to speculation, are not demonstrated by scientific experimentation, and claims for comparatively superior efficacies and safety in particular designs are not founded on direct empirical evidence.

• The preliminary efficacy and safety data from the Phase 1/2 clinical study sponsored by MeiraGTx support the utility of the AAV5-hRKp.RPGR in treatment of subjects with RPGR-XLRP. A Phase 3 pivotal clinical study is currently enrolling patients.

Declaration of interest

The authors are affiliated with MeiraGTx. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was not funded.