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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=iemd20 Expert Opinion on Emerging Drugs ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/iemd20 Response: ‘letter to the editor: emerging gene therapy products for RPGR-associated X-linked retinitis pigmentosa’ Cristina Martinez-Fernandez de la Camara, Jasmina Cehajic-Kapetanovic & Robert E. MacLaren To cite this article: Cristina Martinez-Fernandez de la Camara, Jasmina Cehajic-Kapetanovic & Robert E. MacLaren (2022) Response: ‘letter to the editor: emerging gene therapy products for RPGR-associated X-linked retinitis pigmentosa’, Expert Opinion on Emerging Drugs, 27:4, 449-450, DOI: 10.1080/14728214.2022.2152212 To link to this article: https://doi.org/10.1080/14728214.2022.2152212 Published online: 23 Dec 2022. Submit your article to this journal Article views: 926 View related articles View Crossmark data https://www.tandfonline.com/action/journalInformation?journalCode=iemd20 https://www.tandfonline.com/journals/iemd20?src=pdf https://www.tandfonline.com/action/showCitFormats?doi=10.1080/14728214.2022.2152212 https://doi.org/10.1080/14728214.2022.2152212 https://www.tandfonline.com/action/authorSubmission?journalCode=iemd20&show=instructions&src=pdf https://www.tandfonline.com/action/authorSubmission?journalCode=iemd20&show=instructions&src=pdf https://www.tandfonline.com/doi/mlt/10.1080/14728214.2022.2152212?src=pdf https://www.tandfonline.com/doi/mlt/10.1080/14728214.2022.2152212?src=pdf http://crossmark.crossref.org/dialog/?doi=10.1080/14728214.2022.2152212&domain=pdf&date_stamp=23 Dec 2022 http://crossmark.crossref.org/dialog/?doi=10.1080/14728214.2022.2152212&domain=pdf&date_stamp=23 Dec 2022 Letter to the Editor Response: ‘letter to the editor: emerging gene therapy products for RPGR- associated X-linked retinitis pigmentosa’ Cristina Martinez-Fernandez de la Camaraa,b, Jasmina Cehajic-Kapetanovica,b and Robert E. MacLarena,b aDepartment of Clinical Neurosciences, Nuffield Laboratory of Ophthalmology, University of Oxford, John Radcliffe Hospital, Oxford, UK; bOxford Eye Hospital, Oxford University Hospitals NHS Trust, John Radcliffe Hospital, Oxford, UK KEYWORDS AAV; gene therapy; eye; ciliopathy; RP3; glutamylation We are grateful for the opportunity to respond to the letter [1] from anonymous employees of MeiraGTx in relation to our ‘Opinion’ article, in which we discuss gene therapy approaches for RPGR-related retinitis pigmentosa (RP) [2]. The RPGR vector being tested by MeiraGTx in human clinical trials contains a large in-frame deletion (378 base pairs), resulting in a loss of just over one-third of the entire ORF15 coding sequence [3]. The mouse is not an ideal model in which to test the efficacy of a shortened human RPGR-ORF15 vector for two reasons. First, the degeneration in the mouse is very mild compared to humans and so might be ameliorated by a weaker vector. Second, the mouse ORF15 region is shorter than the human equivalent. Hence, a deleted human ORF15 may be closer to the mouse sequence than that of the human. Since a larger in- frame deletion of the ORF15 region fails to rescue even the mouse phenotype [3], then it is natural to ask whether or not the 378 base pair deleted variant is as effective as the full- length RPGR sequence in humans. Our article is biased only in the sense that we could not find any beneficial reason for removing this critical part of the RPGR coding sequence before testing it in patients. MeiraGTx has provided no argument as to how this shor- tened variant might be better than or even as good as the full-length protein. The vectors developed by AGTC and Biogen both contain the full-length RPGR sequence. Whilst it is true that small in-frame deletions of up to 36 base pairs have been detected on rare occasions [4], the effect of an in-frame deletion 10 times larger than this is unknown, because deletions of this size have never been detected in the human population. There is also no logic as to how this might stabilize the RPGR coding sequence, because repeti- tive purine (GA) sequences will still exist flanking the dele- tion and the intron 15 splice donor site (which might lead to aberrant splicing [5]) has not been inactivated. Shortened RPGR coding sequences with in-frame ORF15 deletions fre- quently occur as a result of cloning errors due to the repetitive purine repeats in this region. Of course, we do not in any way suggest that the deleted MeiraGTx sequence arose due to a cloning error, but we do note that AGTC tested a shortened version of hRPGR (with only a 45 bp deletion within ORF15) in their preclinical program, which was claimed to have arisen during the cloning process [6,7]. AGTC decided to improve on the shortened RPGR by rational design of the coding sequence using codon opti- mization in order to create a full-length stable transcript. A side-by-side comparison of both transgenes in RPGR- mutant dogs favored the codon optimized full-length ver- sion for their clinical trial [8]. The ORF15 region of RPGR encodes glutamate and gly- cine residues in the RPGR protein. This region undergoes extensive post-translational glutamylation, which is believed to be essential for cone function. Absence of RPGR gluta- mylation, as occurs in deficiency of the TTLL5 enzyme, leads to cone or cone-rod dystrophy [9,10]. We have also noted that RPGR patients who have the cone dystrophy pheno- type have almost full-length RPGR protein but with mark- edly reduced glutamylation [11]. An RPGR vector containing a deletion similar to the vector developed by MeiraGTx also has significantly reduced glutamylation compared to the wildtype sequence (~70% reduction, as shown in Sun et al., 2016 [12] – Figure 5C, sample 4), and naturally this raises questions about its efficacy in cone photoreceptors. Independent improvement in cone function has not yet been shown in any of the preclinical animal models because they lack a macula and cone function will be sustained indirectly by secondary mechanisms when rod degeneration is slowed. Independent improvements in cone function fol- lowing RPGR gene therapy have so far only been confirmed when applying the full-length RPGR sequence in the human macula [13]. Hence, it will be critically important to monitor the outcomes of the clinical trials to determine if the deleted RPGR vector has an equally positive effect on human cone photoreceptors. In any case, we believe that investigators and patients being recruited into clinical trials should be made aware that the MeiraGTx RPGR sequence being administered to them has a significant deletion within it and that the consequences of this are currently unknown. CONTACT Robert E. MacLaren enquiries@eye.ox.ac.uk Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Level 5 & 6, West Wing, Headley Way, Oxford OX3 9DU, UK EXPERT OPINION ON EMERGING DRUGS 2022, VOL. 27, NO. 4, 449–450 https://doi.org/10.1080/14728214.2022.2152212 http://www.tandfonline.com https://crossmark.crossref.org/dialog/?doi=10.1080/14728214.2022.2152212&domain=pdf&date_stamp=2022-12-22 Funding This paper was not funded. Declaration of interest RE MacLaren has previously received grant funding from Biogen and has previously provided independent consultancy advice on X-linked retinitis pigmentosa to Biogen Inc. and Janssen Pharmaceuticals. RE MacLaren is also listed as an inventor on a patent for X-linked retinitis pigmentosa gene therapy owned by the University of Oxford. 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 discussedin the manuscript apart from those disclosed. Reviewer disclosures Peer reviewers on this manuscript have no relevant financial or other relationships to disclose. References 1. Georgiadis A, Smith AJ, Michaelides M, et al. Letter to the editor: “Emerging gene therapy products for RPGR-associated X-linked retinitis pigmentosa”. Expert Opin Emerg Drugs. 2022;27(4):443-445. DOI:10.1080/ 14728214.2022.2152202 2. Martinez-Fernandez de la Camara C, Cehajic-Kapetanovic J, MacLaren RE. Emerging gene therapy products for RPGR-associated X-linked retinitis pigmentosa. Expert Opin Emerg Drugs. 2022;27(4):429-441. DOI:10.1080/ 14728214.2022.2152003 3. Pawlyk BS, Bulgakov OV, Sun X, et al. Photoreceptor rescue by an abbreviated human RPGR gene in a murine model of X-linked retinitis pigmentosa. Gene Ther. 2016;23(2):196–204. 4. Karra D, Jacobi FK, Broghammer M, et al. Population haplotypes of exon ORF15 of the retinitis pigmentosa GTPase regulator gene in Germany: implications for screening for inherited retinal disorders. Mol Diagn Ther. 2006;10(2):115–123. 5. Fischer MD, McClements ME, Martinez-Fernandez de la Camara C, et al. Codon-optimized RPGR improves stability and efficacy of AAV8 gene therapy in two mouse models of X-linked retinitis pigmentosa. Mol Ther. 2017;25(8):1854–1865. 6. Deng WT, Dyka FM, Dinculescu A, et al. Stability and safety of an AAV vector for treating RPGR-ORF15 X-linked retinitis pigmentosa. Hum Gene Ther. 2015;26(9):593–602. 7. Beltran WA, Cideciyan AV, Boye SE, et al. Optimization of retinal gene therapy for X-linked retinitis pigmentosa due to RPGR mutations. Mol Ther. 2017;25(8):1866–1880. 8. Song C, Dufour VL, Cideciyan AV, et al. Dose range finding studies with two RPGR transgenes in a canine model of X-linked retinitis pigmentosa treated with subretinal gene therapy. Hum Gene Ther. 2020;31(13–14):743–755. 9. Sergouniotis PI, Chakarova C, Murphy C, et al. Biallelic variants in TTLL5, encoding a tubulin glutamylase, cause retinal dystrophy. Am J Hum Genet. 2014;94(5):760–769. 10. Smirnov V, Grunewald O, Muller J, et al. Novel TTLL5 variants associated with cone-rod dystrophy and early-onset severe retinal dystrophy. Int J Mol Sci. 2021;22(12):6410. 11. Cehajic-Kapetanovic J, Martinez-Fernandez de la Camara C, Birtel J, et al. Impaired glutamylation of RPGRORF15 underlies the cone dominated phenotype associated with truncating distal ORF15 variants. Proc Natl Acad Sci U S A. 2022. In press. 12. Sun X, Park JH, Gumerson J, et al. Loss of RPGR glutamylation under- lies the pathogenic mechanism of retinal dystrophy caused by TTLL5 mutations. Proc Natl Acad Sci U S A. 2016;113(21):E2925–2934. 13. Cehajic-Kapetanovic J, Xue K, Martinez-Fernandez de la Camara C, et al. Initial results from a first-in-human gene therapy trial on X-linked retinitis pigmentosa caused by mutations in RPGR. Nat Med. 2020;26(3):354–359. 450 C. MARTINEZ-FERNANDEZ DE LA CAMARA ET AL. https://doi.org/10.1080/14728214.2022.2152202 https://doi.org/10.1080/14728214.2022.2152202 https://doi.org/10.1080/14728214.2022.2152003 https://doi.org/10.1080/14728214.2022.2152003 Funding Declaration of interest Reviewer disclosures References
