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Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-Associated X-Linked Retinitis Pigmentosa.

ABSTRACT: In a screen of 1,000 consecutively ascertained families, we recently found that mutations in the gene RPGR are the third 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 adeno-associated virus (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 Investigational New Drug application.

SUBMITTER: Giacalone JC

PROVIDER: S-EPMC6703244 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-Associated X-Linked Retinitis Pigmentosa.

Giacalone Joseph C JC Andorf Jeaneen L JL Zhang Qihong Q Burnight Erin R ER Ochoa Dalyz D Reutzel Austin J AJ Collins Malia M MM Sheffield Val C VC Mullins Robert F RF Han Ian C IC Stone Edwin M EM Tucker Budd A BA

Human gene therapy 20190801 8

In a screen of 1,000 consecutively ascertained families, we recently found that mutations in the gene RPGR are the third 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 adeno-associated virus (AAV) vectors, RPGR is an obvious early target for AAV-based ocular gene therapy. In generating plasmids for this application, we discovered that those containin ...[more]

PMID: 31106594

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Project description:ImportanceFor patients with X-linked retinitis pigmentosa and clinicians alike, phenotypic variability can be challenging because it complicates counseling regarding patients' likely visual prognosis.ObjectiveTo evaluate the clinical findings from patients with X-linked retinitis pigmentosa with 13 distinct RPGR mutations and assess for phenotypic concordance or variability.DesignRetrospective medical record review of data collected from 1985 to 2011.SettingKellogg Eye Center, University of Michigan.PatientsA total of 42 patients with X-linked retinitis pigmentosa with mutations in RPGR. Age at first visit ranged from 4 to 53 years, with follow-up ranging from 1 to 11 visits (median follow-up time, 5.5 years; range, 1.4-32.7 years, for 23 patients with >1 visit).Main outcomes and measuresClinical data assessed for concordance included visual acuity (VA), Goldmann visual fields (GVFs), and full-field electroretinography (ERG). Electroretinography phenotype (cone-rod vs rod-cone dysfunction) was defined by the extent of photopic vs scotopic abnormality. Qualitative GVF phenotype was determined by the GVF pattern, where central or peripheral loss suggested cone or rod dysfunction, respectively. Goldmann visual fields were also quantified and compared among patients.ResultsEach mutation was detected in 2 or more related or unrelated patients. Five mutations in 11 patients displayed strong concordance of VA, while 4 mutations in 16 patients revealed moderate concordance of VA. A definitive cone-rod or rod-cone ERG pattern consistent among patients was found in 6 of 13 mutations (46.2%); the remaining mutations were characterized by patients demonstrating both phenotypes or who had limited data or nonrecordable ERG values. Concordant GVF phenotypes (7 rod-cone pattern vs 4 cone-rod pattern) were seen in 11 of 13 mutations (84.6%). All 6 mutations displaying a constant ERG pattern within the mutation group revealed a GVF phenotype consistent with the ERG findings.Conclusions and relevanceWhile VA and ERG phenotypes are concordant in only some patients carrying identical mutations, assessment of GVF phenotypes revealed stronger phenotypic conservation. Phenotypic concordance is important for establishing proper counseling of patients diagnosed as having X-linked retinitis pigmentosa, as well as for establishing accurate patient selection and efficacy monitoring in therapeutic trials.

Dataset Information

Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-Associated X-Linked Retinitis Pigmentosa.

Publications

Development of a Molecularly Stable Gene Therapy Vector for the Treatment of <i>RPGR</i>-Associated X-Linked Retinitis Pigmentosa.

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