Journal article
Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-Associated X-Linked Retinitis Pigmentosa
Human gene therapy, Vol.30(8), pp.967-974
08/01/2019
DOI: 10.1089/hum.2018.244
PMCID: PMC6703244
PMID: 31106594
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.
Details
- Title: Subtitle
- Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-Associated X-Linked Retinitis Pigmentosa
- Creators
- Joseph C Giacalone - Department of Ophthalmology and Visual Sciences, Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IowaJeaneen L Andorf - Department of Ophthalmology and Visual Sciences, Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IowaQihong Zhang - Department of Ophthalmology and Visual Sciences, Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IowaErin R Burnight - Department of Ophthalmology and Visual Sciences, Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IowaDalyz Ochoa - Department of Ophthalmology and Visual Sciences, Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IowaAustin J Reutzel - Department of Ophthalmology and Visual Sciences, Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IowaMalia M Collins - Department of Ophthalmology and Visual Sciences, Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IowaVal C Sheffield - Department of Ophthalmology and Visual Sciences, Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IowaRobert F Mullins - Department of Ophthalmology and Visual Sciences, Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IowaIan C Han - Department of Ophthalmology and Visual Sciences, Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IowaEdwin M Stone - Department of Ophthalmology and Visual Sciences, Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IowaBudd A Tucker - Department of Ophthalmology and Visual Sciences, Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, Iowa
- Resource Type
- Journal article
- Publication Details
- Human gene therapy, Vol.30(8), pp.967-974
- DOI
- 10.1089/hum.2018.244
- PMID
- 31106594
- PMCID
- PMC6703244
- NLM abbreviation
- Hum Gene Ther
- ISSN
- 1043-0342
- eISSN
- 1557-7422
- Publisher
- Mary Ann Liebert, Inc., publishers
- Language
- English
- Date published
- 08/01/2019
- Academic Unit
- Stead Family Department of Pediatrics; The University of Iowa Institute for Vision Research; Iowa Neuroscience Institute; Medical Genetics and Genomics; John and Marcia Carver Nonprofit Genetic Testing Laboratory; Ophthalmology and Visual Sciences
- Record Identifier
- 9984070578202771
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