June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Allele-specific gene editing for the treatment of autosomal dominant Retinitis Pigmentosa
Author Affiliations & Notes
  • Gene Liau
    Precision Biosciences, Durham, North Carolina, United States
  • Kollu Nageswara Rao
    Department of Ophthalmology, Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, United States
  • Victor V Bartsevich
    Precision Biosciences, Durham, North Carolina, United States
  • Jeff Smith
    Precision Biosciences, Durham, North Carolina, United States
  • Derek Jantz
    Precision Biosciences, Durham, North Carolina, United States
  • Matthew Hirsch
    Department of Ophthalmology, Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, United States
  • Footnotes
    Commercial Relationships   Gene Liau, Precision Biosciences (E); Kollu Rao, Precision Biosciences (F); Victor Bartsevich, Precision Biosciences (E); Jeff Smith, Precision Biosciences (E); Derek Jantz, Precision Biosciences (E); Matthew Hirsch, Precision Biosciences (F)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4473. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Gene Liau, Kollu Nageswara Rao, Victor V Bartsevich, Jeff Smith, Derek Jantz, Matthew Hirsch; Allele-specific gene editing for the treatment of autosomal dominant Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4473.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose : Targeted gene knockout is an emerging approach for the treatment of autosomal-dominant disorders such as P23H RHO mediated autosomal dominant retinitis pigmentosa (adRP), a common form of inherited blindness. A single nucleotide distinguishes P23H RHO from the WT copy which collectively present in a heterozygous manner. Previous works have suggested that down-regulation of the mutant allele in presence of the WT copy prevents retinal degeneration. Therefore, a 930nt I-CreI based designer homing endonuclease (HE) to specifically knockout the mutant RHO allele was engineered and validated in a self-complementary AAV vector. The specificity of this HE for P23H RHO was demonstrated in vitro. Preclinical evaluations in a human P23H RHO adRP mouse model were undertaken to determine if this approach demonstrates clinical potential for preventing vision loss in adRP patients.

Methods : A proprietary ARCUS platform was employed to generate a HE that can specifically target the human RHO allele carrying the P23H mutation. The targeted 22 nucleotide sequence differs from the wild type allele only by a single base pair. Specificity of the nuclease was first evaluated in CHO cell lines engineered to contain a single strand annealing GFP reporter that harbors either the WT or P23H RHO sequence. Gene editing efficiency was evaluated by flow cytometry and immunoblotting. To evaluate HE activity in vivo, a scAAV5 vector harboring P23H RHO HE cDNA under control of a photoreceptor specific promoter was administered to the human P23H RHO adRP mouse model via sub-retinal injection. scAAV5-GRK-HE cleavage efficiency of the mutant RHO allele was calculated using sequencing and by digital PCR. In vivo retinal function was evaluated by electroretinography and the subcellular localization of rhodopsin in photoreceptors by histology.

Results : We have generated a HE that is highly specific for the mutant P23H RHO allele. Sub-retinal delivery of scAAV5-GRK-HE decreases the amount of rhodopsin in outer nuclear layer in transduced photoreceptors when compared to non-transduced cells.

Conclusions : This is the first study to demonstrate that a single chain HE can efficiently discriminate a one nucleotide difference and specifically target the P23H RHO allele. This result coupled with decreased rhodopsin mislocalization in an adRP model, foreshadows possible use of scAAV HE-based gene editing of P23H RHO to treat adRP patients.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×