July 2018
Volume 59, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2018
CRISPR-Cas9 RNP complex, delivered via lipid nanoparticle, is effective in disruption of RHO gene in vitro
Author Affiliations & Notes
  • Amirmohsen Arbabi
    USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, United States
  • Christine Spee
    USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, United States
  • David R Hinton
    USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, United States
  • Peter Z. Qin
    Chemistry, University of Southern California, Los Angeles, California, United States
  • Cheryl Mae Craft
    USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, United States
  • Sarah F Hamm-Alvarez
    USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, United States
  • Hossein Ameri
    USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Amirmohsen Arbabi, None; Christine Spee, None; David Hinton, None; Peter Z. Qin, None; Cheryl Craft, None; Sarah Hamm-Alvarez, None; Hossein Ameri, None
  • Footnotes
    Support  Departmental grant from Research to Prevent Blindness
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 26. doi:
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      Amirmohsen Arbabi, Christine Spee, David R Hinton, Peter Z. Qin, Cheryl Mae Craft, Sarah F Hamm-Alvarez, Hossein Ameri; CRISPR-Cas9 RNP complex, delivered via lipid nanoparticle, is effective in disruption of RHO gene in vitro. Invest. Ophthalmol. Vis. Sci. 2018;59(9):26.

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

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Abstract

Purpose : Retinitis pigmentosa (RP) is a hereditary retinal disease that presents with nyctalopia and visual field defect and leads to central vision loss in late stages. Currently, the only approved treatment for RP is the use of retinal prosthesis, which is only suitable for patients with advanced disease and profound vision loss. Gene therapy is a promising approach that could potentially modify the course of the disease. However, gene therapy for autosomal dominant RP (ADRP) is challenging as it requires elimination of toxic products produced by dominant negative effects of the mutated allele. RHO is the most common gene involved in ADRP. Our approach uses CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated system9 (Cas9)) gene editing technology to silence the human RHO gene. We used lipofectamine CRISPRMAX lipid nanoparticle to directly deliver CRISPR-Cas9 Ribonucleoprotein (RNP) complex into cells. When optimized, this approach may enable intravitreal delivery of CRISPR-Cas9 for gene editing in ADRP without the need for viral vectors or electroporation. Following disruption of the mutated gene, a normal copy of the gene can be inserted via viral vector delivery.

Methods : 24 hours after seeding, Human Embryonic Kidney 293 (HEK293) cells were transfected with CRISPR-Cas9 RNP complexed with Lipofectamine CRISPRMAX. Guide RNA was designed to target a region of exon 1, encoding for amino acids 98 through 104, with high on-target and off-target scores of 73 and 74 respectively. Genomic Cleavage Detection Kit was used to verify the gene editing efficacy 48 hours after transfection.

Results : Using PCR, with the amplicon length of 421 bp, two bands with 307 bp and 114 bp length were detected on the agarose gel. The presence of the 2 expected bands confirmed that the RHO gene was successfully disrupted at the desired region.

Conclusions : CRISPR-Cas9 RNP complex, delivered via lipofectamine CRISPRMAX lipid nanoparticle can successfully disrupt the RHO gene in vitro in HEK293 Cells. This mutation independent gene editing may allow silencing of both normal and mutated alleles of the RHO gene, paving the way for a successful treatment of RP patients with mutations in RHO gene.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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