September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Utility of Commercially Available Cationic Lipid-based Delivery of CRISPR-Cas9 to the Retina in vivo
Author Affiliations & Notes
  • Sandy Shen-Chi Hung
    Neuroregeneration Unit, Centre for Eye Research Australia, EAST MELBOURNE, Victoria, Australia
  • Vicki Chrysostomou
    Glaucoma Research Unit, Centre for Eye Research Australia, Melbourne, Victoria, Australia
  • Hsin-Hui Shen
    Department of Microbiology, Monash University, Melbourne, Victoria, Australia
  • Fan li
    Menzies Institute for Medical Research, Tasmania, Tasmania, Australia
  • Anna E King
    Menzies Institute for Medical Research, Tasmania, Tasmania, Australia
  • Jonathan G Crowston
    Glaucoma Research Unit, Centre for Eye Research Australia, Melbourne, Victoria, Australia
  • Alice Pébay
    Neuroregeneration Unit, Centre for Eye Research Australia, EAST MELBOURNE, Victoria, Australia
  • Bang V Bui
    Optometry and Vision Sciences, University of Melbourne, Melbourne, Victoria, Australia
  • Guei-Sheung Liu
    Cytoprotection Unit, Centre for Eye Research Australia, Melbourne, Victoria, Australia
  • Alex W Hewitt
    Clinical Genetics Unit, Centre for Eye Research Australia, Melbourne, Victoria, Australia
    Menzies Institute for Medical Research, Tasmania, Tasmania, Australia
  • Footnotes
    Commercial Relationships   Sandy Hung, None; Vicki Chrysostomou, None; Hsin-Hui Shen, None; Fan li, None; Anna King, None; Jonathan Crowston, None; Alice Pébay, None; Bang Bui, None; Guei-Sheung Liu, None; Alex Hewitt, None
  • Footnotes
    Support  Childhood Eye Cancer Trust
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4038. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Sandy Shen-Chi Hung, Vicki Chrysostomou, Hsin-Hui Shen, Fan li, Anna E King, Jonathan G Crowston, Alice Pébay, Bang V Bui, Guei-Sheung Liu, Alex W Hewitt; Utility of Commercially Available Cationic Lipid-based Delivery of CRISPR-Cas9 to the Retina in vivo. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4038.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Given its accessibility and the number of inherited diseases caused by mutations in genes with very distinct spatial and stoichiometric expression, the eye is a particularly attractive organ for the clinical translation of Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mutation correction in vivo. Cationic-lipid based delivery has been successfully used for CRISPR knockout in mouse inner ear (Zuris et al. Nat Biotech; 2015: 33: 73-80). Here, we investigate the efficiency and efficacy of a cationic lipid-based CRISPR-Cas9 delivery system to the retina in vivo.

Methods : A YFP mouse model was used given the ability to readily detect and monitor CRISPR activity in vivo through observed knockout of YFP fluorescence in the retina. CRISPR guide RNAs (gRNA) were designed against the YFP sequence and synthesized using T7 polymerase. gRNA against LacZ was used as a negative control. Cas9 recombinant protein was generated from His-tagged Cas9 synthesized in E. coli BL21 STAR (DE3)-competent cells and isolated through a His-Pur Ni-NTA resin. YFP gRNA and recombinant Cas9 was packaged in cationic lipids- Lipofectamine 2000, RNAiMAX, invivofectamine 2.0. Intravitreal injections were performed in anesthetised (60:5 mg/kg, ketamine:xylazine) adult Tg(Thy1-YFP) 16Jrs mice. Electroretinography and optical coherence tomography measurements were conducted in anesthetised mice 3 weeks post-injection. Eyes were dissected post-mortem and retinal whole mounts examined using fluorescent microscopy.

Results : Cas9 precipitation was observed with invivofectamine 2.0; however concentrated Cas9 (200mM) could be used with lipofectamine 2000 and RNAiMAX. Following in vitro validation, intravitreal delivery of lipofectamine 2000 and RNAiMAX packaged or nude Cas9 and sgRNA was undertaken for at least 5 mice per experimental arm. Overall poor knockout efficiencies were observed with in delivery conditions with ~32% YFP:DAPI ratios observed in all cases. Packaging using RNAiMAX was found to cause retino-uveitis

Conclusions : Our current studies suggest the use of commercial cationic lipid delivery systems do not result in marked retinal cell gene knockout out. In particular RNAiMAX is not recommended for intravitreal injections in this context. Ongoing work investigating alternative cationic lipid packages as well as different routes, such as subretinal delivery, should be explored.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

×
×

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.

×