April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Lentiviral Vectors Carrying Dual Promoters and 2A Cleavage Peptides Permit Targeted Delivery of Multiple Proteins to Neural Retina
Author Affiliations & Notes
  • S. L. Semple-Rowland
    Neuroscience,
    University of Florida, Gainesville, Florida
  • W. E. Coggin
    Neuroscience,
    University of Florida, Gainesville, Florida
  • M. Geesey
    Neuroscience,
    University of Florida, Gainesville, Florida
  • M. Hochman
    Neuroscience,
    University of Florida, Gainesville, Florida
  • L. Abraham
    Neuroscience,
    University of Florida, Gainesville, Florida
  • E. Walling
    Neuroscience,
    University of Florida, Gainesville, Florida
  • A. Zorrilla
    Neuroscience,
    University of Florida, Gainesville, Florida
  • R. Ludlow
    Neuroscience,
    University of Florida, Gainesville, Florida
  • J. D. Verrier
    Neuroscience,
    University of Florida, Gainesville, Florida
  • W. C. Smith
    Ophthalmology,
    University of Florida, Gainesville, Florida
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 3110. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      S. L. Semple-Rowland, W. E. Coggin, M. Geesey, M. Hochman, L. Abraham, E. Walling, A. Zorrilla, R. Ludlow, J. D. Verrier, W. C. Smith; Lentiviral Vectors Carrying Dual Promoters and 2A Cleavage Peptides Permit Targeted Delivery of Multiple Proteins to Neural Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3110.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: : Treatment of many inherited photoreceptor diseases will require development of multi-protein therapies that correct the genetic defects linked to these diseases and slow the related degenerative phenotypes. To address this problem, we constructed and examined the expression characteristics of lentiviral vectors harboring dual-promoters or 2A cleavage peptides to identify those exhibiting reliable, targeted expression of the proteins they encoded in vivo.

Methods: : Lentivectors were constructed using the following promoters: XOPS1.3, mOP500, mIRBP156, RK, mNRLL, mVIM, mCD44, and mGFAP. Vectors were packaged and injected into the neural tubes of chicken embryos. The activities of the promoters alone, in duplicate, or when paired with a different promoter or 2A cleavage peptide were analyzed in transduced, fully-developed retinas using fluorescent microscopy and western blot.

Results: : IRBP156, NRLL and RK were active in cones and rods while XOPS1.3 was active only in rods. Of the glial promoters, only GFAP activity was restricted to Muller cells; both VIM and CD44 were active in neural as well as Muller cells. Dual-promoter vectors carrying IRBP156 and RK or XOPS1.3 and mOP500 in the order listed, robustly expressed both reporter transgenes in cones and rods or rods only, respectively. Expression of the upstream transgene was much lower than the downstream transgene in dual-promoter vectors constructed using two copies of RK or IRBP156. CD44 and VIM paired in the order listed increased the specificity of the VIM promoter, limiting its activity to Muller cells, but little CD44 activity was observed. Vectors carrying 2A cleavage peptides reliably produced two proteins, but not all of the fused protein was cleaved.

Conclusions: : We identified two dual-promoter vectors that specifically target cones and rods or rods alone and reliably express both proteins encoded by the transgenes. When two well-matched promoters are not available, our results suggest that targeted expression of two or more proteins to single cells may be achieved using 2A cleavage peptides.

Keywords: gene transfer/gene therapy • photoreceptors • retinal degenerations: hereditary 
×
×

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.

×