Related Experiment Video
Updated: May 29, 2026
Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium
Published on: August 7, 2015
Lentivirus-mediated expression of cDNA and shRNA slows degeneration in retinitis pigmentosa
Joaquin Tosi1, Javier Sancho-Pelluz, Richard J Davis
1Bernard & Shirlee Brown Glaucoma Laboratory, Department of Pathology & Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Abstract:
Mutations in Pde6b lead to high levels of signaling molecules cyclic guanosine monophosphate (cGMP) and Ca(2+), which ultimately result in photoreceptor cell death in certain forms of retinitis pigmentosa (RP). The level of cGMP, which is controlled by opposing activities of guanylate cyclase (GUCY) and photoreceptor phosphodiesterase-6 (PDE6), regulates the opening of cyclic nucleotide-gated ion channels [CNG] and thereby controls Ca(2+) influx into the outer segments. Using a lentiviral gene therapy approach, we have previously shown that degeneration can be temporarily slowed either by introducing wild-type PDE6β or knocking down expression of GUCY2E and CNGA1 in photoreceptors of Pde6b(H620Q), a mouse model for RP. Rescue was transient with either approach. Therefore, we tested a novel combination therapy using bipartite lentiviral vectors designed to both introduce wild-type PDE6β expression and knockdown GUCY2E or CNGA1. Immunoblot analysis shows simultaneous increases in PDE6β and decreases in GUCY2E or CNGA1 in retinas transduced by the vectors, indicating successful transduction. In Pde6b(H620Q) mutants, we observe rescue of photoreceptor function and an increase in photoreceptor rows as compared with untreated controls. However, no evidence of prolonged rescue beyond the limit of the previously tested single therapy was observed.
Insights
Gene therapy combining PDE6β expression and GUCY2E/CNGA1 knockdown shows transient rescue of photoreceptor degeneration in a retinitis pigmentosa mouse model.
Area of Science:
- Ophthalmology
- Genetics
- Molecular Biology
Background:
- Mutations in Pde6b cause retinitis pigmentosa (RP) by increasing cyclic guanosine monophosphate (cGMP) and Ca(2+), leading to photoreceptor cell death.
- cGMP levels, regulated by guanylate cyclase (GUCY) and phosphodiesterase-6 (PDE6), control cyclic nucleotide-gated ion channels (CNG) and Ca(2+) influx.
Purpose of the Study:
- To investigate the efficacy of a novel combination gene therapy using bipartite lentiviral vectors.
- To simultaneously introduce wild-type PDE6β and knockdown GUCY2E or CNGA1 in photoreceptors of Pde6b(H620Q) mice.
Main Methods:
- Development of bipartite lentiviral vectors for combined gene expression and knockdown.
- Immunoblot analysis to confirm successful transduction and changes in protein levels (PDE6β, GUCY2E, CNGA1).
- Assessment of photoreceptor function and structure in the Pde6b(H620Q) mouse model.
Main Results:
- Successful simultaneous increase in PDE6β and decrease in GUCY2E or CNGA1 expression confirmed by immunoblot.
- Observed rescue of photoreceptor function and increased photoreceptor rows in treated Pde6b(H620Q) mice.
- The combination therapy did not provide prolonged rescue beyond the duration of single-therapy approaches.
Conclusions:
- Combination gene therapy targeting PDE6β, GUCY2E, and CNGA1 is feasible and shows transient benefits in a mouse model of RP.
- Despite successful transduction and initial rescue, the therapeutic effect was limited in duration.
- Further strategies are needed to achieve long-term rescue of photoreceptor degeneration in RP.
