Innate Immunity and the Retinal Pigment Epithelium in Gene Therapy for Inherited Retinal Disorders

  • Author / Creator
    Casey, Geoffrey A
  • Background: The photoreceptors are post-mitotic (no longer dividing), so the eye has evolved several unique immunological mechanisms to protect the photoreceptors and prevent vision loss. Inherited retinal disorders occur when mutations disrupt normal retinal function and photoreceptors die. Choroideremia is an inherited retinal disorder that is currently incurable, although several therapies are in development. Choroideremia and other inherited retinal disorders are candidates for viral gene therapy, where engineered viruses deliver therapeutic “wild-type” (normal) copies of mutated genes to target cells, restoring normal function. Many such therapies are now in clinical trials, but the results of some early trials suggest that the immune response of the retina against viral gene therapy vectors is not well understood and is likely limiting efficacy and decreasing the safety of the treatment.
    Purpose: This study was designed to evaluate the innate immune response of the retinal pigment epithelium (RPE) against adeno-associated virus (AAV) and equine infectious anemia virus (EIAV) vectors, two vector platforms commonly used in ocular gene therapy. I hypothesize that AAV and EIAV vectors will stimulate an innate immune response in human RPE when detected by Toll-like receptor 9 and Toll-like receptor 3, respectively.
    Methods: RPE derived from induced pluripotent stem cells (iPSC-RPE) was grown in culture until it demonstrated markers of functional, mature RPE, including pigmentation, polarization, polygonal morphology, and fluid pumping capability. Cells were treated with analogues of viral RNA and DNA (Poly(I:C) and CpG-DNA, respectively), the anti-viral cytokine IFNγ, or viral vectors. Levels of secreted immune response proteins (IL6, IL8, or CCL2) were measured via enzyme-linked immunosorbent assay (ELISA) analysis of harvested cell culture media. At the terminal time point for each sample, RNA was extracted and purified before RT-qPCR expression level analysis of several pro-inflammatory and anti-viral genes (IL6, IL8, CXCL10, RSAD2, and others).
    Results: While Poly(I:C) and IFNγ stimulated upregulation of several pro-inflammatory and anti-viral genes and increased secretion of IL6, IL8, and CCL2 to varying degrees, treatment with viral vectors resulted in very low transduction efficiency (GFP transgene expression observed in an estimated <1% of all cells). As a result, most changes in gene expression failed to reach statistical significance.
    Conclusions: To increase the predictive capability of this study, it must be repeated with a higher ratio of viral particles to target cells (multiplicity of infection or “MOI”) before conclusions can be drawn. While many trends in the data agree with previously published findings from other cell types in vitro and model organisms in vivo, the low transduction efficiency prevents deeper analysis.

  • Subjects / Keywords
  • Graduation date
    Fall 2021
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.