Towards the Development of Ocular Gene Therapy.
therapy has shown promise in the treatment of retinal degeneration in clinical trials.
This study aims to advance the field of ocular gene therapy for the treatment of retinal
degeneration by exploring the use of a novel therapeutic gene and non-viral delivery
methods. Adeno-associated viruses (AAVs) show great potential but have limited transgene
capacity. Non-viral methods ... read moreavoid complications associated with viruses but have
decreased efficiency and longevity. This study begins by investigating the use of the
POD (Peptide for Ocular Delivery) for longer-term gene expression to the retinal pigment
epithelium (RPE) of adult mice. By optimizing the nanoparticle dose, significant gene
expression is seen for 70 days in vivo. The biological efficacy of this dose is then
tested using POD-Glial cell line-derived neurotrophic factor (GDNF) nanoparticles in a
light-induced model of retinal degeneration. Degeneration is assessed by
electroretinograms (ERGs) and thickness of the outer nuclear layer (ONL). POD-GDNF
nanoparticles mediate functional rescue for 30 days and histological rescue for 70 days.
To address the issue of efficiency, Nucleolin Binding Protein (NBP) is tested. NBP has
been shown to bind to cell surface nucleolin and transport directly into the nucleus,
avoiding endosomal sequestration, a limitation of POD-mediated delivery. The ability of
NBP to deliver fluorophores, protein, and DNA to ocular tissues is tested in vivo. NBP
delivers small and large molecules into retinal and corneal cells and plasmid DNA into
RPE cells in vivo. However, NBP is not a more efficient than POD. Lastly, the
neurotrophic factor artemin is applied to the light-induced retinal degeneration model.
Artemin is a member of the GDNF family of neurotrophic factors and less promiscuous than
GDNF. Artemin activity in the retina has not been tested in vivo. Because non-viral
delivery methods are not yet optimized but gene delivery by AAVs is well established and
highly efficient, AAVs are used in this initial study. Rescue is evaluated using TUNEL
staining, ONL thickness, and ERGs. Artemin-mediated rescue is compared to GDNF-mediated
rescue. AAV-Artemin and GDNF lead to equivalent histological rescue in this model when
delivered using AAVs.
Thesis (Ph.D.)--Tufts University, 2014.
Submitted to the Dept. of Genetics.
Advisor: Rajendra Kumar-Singh.
Committee: Janis Lem, Philip Hinds, and Alan Kopin.
Keywords: Genetics, and Ophthalmology.read less