Optimisation of therapies for inherited retinal and mitochondrial diseases
Embargo End Date:2023-04-01
Citation:Killian Hanlon, 'Optimisation of therapies for inherited retinal and mitochondrial diseases'
Thesis_corrected_Hanlon (1).pdf (PDF) 120.5Mb
Inherited retinal degenerations (IRDs) are the most frequent cause of vision loss in people of working age. They have highly varied causes and pathophysiologies, and are typically incurable. Gene therapy has emerged as a powerful tool for correcting IRDs. Mitochondrial retinal diseases represent a subgroup of IRDs caused by mutations in genes governing mitochondrial function. These can be nuclear genes required for healthy mitochondria or genes encoded by the mitochondrial genome. Studies have shown that retinal ganglion cells (RGCs) are seen to be dysfunctional in several mitochondrial diseases, and so the ability to effectively target these with relevant therapeutics is of significant importance. In Chapter 2 of this work, a codon-optimised version of the yeast gene Ndi1 (termed ophNdi1) was tested for its efficacy in protecting against RGC loss caused by rotenone-induced Complex I deficiency. Ndi1 is a single-subunit, nuclear gene encoded by S. cerevisiae that has previously been shown to be effective in treating a rotenone-mediated Complex I deficiency model of LHON. ophNdi1 was found to be effective at a viral dose administered as low as one sixth that of the original Ndi1 viral vector. ophNdi1 and Ndi1 both demonstrated significant protection against rotenone in terms of RGC survival, optic nerve thickness, and also visual acuity threshold as measured by optokinetic response (OKR). The protective effect of ophNdi1 was equal to or greater than that of Ndi1, even with a lower viral titre. To develop gene therapy for RGCs further, promoters with high RGC specificity suitable for use in AAV are required. In Chapter 3, a pipeline was developed, the goal of which was to streamline the selection process for novel promoters using a combination of expression data and conservation of upstream sequence. Following from this, one putative promoter sequence, Nefh, was tested in an AAV2/2 vector. It was found that Nefh offered highly preferential expression of EGFP in RGCs when injected intravitreally into adult mouse eyes, as compared to an AAV2/2-CMV-EGFP vector. Two minimal versions of the Nefh promoter sequence were designed, utilising only the highly conserved regions of the Nefh sequence. These were tested in AAV2/2 vectors, and while it was found that initial data suggests that Nefh_short drives some degree of expression at the RNA level, the msMin_Nefh¬ promoter appears to lack both the expression level of Nefh and its specificity. It has been shown that neuronal DNA may contain multiple megabase-scale copy number variants (CNVs) that could potentially vary significantly between individual neurons. Next generation sequencing (NGS) has enabled researchers to examine even the minute quantities of DNA that come from a single cell. It would be of interest to understand if and to what extent these CNVs occur in the retina. To accomplish this, in Chapter 4 single rod photoreceptors were isolated and sequenced, with the goal of determining whether megabase-scale CNVs exist in retinal neurons. Rods were isolated using flow cytometry, making use of a Rho-EGFP mouse line in which rods exclusively express EGFP. DNA from these single cells was amplified and prepared for sequencing on an Illumina MiSeq. The resultant data was analysed by dividing the mouse genome into windows of 500kb uniquely mappable sequence, with sequence reads binned into those windows. Circular binary segmentation (CBS) was used to detect changepoints in the read count data that indicates the presence of a CNV. 40% of samples sequenced showed evidence of CNVs at least 3mb in size (n=20), with as many as five CNVs found per sample.
Author: Hanlon, Killian
Type of material:Thesis
Availability:Full text available