Biology subjects

Tilakaratna, V.

Publications and source records attributed to Tilakaratna, V..

3 recordsLinked to original sources

bioRxiv2025

CTx001 for geographic atrophy: a gene therapy expressing soluble, truncated complement receptor 1 (mini-CR1)

Over-activation of the complement system is strongly associated with geographic atrophy (GA), a late-stage form of age-related macular degeneration (AMD) and major cause of blindness. Here we report the development of a candidate GA treatment that is a potent complement modifier (mini-CR1) that addresses consequences of complement over-activation including membrane attack complex (MAC) formation. Mini-CR1 acts a potent cofactor for factor I driven proteolysis of C3b leading to iC3b and then C3dg formation; iC3b itself remains a potent opsonin. As well as inhibiting the alternative complement pathway, mini-CR1 acts as a cofactor for C4b degradation thereby inhibiting the classical pathway. Mini-CR1 prevents MAC formation in activated human serum with an IC50 of 125 nM. Mini-CR1 was shown to cross human Bruchs membrane ex vivo, implying the ability to cross into the choroidal space. Transduction of RPE cell lines with rAAV2-mini-CR1 (CTx001) resulted in dose-dependent transcription, and both basolateral and apical secretion of mini-CR1 by monolayers of human iPSC derived RPE cells. Mini-CR1 transduction of ARPE19 cells resulted in increased consumption of C3b and iC3b1 in activated culture media and decreased MAC formation on the cell surfaces. Subretinal injection of CTx001 in rats resulted in dose-dependent mini-CR1 production as demonstrated by solid-phase immunoassay. MAC formation following laser induced CNV in a rat model was reduced by 75% in CTx001-treated animals relative to null vector (p<0.01). This first generation of CTx001 represents a potent single administration complement modifier capable of effectively addressing pathologic complement amplification in the retina/choroid.

biochemistry↗

bioRxiv2020

FHL-1 interacts with human RPE cells through the α5β1 integrin and confers protection against oxidative stress

Retinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruchs membrane, are essential for RPE cell health and function, but the signals induced by Bruchs membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruchs membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the 5 and {beta}1 integrin subunits. The results obtained from primary RPE cells were replicated using the hTERT-RPE cell line. RNAseq expression analysis of hTERT-RPE cells bound to FHL-1 showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death showed hTERT-RPE cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.

cell biology↗

bioRxiv2016

Habitat predicts levels of genetic admixture in Saccharomyces cerevisiae

Genetic admixture can provide material for populations to adapt to local environments, and this process has played a crucial role in the domestication of plants and animals. The model yeast, Saccharomyces cerevisiae, has been domesticated multiple times for the production of wine, sake, beer and bread, but the high rate of admixture between yeast lineages has so far been treated as a complication for population genomic analysis. Here we make use of the low recombination rate at centromeres to investigate admixture in yeast using a classic Bayesian approach and a more conservative locus by locus phylogenetic approach developed here. Using both approaches, we find that S. cerevisiae from stable oak woodland habitats are less likely to show recent genetic admixture compared with those isolated from transient habitats such as fruits, wine or human infections. When woodland yeast strains do show recent genetic admixture, the degree of admixture is lower than in strains from other habitats. Furthermore, S. cerevisiae populations from oak woodlands are genetically isolated from each other, with only occasional migration between woodlands and local fruit habitats. Application of our phylogenetic approach suggests that there is a previously undetected population in North Africa that is the closest outgroup to the European S. cerevisiae, including the domesticated Wine population. Thorough testing for admixture in S. cerevisiae therefore leads to a better understanding of the underlying population structure of the species and will be important for understanding the selective processes underlying domestication in this economically important species.

evolutionary biology↗