The term Autosomal Recessive Congenital Ichthyosis (ARCI) covers a collection of genetic disorders characterized by difficulties in the process of keratinization. Patients with ichthyosis experience a compromised skin barrier function, resulting in reduced capacity to defend against bacterial, chemical, and mechanical attacks, as well as to prevent the loss of water through the skin. While several genes have been found to have mutations, TGM1 and ALOX12B mutations account for 50% of ARCI cases. Unfortunately, palliative care is usually used to manage the ARCI phenotype, due to the lack of cures and treatments. Current studies are concentrating on developing a causal therapy utilising skin models that replicate the ARCI phenotype and enzyme replacement therapy with recombinant transglutaminase 1, a protein involved in crosslinking the cornified envelope in the stratum corneum of the skin. As part of a longer-term strategy to assess the efficacy of enzyme replacement therapy, a series of knockdowns of TGM1 were first completed in primary HKC18 keratinocyte cells using small interfering siRNA. RQ values and CT values were obtained from the real-time PCR analysis. The initial aim was to generate an experimental model of TGM1 deficiency which could subsequently be used to assess the effect of transfecting TGM1 into keratinocyte cells and use immunocytochemistry to visualise the protein's cellular location. However, the results focused on improving the efficiency of TGM1 knockdown using siRNA.In this study, the results of the series of TGM1 knockdowns were as follows. Following the first run, the treatment of HKC18 cells with TGM1 siRNA led to a reduction in TGM1 expression of 16% (RQ-0.839) whereas the expression of ALOX12B and ALOXE3 increased to an RQ value of 2.462 and 1.654 respectively (The percentage of knockdown was calculated for RQ values below 1, therefore these values could not be calculated). Similarly in run 2, there was a reduction in TGM1 expression of 45% (RQ-0.55) and the expression of ALOX12B and ALOXE3 increased to an RQ value of 1.305 and 1.811 respectively. Run 3 did not produce any results as the cells lost viability following the inclusion of calcium prior to transfection with siRNA. Finally, run 4 generated results where there was a reduction in TGM1 expression of 59% (RQ-0.408) and the expression of ALOX12B and ALOXE3 increased to an RQ value of 0.702 and 0.708, showing a knockdown percentage of only 29% respectively. The target reduction in TGM1 expression was 90% and as this wasn’t achieved, the project could not be progressed to investigate the efficacy of enzyme replacement therapy in this model. In conclusion, whilst this study encountered technical difficulties, modifications to the methodology did result in increased TGM1 knockdown efficiency in HKC18 cells but it was less than the target of 90% reduction in TGM1 expression required for the project to progress to the next stage. It is not known whether the lack of knockdown efficiency is cell line dependent but further improvements in methodology are required. Other approaches to disrupt the TGM1 gene may prove more fruitful and additional studies are required to explore alternative approaches.
| Date of Award | 2 Jun 2025 |
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| Original language | English |
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| Supervisor | Roger Phillips (Main Supervisor) |
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