(2017/18) Post PhD: MBBS Year 6 - Preparation for Practi
Immune and huntingtin biology in Huntington's disease
Professor Sarah Tabrizi - Institute of Neurology
Rosetrees Charitable Trust and MRC DTG
Description of Project:
Huntington’s disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. Expression of the mutant protein (mHTT) is associated with disease pathology, so lowering cellular HTT levels is a promising therapeutic strategy. Non-selective suppression of wild-type and mHTT has been demonstrated successfully in human HD cells. However, wild-type HTT has important roles in cellular function, and an ideal therapy would selectively lower mHTT levels while preserving those of the wild-type. This project demonstrates for the first time the allele-selective suppression of mHTT in primary human ex vivo cells using small interfering RNA (siRNA) targeted to single nucleotide polymorphisms (SNPs) in the HTT gene. HD patients were genotyped to determine SNP heterozygosity before SNPs were linked to the wild-type or mutant HTT alleles. Myeloid cells were then isolated from patient blood samples and transfected by packaging the siRNA into yeast-derived glucan shells which are readily phagocytosed. Finally, huntingtin knockdown was assessed using qPCR to determine both potency and allele-selectivity of the siRNAs.
Comparison of a panel of siRNAs has revealed varying levels of allele-selectivity when targeting different SNP sites. This has allowed the identification of promising targets, of which rs362331 in exon 50 is notable. Further work to enhance the selectivity of HTT knockdown by varying the dose of siRNA is currently ongoing, with functional studies to follow. These findings represent the first steps in evaluating siRNA-mediated allele-selective suppression in primary human HD cells, while demonstrating the feasibility of tailoring treatments to a genotype. In time it is hoped this will also enable further investigation into the cellular function, synthesis and clearance of wild-type and mHTT in human cells.
Furthermore, the innate immune system has been shown to be altered in HD. Our group has previously demonstrated NFκB pathway dysregulation in HD myeloid cells; however the complexity of cellular signalling means multiple pathways are likely to be involved. 150 HD and control samples have been collected to investigate changes across the entire HD monocyte transcriptome using RNA deep-sequencing technology. Analysis and functional validation of this dataset is currently ongoing, and it is hoped that the information gained from this study will enable us to dissect in detail the mechanisms underlying HD innate immune dysfunction. Investigation of the HD adaptive immune system has also been carried out with a focus on T lymphocytes, and these experiments provide considerable new insight into HD as a disease of the whole body.
Miller JR, Träger U, Andre R, Tabrizi SJ: “Mutant Huntingtin Does Not Affect the Intrinsic Phenotype of Human Huntington’s Disease T Lymphocytes.” PLoS ONE. 2015 Nov 3. 10(11): e0141793. doi: 10.1371/journal.pone.0141793
Tochiki KK, Maiarù M, Miller JR, Hunt SP, Géranton SM: “Short-term anesthesia inhibits formalin-induced extracellular signal-regulated kinase (ERK) activation in the rostral anterior cingulate cortex but not in the spinal cord.” Mol Pain. 2015 Aug 14;11(1):49. doi: 10.1186/s12990-015-0052-z.
Wild EJ, Boggio R, Langbehn D, Robertson N, Haider S, Miller JR, Zetterberg H, Leavitt BR, Kuhn R, Tabrizi SJ, Macdonald D, Weiss A: “Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington's disease patients.” J Clin Invest. 2015 Apr 6. pii: 80743. doi: 10.1172/JCI80743.
Träger U, Andre R, Magnusson-Lind A, Miller JR, Connolly C, Weiss A, Grueninger S, Silajdžić E, Smith DL, Leavitt BR, Bates GP, Björkqvist M, Tabrizi SJ: “Characterisation of immune cell function in fragment and full-length Huntington's disease mouse models.” Neurobiol Dis. 2015 Jan; 73: 388–398.
Miller JR & Andre R: “Quantitative polymerase chain reaction”. Br J Hosp Med (Lond). 2014 Dec;75(12):C188-92.
Awards & Prizes:
- UCL Faculty of Brain Sciences Poster Symposium Runner Up 2015
- Guarantors of Brain Travel Grant (HD2014 conference) 2014
- Merit in MBBS 4th Year Examinations 2013
- Wolfson Foundation Intercalated BSc Award 2011-2012
- UCL Undergraduate Faculty Scholarship for Excellence 2011-2012
- Cordwainers’ Scholarship for Overall Performance in MBBS Year 2 2011
- The Meyerstein Scholarship for Overall Performance in Years 1 & 2 2011
- The Cluff, Dixon & Paraskevaides Prize for First Place in MBBS Year 2 2011
- UCL Undergraduate Faculty Scholarship for Excellence 2010-2011
- The Paraskevaides Foundation Prize for Overall Performance in Year 1 2010
- 1st Drummond and Thomas Yeates Prize for Circulation & Breathing 2010