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Supervisors:
Dr Jennie Chandler and Professor David Long
Project Description:
Background:
Polycystic kidney disease (PKD) is characterised by the gradual formation of fluid-filled cysts in the kidney tubules, eventually leading to end-stage kidney disease, requiring dialysis or transplantation. Autosomal dominant PKD (ADPKD; 1:400-1:1000 worldwide) is commonly associated with mutations in PKD1 (encoding Polycystin-1); accountable for ~85% of ADPKD1. More than 200 PKD1 mutations have been described, most of which result in truncated forms of the protein, consistent with inactivation of one allele. There is only one treatment available to ADPKD patients (Tolvaptan) which has significant side-effects. Gene therapy offers an alternative strategy2, however progress in this area has been hindered by poor adeno-associated virus (AAV) transduction in the kidney and AAV packaging constraints preventing the delivery of large genes such as PKD1 (12,911nt). This project intends to overcome these issues and develop an alternative AAV gene therapy approach for ADPKD.
Aims and Methods:
During this project, the student will:
Aim 1: Determine which AAVs best transduce human kidney tubules, the cell type affected in ADPKD, using a screen of 68 serotypes. The top three will be examined in more detail to assess off-target biodistribution in human cell lines from lung, liver, heart and spleen.
Aim 2: Design single guide RNAs (sgRNA) targeted to the PKD1 promotor which will be trialled with CRIPSRa-Cas93 technology to determine optimal conditions. Chip-seq and RNA-seq4 will be used to assess the specificity of CRIPSR activation.
Aim 3: The optimal sgRNA and AAV capsid will then be used to assess the therapeutic benefit of PKD1 rescue in heterozygous PKD1 mutant tubular cells5 using a 3D-culture, cyst-formation assay to assess cyst size and number.
Collectively, this project will optimise and provide proof-of-concept for a novel gene therapy approach to treat ADPKD in a relevant human cell line.
Timeline: Aim 1, 0 - 6 months; Aim 2, 6 - 20 months; Aim 3, 20 - 30 months; Thesis write up and manuscript publication, 30 - 36 months
References:
1. Huang et al. JASN. 2016; 27(1): 69-77
2. Mariot et al. Mol Ther Methods Clin Dev; 2020; 24;18:415-421
3. Matharu et al. Science. 2019; 18;363(6424):eaau0629
4. Chandler et al. Journal of Pathology. 2024; doi.10.1002/path.6339
5. Perretta-Tejedor et al. Methods in Molecular Biology. 2019; p 323–34
Contact Information:
Jennie Chandler