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Towards personalized medicine- customized oligonucleotide therapy for rare metabolic disorders

Supervisors: Dr Haiyan Zhou, Professor Paul Gissen

Background:
Antisense oligonucleotide (AON) technology offers great potential as a therapeutic strategy for conditions that cannot be treated by traditional drugs. These short synthetic single stranded nucleotides can regulate the target gene’s expression in a gene-specific manner. This technology has been approved to be successful in a number of neuromuscular and metabolic diseases, as exemplified by the FDA approvals of a number of antisense drugs in the last decade.[1] The AON technology also provides a new avenue for drug discovery and development for personalized medicine by correcting the specific genetic defect. The Zhou’s laboratory has been substantially involved in the preclinical development of AON drugs in a number of genetic disorders, in areas of new target identification, novel approach development and the in vivo validations in murine models.[2,3]

There are more than 60 different inherited lysosomal storage disorders (LSD) with overall incidence of 1 in 5000 births.  Most of them are life-limiting and extremely debilitating. For most of these conditions either there are no established therapies or only partially effective treatments. In this study, we will focus on developing AON therapy for one of the LSDs, the Athrogryposis, Renal dysfunction and Cholestasis (ARC) syndrome, a severe and rare autosomal recessive multi-system disorder, affecting kidneys, liver, skin, bones and blood cells. Currently there is no treatment available. As described previously by Prof Gissen’s group, the majority of ARC cases are caused by loss-of-function mutations in the VPS33B gene, which is involved in intracellular protein trafficking and membrane fusion.[4,5]

Aims/Objectives:
The overarching aim of this project is to develop a novel RNA therapy for children with rare metabolic disorders. In this 3-year PhD program, we will focus on developing AON approaches to target loss-of-function mutations and using ARC syndrome as the disease model.

Methods:
1.  Design and validation of AON approaches to target a common nonsense mutation in ARC patients. Various AON strategies will be investigated, including AONs to inhibit the nonsense mediated mRNA decay in a gene-specific way, which will also be developed together with read-through small molecule compounds (i.e G418). and exon-skipping AON approach to correct the reading frame and restore the major protein product.
2.  Skin fibroblast cultured from an ARC patient carrying the target mutation, currently available in our laboratories, will be used as the cellular model for the in vitro validation.
3.  Outcome measures on the effects of AONs on VPS33B expression will be measured at mRNA and protein levels and functional rescue in collagen homeostasis. 

The student is expected to gain a broad experience in the following fields and techniques: understanding different mechanism of action of AON therapies, the design principle of each AON approach and the usage of various bioinformatics in silicon assay; the in vitro validating system of AON therapies; expression analyses at the RNA and protein levels (quantitative RT-PCR, western blotting, immunochemistry, mass spectrometry, mRNA next generation sequencing).

Timeline:
Month 1-18: The development of an AON approach to correct the genetic defect in VPS33B gene on mRNA and protein expression.
Month 19-30: Further validation of the AON approach at functional level.
Month 31-36: Data analysis, manuscript preparation and thesis write-up         

References:
1.  Sardone V et al. Molecules. 2017 Apr 5;22(4) pii: E563.
2.  Zhou H et al. Nuc Acid Res. 2011;39:7194-208.
3.  Marrosu E et al. Mol Ther Nuc Acid. 2017;8:416-427.
4.  Gissen P et al. Nat Genet. 2004;36:400-4.
5.  Hanley J et al. J Hepatol. 2017;66:1001-11.