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Title: Optimisation of lentiviral vectors for gene therapy of spinal muscular atrophy
Author: Ali Mohammadi Nafchi, Neda
ISNI:       0000 0004 8499 6270
Awarding Body: Royal Holloway, University of London
Current Institution: Royal Holloway, University of London
Date of Award: 2017
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Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease particularly characterised by degeneration of motor neurons from the ventral horn of the spinal cord. Such degeneration leads to muscle weakness and wasting (atrophy). Survival motor neuron (SMN) 1 gene is the SMA-determining gene, being absent or mutated in most people affected. SMN2 is a highly similar gene which produces low levels of SMN protein and allows survival in the absence of SMN1. Full-length SMN is a ubiquitous and essential cellular protein, and low levels of it result in a wide range of systemic pathologies in affected individuals. One avenue to treat SMA is gene therapy, a technology that uses genetic material to alter gene expression and can be applied to both inherited and acquired disorders. Gene addition therapy to replace the faulty SMN1 gene and thus increase the levels of SMN protein to a therapeutic threshold is one of the main strategies for treating SMA. Adeno-associated virus (AAV) vectors are currently being used in clinical trials to attempt such enhancement of SMN levels. A possible alternative may be provided by lentiviral vectors, which are widely used in basic research and clinical applications for gene transfer, including long-term expression. A previous study in Prof. Yáñez's lab demonstrated that lentiviral vectors were significantly more effective for in utero transduction of motor neurons than AAV. Therefore, this study is focused on optimising a lentiviral expression system for SMN1 expression. A variety of integration-proficient and integration-deficient lentiviral vectors (IPLV and IDLV, respectively) with different promoters -either cytomegalovirus (CMV) or human synapsin (hSYN)- and transgenes -either wild-type or a novel codon optimised hSMN1- were produced to compare the efficiency of transgene expression and thus determine the lentiviral configuration resulting in the highest production of full-length SMN. Vectors were tested in a variety of proliferating and quiescent in vitro cell culture models for quantitative estimation of full-length SMN production. The results of these experiments revealed that using integrating, CMV-driven, codon-optimised hSMN1 lentivector resulted in highest production of full-length SMN protein. In general, a comparison of expression levels revealed that IPLVs produced more transgenic protein than IDLVs. However, IDLVs achieved significant transgene expression levels, particularly in quiescent or growth-arrested cells, and they are expected to be significantly safer than IPLVs. Of note, overexpression by hSMN1 lentivectors restored gems in transduced type I SMA fibroblasts in a dose-dependent manner. Encouraged by the results of the cell culture work, we performed very preliminary in vivo experiments. As SMN-replacement therapy is particularly effective at early stages of post-natal development, we hypothesised that earlier treatment may be advantageous. We thus attempted to deliver control and our novel SMN1 lentivectors to the developing embryo of wild-type and SMA mice. While it is clear that this part of the work is at a very early phase, the outcome showed encouraging viability and lack of toxicity in treated animals. In conclusion, the results of this study suggest that lentiviral vectors are potent agents for transgenic expression of codon-optimised hSMN1, and that in utero delivery has significant potential as a therapeutic strategy for SMA.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biomedical science ; Gene therapy ; Gene addition ; Lentiviral vectors ; In utero ; Spinal muscular atrophy ; Survival motor neuron gene