Gene Therapy
Dr Richard Harbottle, Group Leader
Gene therapy vectors based on modified viruses are unquestionably the most effective gene delivery systems in use today. Their efficacy at gene transfer is, however, tempered by their potential toxicity [1,2]. An ideal vector for human gene therapy must deliver sustainable therapeutic levels of gene expression without compromising the viability of the host (at either the cellular or somatic level) in any way. Permanently maintained extra-chromosomal gene expression vectors, which comprise entirely human elements, provide an ideal method of achieving this. Non-viral vectors are attractive alternatives to viral gene delivery systems because of their low toxicity, relatively easy production and great versatility. However, their efficiency is still below the requirements for realistic in vivo gene therapy. Fundamental to this inadequacy is deficient delivery exacerbated by the merely transient gene expression of plasmid DNA in vivo.
The development of safer, more efficient, easily and economically prepared, persistently expressing gene delivery vectors therefore remains one of the main strategic tasks of gene therapy research and is the crucial prerequisite for its successful clinical application.
We have produced a novel plasmid vector, which addresses all of these issues providing clinically relevant levels of persistent transgene expression, in vivo, without vector toxicity. The primary aim of the Gene Therapy group is to take the development of this vector system forward as a proof of principle for clinical application.
- Episomal replication without viral proteins
- Prototype gene expression plasmid vector
- Minicircle S/MAR vectors
- Utilising Non-Viral S-MAR Plasmid Vectors to Develop Prophylactic Gene Therapy for Birt-Hogg-Dubé Syndrome
Episomal replication without viral proteins
A vector which does not require transforming viral gene products for episomal maintenance was generated by a collaborator Professor Hans Lipps; the functional element on this plasmid is the scaffold/matrix attachment region (S-MAR) from the human β-interferon gene cluster [3].
The utility that this element provides to plasmid molecules in in vitro systems is threefold:
- plasmids harbouring an S/MAR motif are rendered resistant to integration
- their expression cassettes are not subject to epigenetic silencing [4]
- they exhibit extrachromosomal, mitotic stability [5]
Prototype gene expression plasmid vector
We recently developed the S/MAR vector system for in vivo application and demonstrated its utility to sustain transgene expression in vivo in the livers of mice[6]. We reported that our prototype plasmid vector, which contains an S-MAR domain and the luciferase reporter gene, showed transgene expression for at least six months following a single administration. Subsequently, we have observed that this expression is sustained for the lifetime of the animal. This vector system can readily be adapted for use in other tissues and is ideally suited for application to the kidney with the use of kidney specific or ubiquitous non-viral promoters.
We are also continuously developing and improving our vector systems. We hypothesised that by altering the composition of our vectors we might reduce their toxicity and improve their performance.
Plasmids contain sequences, which are essential for propagation in bacteria but are unnecessary for expression in mammalian cells. These bacterial components have been shown to be responsible the genetic silencing of the vector and toxicity when applied in vivo.
We have developed a minimally sized S/MAR vector, which is devoid of extraneous bacterial sequences and merely comprises an expression cassette and an S/MAR moiety. We demonstrate that it is capable of providing higher and more sustained transgene expression both in vitro and in vivo. Typically, levels of transgene expression from hydrodynamically injected plasmids drop significantly following administration. This is most likely due to the toxicity of the vector and the procedure. The expression from our original S-MAR plasmid drops markedly to 10% of its initial expression within 25 days. In contrast S-MAR minicircle vectors derived from this plasmid not only produce increasing levels of expression following administration but after 25 days show sustained levels which are twice as high as those found immediately after administration and higher than those derived from the original plasmid vector.
Birt–Hogg–Dubé (BHD) syndrome is a genetic disorder whose principle life-threatening manifestation is an increased risk of renal neoplasia [7-9]. BHD is caused by mutations in the BHD gene, which encodes folliculin, a putative tumour-suppressor protein. BHD carriers, who are born with a mutation in one of their BHD alleles, are at risk of “second-hit” renal mutations that are the triggering mechanism of tumour development.
Much is yet to be learnt about the molecular mechanisms of this disease. Although great breakthroughs have been made recently in the identification of its genetic cause the only current treatment for the renal tumours is repetitive surgical intervention. Unfortunately, even with a complete resection of the tumour the risk of spontaneous oncogenesis in the remaining renal tissue remains.
Our research is based on the hypothesis that if a protective, mutation-proof copy of the folliculin gene could be introduced into the renal cells of BHD patients these cells would become resistant to BHD-associated renal neoplasia.
Selected Publications
- Wong SP; Argyros O; Howe SJ; Harbottle RP. (Dec 2010). Systemic gene transfer of polyethylenimine (PEI)-plasmid DNA complexes to neonatal mice. Journal of controlled release : official journal of the Controlled Release Society. Publisher weblink DOI.
- Wong SP; Argyros O; Coutelle C; Harbottle RP. (Jan 2011). Non-viral S/MAR vectors replicate episomally in vivo when provided with a selective advantage. Gene Ther. 18:82-87. DOI.
- Argyros O; Wong SP; Niceta M; Waddington SN; Howe SJ; Coutelle C; Miller AD; Harbottle RP. (Dec 2008). Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector. Gene Ther. 15:1593-1605. DOI.
