Wave II Viral Vector Development

Transduction of human air–liquid interface (ALIs) cultures with F/HN-SIV-lux
Transduction of human air–liquid interface (ALIs) cultures with F/HN-SIV-lux

Recombinant viral vectors are gene therapy vectors which are derived from viruses. Many viruses such as adenovirus, adeno-associated virus & Sendai virus have evolved over millions of years to become efficient agents at infecting human airway cells.

Using an array of molecular biology tools methods have been developed to allow virus particles to be modified such that they contain no infectious material or replicative capacity but instead they package a gene therap construct within their viral structure. In this way it is hoped to use the virus's ability to get into cells and deliver a therapeutic gene.

Such methods have been heavily investigated for CF gene therapy and indeed most CF gene therapy trials that have been conducted to date have used viral vectors. However, viral vectors induce an immune response from the host (human) organism as their viral particles are recognised as foreign material. This neutralising antibody reponse means that to date no viral vector has been successfully repeat administered without loss of activity.

Production of lentiviral vectors in suspension cell culture.

For several years now the Consortium has been collaborating with DNAVec a Japanese Biotech company to develop a new viral vector platform for CF Gene therapy. Lentivirus-based vectors hold the promise of long duration of gene expression and low immunogenicity and are therefore particularly attractive for airway gene therapy. Previously however, it had been shown that the traditionally used Lentivirus envelope (VSVG-pseudotype) was inefficient at transducing the airway epithelium, unless the epithelium efficiently had been deliberately damaged, or had the epithelial tight junctions opened to provide access to the virus. As an alternative, DNAVEC proposed to pseudotype the lentivirus vector with the F and HN proteins from Sendai Virus (Kobyashi et al 2003) to increase the efficiency of airway transduction without the need for pre-conditioning agents to damage the epithelium. In close collaboration with DNAVEC we assessed the novel F/HN-pseudotyped lentivirus in mice in vivo and in various ex vivo lung models. As reported recently (Mitomo et al 2010 & Griesenbach et al 2010) we have shown that:

a) the SIV-F/HN vector can transduce the respiratory epithelium of the murine airways via the apical membrane in vivo at levels that may be relevant for clinical benefit in CF.

b) this can be achieved in a single formulation, and without the need for pre-conditioning with additional chemicals.

c) stable expression levels can be achieved for the life-time of the mouse.

d) re-administration to nasal epithelium and lung is feasible

e) the SIV-F/HN vector can transduce human air-liquid interface (ALI) cultures and generate stable, long-term gene expression.

f) the SIV-F/HN vector transduces human lung slices ex vivo and functional CFTR chloride channels can be generated in vitro.

These very promising results need a significant further investment of time and effort (minimum of 3 years) before it would be realistic to expect clinical trials to start. If further studies in mice and some limited studies in sheep continue to prove encouraging, then this SIV-F/HN Wave 2 product could potentially be ready to follow on closely behind the completion of the Wave 1 study. The Consortium's enthusiasm for developing Lentiviral vectors for CF gene therapy further is based on several important findings: firstly, in every model studied so far, the levels of Lentivirus-mediated gene transfer are log-orders higher than the current Wave 1 product. Secondly, Lentiviruses integrate into the cell genome, which leads to prolonged and stable expression (in mice for the lifetime of the animal after a single dose). Finally, the virus does not induce effective immune responses after repeat administration. Importantly, a collaborative research agreement was recently agreed between DNAVEC and the Consortium to provide the opportunity for rapid further development of the SIV-F/HN vector for fast progression into a clinical trial.


Previous Viral Vector Development - AAV


A common strategy for the delivery of genes for therapeutic purposes is to utilise the biology of viruses in the form of viral vectors. We have been looking at one particular type of vector, derived from Adeno Associated Virus serotype 5 (AAV5). This is one of a growing family of vectors that have demonstrated long-term expression of a variety of genes in pre-clinical studies, and is now being taken through to clinical trials.

We were interested in AAV5 because of its reported access into cells via receptors that are present at the surface of airway cells in several species.

Our work demonstrated that AAV5, like other members of its family, does indeed lead to very long-term (at least one year) gene expression in rodent models of gene transfer.

One of the general concerns about the clinical application of viral vectors including AAV5 however is that chronic diseases are likely to require repeat administration, and viruses tend to be prone to immune responses which limit the efficiency of repeated doses.

We investigated this in our models of airway gene delivery, and found that the immune response to AAV5 vectors is potent enough to significantly knock down any gene transfer on the second or third exposure under the conditions that we tested. This is due to an antibody response, which it may be possible to blunt using short-term immune repression, however the issue of whether this would be acceptable in a chronic infection setting such as CF, would probably be problematic.

This project has led to the preparation of two papers, one published in 2006 and 2007


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