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Nonviral Vector Development

Plasmid DNA molecules are one of the most widely used tools in molecular biology research. They are derived from naturally occurring molecules in microbes.

These circular double stranded DNA molecules have been manipulated by molecular biologists to allow for the propogation and study of interesting pieces of DNA from a wide variety of sources. Plasmid molecules are available to meet a wide range of needs.

Whether the plasmid is required for a small piece of work, or if it is intended for future clinical use, the process always starts by transforming the molecule into E. coli so it can be replicated by the microbial machinery.

Restriction endonuclease enzymes are used cut up DNA and individual fragments can be readily visualised and purified on gels.

This has allowed us to create a series of modular plasmid molecules comprised of interchangeable bacterial bacbones, promoter/enhancer regions and transgenes. This has allowed our group to optimise the elements within our plasmids so they are suitable for clinical use.

Restriction endonuclease analysis of plasmid DNA.Over the past few years we have put tremendous effort into improving the design of our plasmid DNA molecules to make them more suitable for clinical studies.

We have developed a range of promoter elements that direct long-term expression in-vivo and have reduced the toxicity of the plasmid by removing elements which cause inflammation in human cells.

This process culminated in the development of our current clinical trial plasmid, pGM169.

Aerosol Delivery of Gene Transfer Vectors


Gene therapies which utilise modified viruses or plasmid DNA (pDNA), to re-introduce functional copies of defective or mutated genes, are being investigated for the treatment of a wide range of lung diseases including cystic fibrosis (CF), cancer and alpha-1 anti-trypsin deficiency (Gill et al., 2004).

The relative accessibility of the pulmonary epithelium makes aerosol delivery of gene therapy formulations an attractive possibility, allowing non-invasive application to target cells within the lung whilst minimising the risks associated with systemic delivery.

Following identification of the gene responsible for CF in 1989 (Riordan et al., 1989) the disease has served as a paradigm for gene therapy in general and aerosol gene therapy in particular. To date there have been ~30 Phase I/II gene therapy clinical trials for CF including 8 trials incorporating aerosol delivery (of gene transfer agents) to the lungs of CF patients as a key component of the study (Griesenbach et al., 2009).


Typical clinical nebuliser generating an aerosol


Challenges to Aerosol Gene Delivery.

Although the field of gene therapy proceeds at pace, transfer of technological developments to aerosol gene therapy has been limited by the additional constraints placed upon formulations for nebulisation and the associated costs of developing and testing aerosol formulations in relevant animal model systems.

To date, only 3 gene transfer agents, recombinant adenovirus (Ad) (Perricone et al., 2001), adeno-associated virus (AAV)(Moss et al., 2007) and pDNA complexed to the cationic lipid Genzyme Lipid 67A (GL67A) (Alton et al., 1999) have been aerosolized to the lungs of patients and whilst a huge variety of gene transfer agents are now available, few have proven suitable for aerosolisation.

A recent study by the Consortium identified shear related degradation of pDNA during nebulisation as the primary limitation in the progression of novel non-viral gene therapy formulations towards the clinic. Only 10% of tested formulations demonstrating efficacy following jet nebulisation.

Consequently, potential aerosol gene therapy applications are currently limited both by the choice of suitable vectors and available delivery devices. Evidence from in-vivo studies and human clinical trials has indicated that lung gene transfer mediated commitment  by aerosolisable vectors remains inefficient and gene expression is transient in nature (Rochat et al., 2002).

As a result, it is likely that repeated administration of gene therapy formulations will be required to observe clinical benefit.


A Next Generation Cascade Impactor (NGI). Used for measuring the droplet sizes in aerosol formulations.


Present State of the Art.

Considerable advances have been made to achieve gene delivery via aerosol. More effective nebulisation devices are available to deliver gene therapy formulations efficiently and without destructive effects. Optimisation of gene transfer agents to generate persistent high-level gene expression in the lung will also be of significant benefit.

Lentiviral Vector Development

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. Instead they package a gene therapy construct within their viral structure. In this way it is hoped to use the virus' ability to get into cells and deliver a therapeutic gene.

These methods have been heavily investigated for CF gene therapy and indeed most CF gene therapy trials conducted to date have used viral vectors. However, viral vectors induce an immune response from the host (human) organism. 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.

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. However, previously it had been shown that the traditionally used Lentivirus envelope (VSVG-pseudotype) was inefficient at transducing the airway epithelium, unless the epithelium 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, the Consortium 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:

  • 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.
  • This can be achieved in a single formulation, and without the need for pre-conditioning with additional chemicals.
  • Stable expression levels can be achieved for the life-time of the mouse.
  • Re-administration to nasal epithelium and lung is feasible
  • The SIV-F/HN vector can transduce human air-liquid interface (ALI) cultures and generate stable, long-term gene expression.
  • 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.


A Sendai-Pseudotyped SIV vector combines the long lived transgene expression characteristics of SIV with the efficient airway cell tropism of Sendai Virus.