A programme of development for alternative technology to conventional vaccination is being pioneered by The Welsh School of Pharmacy and sponsored by 3M Drug Delivery Systems. Richard Toon, a research specialist with 3M, explains how the inhaled vaccines are being produced
In the search for more efficacious vaccines, a novel approach using inhaled plasmid DNA (pDNA) is showing great promise. The approach, being pioneered by The Welsh School of Pharmacy and sponsored by 3M Drug Delivery.
Systems, can be used against a variety of pathogens and tumours for which conventional vaccination regimes have failed and is potentially safer than using live vaccines. It not only represents an alternative to conventional vaccination, but also offers significant potential for the prevention and treatment of fatal diseases and infections.
3M Drug Delivery Systems has a 50-year history in the development and manufacture of pressurised metered dose inhalers (pMDIs) for the pulmonary delivery of small molecule Active Pharmaceutical Ingredients (APIs). However, the pipeline of small molecule APIs for many pharmaceutical companies is beginning to dwindle, due to the high costs involved in developing and bringing such entities to the marketplace. As a consequence, the market for small molecule APIs is beginning to transform from a patent protected area into a generic market. At the same time, the market for biologics is expanding due to the high potential rewards in this relatively untapped market.
As a consequence, 3M Drug Delivery Systems has been exploring new research areas to align its capabilities with the changing therapeutic landscape. The pulmonary delivery of pDNA to correct genetic lung disorders, treat cancers and vaccinate against infectious diseases is one such exciting and novel area for investigation.
The DNA sequence required for vaccination or genetic manipulation is cloned into conventional pDNA, which is easily produced in bacterial cells. pDNA is easy to handle and to scale up for each gene of interest with comparatively low production costs.1 The entire production process for a DNA vaccine can be completed within eight months, whereas approximately 20 months are needed for conventional or subunit vaccines.2
Since pDNA is both chemically and biologically stable, no cold chain may be needed during the distribution of vaccine doses, making it highly suitable for distribution both in developing countries and troubled regions. The risk of reversion to virulent forms and cytotoxic effects is not apparent with these DNA-based approaches.3
Two veterinary DNA vaccines have so far been approved for commercial sale. Fort Dodge Animal Health (a division of Wyeth Inc, NJ, US) licensed a gene-based vaccine approved in the US for protecting horses from West Nile Virus (West Nile Innovator).4 Aqua Health Inc (Switzerland) received approval in Canada for a DNA vaccine based on licenced technology from Vical Inc (CA & US) to protect salmon from infectious hematopoietic necrosis virus.5 However, so far no human DNA vaccine has been approved for use.6
The process involved in transfecting cells with pDNA is extremely complicated. The initial stage involves the formation of a suitable pDNA vector and formulation for pulmonary delivery. This is currently the focus of interest at the Welsh School of Pharmacy. Once the pDNA vector has been successfully delivered to the required site, the process of transfection can begin. The pDNA vector can be taken into the cell via a process of endocytosis, forming an endosome within the cell.
The membrane of the endosome must then be made to burst to release the pDNA vector into the interior of the cell. This can be accomplished by formulating the pDNA vector with a transfection agent that is used to disrupt the endosome membrane, often via an opposing charge interaction with membrane components. Such agents include polyethylenimine (PEI), cationic lipids and fusion proteins.
Once the endosome releases the pDNA, it then needs to cross the nuclear membrane to allow access to the machinery required to convert the DNA to its therapeutic product. The pDNA can either be used to replace a faulty portion of DNA, or to replace missing DNA or it can be a template to produce host antigens.
However, formulating the pDNA to transfect is an extremely complex area to investigate. Handling of macromolecules such as DNA requires specialised knowledge. DNA has to be processed in a special way to ensure that it remains chemically stable and retains its tertiary physical structure.
In addition to the processing issues for such macromolecules, the analysis of DNA requires a certain level of expertise to evaluate stability both during processing and after formulation. The Welsh School of Pharmacy at Cardiff University has a patented process for the processing of pDNA nanoparticles and has the relevant expertise in this area to evaluate the stability of pDNA within suspension formulations for pMDIs.
As a consequence, 3M Drug Delivery Systems approached two experts in this area, James Birchall and Glyn Taylor, at the Welsh School of Pharmacy. The processing and formulation of pDNA for incorporation within a pMDI formulation is currently being investigated under a jointly funded research programme.
A replication process is required to generate sufficient pDNA for laboratory use. The required gene is amplified in a strain of E.coli. Researchers at the Welsh School of Pharmacy use the plasmid enhanced green fluorescent protein (pEGFP) reporter gene, which is grown in the DH5a strain of E.Coli. EGFP emits bright green fluorescent light when exposed to UV or blue light. This allows expression of the protein within a cell to be monitored by fluorescence microscopy or flow cytometry without the need for prior harmful staining techniques. An antibiotic (kanamycin) resistant gene is also incorporated.
The strain is then grown on Luria agar containing kanamycin, which allows only the growth of colonies of the required strain. The colonies are then removed and grown in broth containing kanamycin. Isolation of the cells, cell lysis and a clean up and elution procedure then releases and collects the pDNA.
The processing of suitable pDNA nanoparticles for incorporation into a pMDI formulation initially involves incorporation of the pDNA into a microemulsion, which is then snap frozen in liquid nitrogen. The microemulsion is formed via a ternary mixture of a surfactant (lecithin), an organic phase (iso-octane) and water (see Figure 2). The aqueous phase contains a lyoprotectant, such as a sugar, which protects the structural integrity of the pDNA during the snap freezing process.
Once the microemulsion has been snap frozen, the material is then dried under a vacuum while frozen. The lyophilised material is then washed with an organic solvent. This forms suitable surfactant-coated particles for dispersion in an organic solvent within a HFA propellant (see figure 3).
The current research work has been undertaken by Baljinder Bains, a post-graduate student at the Welsh School of Pharmacy, using ethidium bromide gel electrophoresis to assess the structural integrity of pDNA after the snap freezing process. Bains has assessed a number of lyoprotectants, which could be used (Figure 4) and has optimised this stage of the process.
A model system, using surfactant-coated salbutamol sulphate, has been found to be easily dispersed by simply shaking the lyophilised material in a HFA propellant containing a quantity of ethanol (Figure 5).
However, while significant progress has been made towards the development of a pMDI formulation containing pDNA for pulmonary administration, significant hurdles still remain. The current work undertaken at the Welsh School of Pharmacy has proven the initial feasibility of incorporating novel pDNA particles into a pMDI formulation. Initially, the work involved the incorporation of a pEGFP gene. However, at some point, a gene specific for a required condition will need to be incorporated to undertake clinical assessments.
At this stage, the biological functionality of the pDNA must be optimal and unaffected by processing. In addition, the processing of the pDNA particles has been undertaken only on a laboratory scale. This will need to be scaled up to allow a sufficient quantity of pDNA particles to be processed.
Pulmonary pDNA delivery for gene-based treatments and genetic vaccination offers substantial promise for the future. While hurdles such as scale-up and clinical assessments remain, the initial research carried out so far has shown promising results.