Using DNA as a drug—Bioprocessing and delivery strategies

chemical engineering research and design 8 7 ( 2 0 0 9 ) 343–348 Contents lists available at ScienceDirect Chemical Engineering Research and Design journal homepage: www.elsevier.com/locate/cherd Using DNA as a drug—Bioprocessing and delivery strategies Ying Han, Shan Liu, Jenny Ho, Michael K. Danquah, Gareth M. Forde ∗ Bio Engineering Laboratory (BEL), Department of Chemical Engineering, Monash University, Clayton, Melbourne 3800, Australia a b s t r a c t DNA may take a leading role in a future generation of blockbuster therapeutics. DNA has inherent advantages over other biomolecules such as protein, RNA and virus-like particles including safety, production simplicity and higher stability at ambient temperatures. Vaccination is the principal measure for preventing influenza and reducing the impact of pandemics; however, vaccines take up to 8–9 months to produce, and the global production capacity is woefully low. With production times as short as 2 weeks, improved safety and stability, bioprocess engineering developments, and the ability to perform numerous therapeutic roles, DNA has the potential to meet the demands of emerging and existing diseases. DNA is experiencing sharp growths in demand as indicated by its use in gene therapy trials and DNA vaccine related patents. Of particular interest for therapeutic use is plasmid DNA (pDNA), a form of non-genomic DNA that makes use of cellular machinery to express proteins or antigens. The production stages of fermentation and downstream purification are considered in this article. Forward looking approaches to purifying and delivering DNA are reported, including affinity chromatography and nasal inhalation. The place that pDNA may take in the preparation for and protection against pandemics is considered. If DNA therapeutics and vaccines prove to be effective, the ultimate scale of production will be huge which shall require associated bioprocess engineering research and development for purification of this large, unique biomolecule. © 2008 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: DNA therapeutics; Plasmid DNA; Bioprocess engineering; Pandemic preparedness Contents 1. 2. 3. 4. 5. 6. 7. 8. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The growing interest in DNA as a therapeutic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . “Let’s take it outside” (of the lab) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plasmid DNA production from bacterial fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Approaches to the purification of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Overview of purification methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Affinity purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Anion-exchange purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Delivery strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pandemic preparedness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corresponding author. Tel.: +61 3 9905 3437. E-mail address: [email protected] (G.M. Forde). Received 10 March 2008; Received in revised form 23 September 2008; Accepted 29 September 2008 0263-8762/$ – see front matter © 2008 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cherd.2008.09.010 ∗ 344 344 344 345 345 345 345 346 346 347 347 347 347 344 1. chemical engineering research and design 8 7 ( 2 0 0 9 ) 343–348 Introduction A number of diseases have not yet been conquered with millions of people dying each year due to the inefficacy of current therapeutic methods (Liu, 2003). Innovative medical treatments, such as DNA vaccines and gene therapy, hold great promise to meet the demands of current and emerging public health problems. DNA vaccines based on plasmid DNA contain specific genes encoding target proteins and can activate both cell-mediated immunity and humoral responses (Prather et al., 2003), yielding a much better overall protective immunity against diseases. Gene therapy aims to treat diseases by making available functional genes to assist or replace defective or mutant genes. Compared to conventional therapeutic methods, using DNA as a drug has many potential advantages, including being cheaper to produce, more stable at ambient temperature and safer to administrate (Manoj et al., 2004). Recently, several DNA vaccines have progressed to the clinical evaluation including those for hepatitis, tuberculosis, HIV, influenza, and malaria. If DNA vaccines prove to be effective, the ultimate scale of production will be huge: prevention of epidemics will require greater than 60% vaccination, hence global vaccination against a pandemic will manufacturing capacity for flu vaccines is currently woefully inadequate at 350 million shots (Kieny et al., 2006) compared to a current global population of over 6.5 billion (United Nations, 2006). DNA production and purification under non-optimized laboratory conditions cannot meet the future needs for industrial scale production. The establishment of plasmid DNA manufacturing and engineering bioprocesses must be developed from laboratory scale through to final control agency licensing and market approval. Additionally, the challenge of efficiency delivering the DNA molecules to the cell nucleus poses new challenges to biochemical engineers. This review article addresses practical considerations for the large scale production and purification of DNA, especially via affinity and anion-exchange chromatography, and strategies for enhanced pDNA delivery. 2. The growing interest in DNA as a therapeutic The demand by research groups for purified DNA in novel vaccine and gene therapy applications has resulted in pressure to make more DNA in less time at reasonable prices. DNA offers to generate a whole new generation of reverse engineered biopharmaceuticals. In particular, plasmid DNA (pDNA) is gaining huge interest for a range of therapeutic applications as it has an excellent safety profile, displays no toxicity and is simpler to develop than current viral systems—this is displayed clearly by the rising demand for DNA in gene therapy clinical trials displayed in Fig. 1 (Ulmer et al., 1996). There has been an explosion in number of patents in the area of DNA vaccines as shown in Fig. 2. In 1990, only 2 patents contained the term “DNA vaccine” or “DNA vaccines”. By the end of 2007, this figure had grown exponentially to 1468 patents (Donnelly et al., 1996). 3. Fig. 1 – The rapidly expanding interest in DNA as the vector of choice in gene therapy trials (Ulmer et al., 1996) indicates that DNA is gaining interest compared to other vectors such as adenoviruses and retroviruses. extensive practical examples of the current use of pDNA in non-laboratory applications: - 416 completed or active trials utilizing DNA were registered with the National Institute of Health (NIH, http://www.nih.gov, accessed 9th July 2008) for applications as wide as cancer treatment and HIV, malaria, prostate cancer, ebola and avian influenza prevention. - Four active HIV DNA vaccine clinical trials at Phase 2 and 10 active HIV DNA vaccine clinical trials at Phase 1 (http://avac.org/, accessed 9th July 2008). - VGX Pharmaceuticals (www.vgxp.com) announced on June 30th 2008 that the U.S. Food and Drug Administration (FDA) approved the company’s application for its DNA vaccine for cervical cancer therapy. - 246 gene therapy trials that utilize plasmid DNA globally (http://www.wiley.co.uk/genetherapy/clinical/, accessed). - Aldevron LLC (ND, USA) opened a facility in 2007 to manufacture kilogram amounts of pDNA, which is sufficient for the creation of up to 2 000 000 DNA vaccine doses assuming that a dose contains 500 ␮g of pDNA. - The world’s first licensed DNA vaccine was released on July 21st 2005 by the US Department of Agriculture (USDA) to immunize. - Horses against West Nile virus (WNV). As a lag time of 10–15 years is expected between the implementation of a new technologies in a veterinary environment to when the technology is employed in commercially available human therapeutic products, it would be expected that the first DNA “drug” is available on the market at around 2015–2020. The above mentioned weight of evidence shows “Let’s take it outside” (of the lab) DNA vaccines offer a real and viable option for the future prevention of old and emerging diseases. This is evidenced by Fig. 2 – The increasing commercial applications of DNA vaccines—number of United States Patent and Trademark Office, http://www.uspto.gov, Accessed 21st January 2008. chemical engineering research and design 8 7 ( 2 0 0 9 ) 343–348 that the development of new up, mid and downstream process technologies and enhanced delivery methods are timely to meet the huge future demand of pDNA. To quote Dr Barney Graham (MD, PhD, chief of the NIH National Inst of Allergy and Infectious Diseases Vaccine Research Center’s Clinical Trials Core Laboratory): “The DNA vaccine’s exploitation of both cellular and antibody immunity offers a potentially potent defence against West Nile virus. In our experience in clinical trials, DNA vaccines generally cause few side effects, making them a promising alternative to conventional vaccines.” (http://www.3.niaid.nih.gov, article released April 18th 2005). This weight of evidence shows that DNA has a predicable and reproducible efficacy for use in health applications. 4. Plasmid DNA production from bacterial fermentation Bacterial fermentation for plasmid DNA production is mostly performed in shake flasks or fermentor in a batch or fedbatch fashion. In all these cases, fermentation proceeds to a defined cell density and the cells are further processed. Both batch and fed-batch fermentation technologies have been successfully employed for plasmid production via E. coli system (Riesenberg, 1991; Zabriskie and Arcuri, 1986; Yee and Blanch, 1992; Lee, 1996). Plasmid DNA production processes that employ simple batch cultivation methodology produce relatively low biomass and plasmid yields. When a rich cultivation medium is employed in batch fermentation, usually the fermentor oxygen transfer capacity is eventually exceeded, and this results in the creation of an oxygen-limited environment. This lack of oxygen triggers the metabolism of E. coli and leads to the production of toxic by-products that severely limit growth and even lead to cell death (Pan et al., 1987; Luli and Strohl, 1990; Cherrington et al., 1990). Fed-batch fermentation technology delivers nutrients over an extended period of time, thereby achieving the control of nutrient availability to a level compatible with the oxygen transfer capacities of the fermentor. This results in higher cell densities, plasmid yields and quality. The feeding of nutrients, usually carbon source, has been extensively researched, and incorporates a range of approaches that span from simple to very elaborate, with each presenting its own advantages and disadvantages. Feeding regimens aimed at maintaining a more constant environment thus maintaining a desired growth-rate, have been successfully implemented. In general, these strategies lead to the accumulation of high biomass levels, with recombinant construct presenting its own limitations. These limitations are probably the result of the extra metabolic burden caused by the over-expression of plasmid and recombinant product. In optimizing plasmid DNA yield for therapeutics, three factors or areas must be considered. The premier factor is the culture mass at the time of harvest. Obviously higher cell density will yield more plasmid per unit volume of culture. The culture mass can be improved through the optimisation of process parameters like pH, dissolved oxygen concentration (DO) and temperature as well as the type and concentrations of nutrients present in the growth medium. The second factor is high plasmid copy number which also leads to high specific yields. Additional significance of maximizing plasmid copy number is that the plasmid obtains high purity since the amount of plasmid is increased relative to contaminants such as genomic DNA, RNA and proteins. This is a very advantageous for downstream processes. Improved seg- 345 regational plasmid stability increases purity of plasmid DNA, since plasmid-free cells will only produce contaminants to the fermentation process stream (O’Kennedy et al., 2000). The third factor that must be considered is plasmid quality. A single plasmid can exist in a number of different forms in a cell. Changes in growth conditions, including temperature, nutrient concentrations, pH, oxygenation and growth phase can affect the existence of these alternate forms (Prather et al., 2003). Expression of a plasmid-harboured gene of interest is undesirable during fermentation process and in some instances plasmid-derived expression may obstruct growth and thereby affect plasmid DNA production. The major features which are imperative for efficient expression vector will still be important for therapeutic plasmid vectors (Zabriskie and Arcuri, 1986; Wang et al., 2001). The purity of the final plasmid DNA product is affected by purification step. 5. Approaches to the purification of DNA 5.1. Overview of purification methods If DNA is applied to clinical trials, high purity supercoiled plasmid DNA is the desired topological form. Precipitation (Murphy et al., 1999) and extraction of plasmid DNA by organic solvents, ultrafiltration, and chromatographic techniques are mostly used for this purpose. Among the four methods, chromatographic techniques are predominantly employed because they have the highest resolution and they are scaleable. For instance, anion-exchange chromatography (AEC) (Lyddiatt and O’Sullivan, 1998; Varley et al., 1999; Ferreira et al., 2000), which effectively removes host protein (Prazeres and Ferreira, 2004) but does not remove all genomic DNA, RNA, or endotoxin (Sofer and Hagel, 1994). Size-exclusion chromatography (SEC), which can be used to remove small molecular weight contaminants such as small RNA molecules and endotoxin (Ferreira et al., 2000; Durland and Eastman, 1998). Reverse-phase chromatography (RPC) (Green et al., 1997), can be used to successfully remove contaminating proteins and endotoxin (Huber, 1998); however, RPC typically uses organic solvents and toxic reagents, all of which present elevated challenges and production costs. 5.2. Affinity purification The high selectivity of affinity binding is an attractive proposition for the purification of biopharmaceuticals such as plasmid DNA. Elegant affinity approaches to the purification of plasmid DNA have been reported using triplex-forming oligonucleotides (Schluep and Cooney, 1998; Wils et al., 1997); although highly selective, this method is generally characterized by slow binding kinetics and is limited to specific DNA base sequences and therefore scale-up costs may be prohibitively high. Interestingly, sequence-specific DNA binding proteins have yet to be fully exploited in the affinity-based purification of plasmid DNA. It is now possible to design and engineer stable affinity ligands capable of discriminating and selectively binding a target biomolecule in the presence of closed related impurities. Practice has shown that a ligand with an optimized dissociation constant will facilitate the successful operation of affinity chromatography for purification of plasmid DNA. The optimal dissociation constant for an affinity binding mechanism for use in a chromatographic system is 10−6 M−1 to 10−8 M−1 . If the dissociation constant is greater 346 chemical engineering research and design 8 7 ( 2 0 0 9 ) 343–348 than this value, non-target molecules can be co-purified as the mechanism is not selective enough for the target. If it is too low, production yields are low. The ligands designed will be specific enough so that the target plasmid DNA is selectively bound without the co-purification of genomic DNA, RNA, protein, and endotoxins with higher yield. Current industrial purification systems normally use several filtration stages and three chromatographic stages, which require a yield of at least 90% (Varley et al., 1999). Obtaining the smart ligands will enable plasmid DNA purification systems to be simplified by combining purification, concentration and clarification into a single process. As a result, the process yield of affinity chromatography only needs to be more than 73% to have economic viability. 5.3. Anion-exchange purification The polyanionic structure of plasmid DNA can be exploited by anion-exchange chromatography using an anion exchange ligand. The overall charge of plasmid DNA depends on the number of nitrogen bases that make up the molecule. The expected elution profiles follow the order of increasing molecular size. However, inversions have been observed in the predicted retention factors in the anion-exchange chromatography of double stranded DNA (dsDNA) fragments on a variety of stationary phases (Huber, 1998; Yamakawa et al., 1996). It was suggested that some dsDNA fragments are retarded because of their high AT content (Yamakawa et al., 1996), which would indicate that the elution profile of nucleic acids on ion-exchange resins is also sequence dependent. A more detailed analysis revealed that peak retardation is not always proportional to the AT content of the dsDNA sequences (Huber, 1998; Yamakawa et al., 1996). Rather, the binding of nucleic acid molecules onto anion exchangers is favoured by the optimal interaction with the curvature of the pores (Colpan and Riesner, 1984). Therefore, it has been proposed that separation is conformation dependent. The flexibility of the nucleic acid promotes a better fit within the pore curvatures, enabling more charges to interact with the solid phase, thus leading to higher retention factors. Another physical explanation for the stronger binding of flexible dsDNA fragments relies on the appearance of a dipole character owing to the local compression of charges at curved regions (Huber, 1998). Bending increases the local charge density and consequently, stronger electrostatic interactions take place at these locations. Consequently, the observed sequence dependent elution profile of dsDNA is an artefact that conceals the phenomenon that is really responsible for peak retardation: sequence bending. The separation of plasmid topoisomers with anion exchangers, mainly the relaxed and supercoiled plasmid isoforms (Prazeres and Ferreira, 2004; Lahijani et al., 1996a,b; Marquet et al., 1995), confirms this conformation dependent retention hypothesis. Supercoiled plasmid DNA is more stretched, compact and bent than its relaxed isoform, and so presents higher charge densities and a better fit with the curvature pores. The flow rate has no significant effect on the resolution of nucleic acids (Colpan and Riesner, 1984), with the sole disadvantage of high flow rates being the dilution of the eluted nucleic acids. However, in process chromatography on particulate support, the flow rate controls the residence time, which is a function of the target molecule diffusivity, sample viscosity and particle size (Sofer and Hagel, 1994). The loss of plasmid DNA in the column flow-through (Prazeres and Ferreira, 2004) can thus be attributed to high flow rates which represent shorter residence times than those required for efficient plasmid binding. 6. Delivery strategies The transfection efficiency of DNA is relatively low compared to other vectors such as viruses, virus-like particles and cellular vectors. Post administration, naked DNA is susceptible to degradation by serum enzymes such as endonucleases, thereby reducing the amount of DNA that is available in hosts to express antigen. Upon entry into a host cell, the transport of DNA across cell membranes is limited by its net negative surface charge and large hydrodynamic diameter (Ledley, 1996). In addition, one possible mechanism of DNA uptake is endocytosis, after which the DNA travels through the endosome–lysosome compartment, where it may be degraded. Thus, improved DNA delivery strategies are needed. Currently, the most commonly used delivery method for DNA vaccines is intramuscular injection. The results varied with both the antigen expressed and the species were studied (Liu, 2003). However, this method has several problems which include the reuse of syringes and needles without sterilization in most nations. DNA vaccines are common delivered by gene gun. This delivery method has gained popularity in terms of efficiently delivery DNA vaccines. However, only a limited amount of gold beads coated with plasmid DNA can be discharged directly into the cellular cytoplasm of skin cells by gene gun (Greenland and Letvin, 2007). Another delivery method is electroporation, previous studies have shown that intradermal or intramuscular injections, followed by electroporation of the injection sites, resulted in higher transfection efficiency and immune responses in mice and pigs (Manoj et al., 2004). However, severe tissue damage and toxicity could company electroporation due to the release of heat during the transfer of electric pulses of high strength into the tissues (Manoj et al., 2004). A very promising delivery method is nasal delivery which is an important arm of the mucosal and systemic immune system. And this revolutionary approach will overcome several of the problems of existing vaccine delivery including the need for medical personnel to administer needles, the cost and logistics of storing and transporting vaccines, and hygienic needle used and disposal (Davis, 2001). Intranasal vaccination offer non-invasive administration and easily accessible for a larger population, thus, will greatly assist the development of global health vaccination particularly in third world countries with the potential to reach greater numbers of people. Nasal delivery system can be achieved via different pharmaceutical forms such as dry powders, solutions or suspensions that been generated as different sized particles or droplets (Kuper et al., 1992). First nasally administered vaccine was approved by the FDA in 2003, and provides another option for protection against influenza (http://www.fda.gov/Fdac/features/2003/503 flu.html). However, the short residence time and low permeability of the formulations within the nasal cavity did contribute to the poor bioavailability reported in previous studies. Indeed, nasal delivery has not yet challenged the transfection efficiencies of other proven delivery methods such as gene gun (as a needle-less delivery option) and liposomal formulation (for injected delivery). Thus, the attention for developing a new generation of delivery options has shifted to encapsulating chemical engineering research and design 8 7 ( 2 0 0 9 ) 343–348 DNA based therapeutic products into biodegradable polymer systems to maintain a sustained release profile over a period of time, ideally to a specific site (Alpar et al., 2005). The design and use of biodegradable polymers for the controlled delivery of entrapped DNA is an approach that holds promise for improving the duration and effectiveness of DNA delivered locally or systematically. Several products based on biodegradable polymer have been approved by FDA as biomaterials. In addition, microencapsulation via ultrasonic atomization has been recently reported to be a viable method for the large scale production of monodispersed biodegradable polymer microspheres for controlled delivery of pDNA (Ho et al., 2008). One of the more exciting uses for plasmid DNA vaccines is for the simultaneous delivery of multivalent vaccines for the creation of vaccines against more complex and challenging diseases such as HIV (Schillinga et al., 2006), malaria (Sahaa et al., 2006) and tuberculosis (Tanghe et al., 2001). A multivalent vaccine is a vaccine designed to elicit an immune response either to more than one infectious agent or to several different antigenic determinants of a single agent. 7. Pandemic preparedness Vaccination is the principal measure for preventing influenza and reducing the impact of pandemics (Infuenza Fact sheet, 2003). However, the United States General Accounting Office (USGAO, 2000) reported that due to the inability to produce the required number of vaccines within the required time period, vaccines may be unavailable, in short supply, or ineffective for certain portions of the population during the first wave of a pandemic (based on a traditional vaccine production cycle taking at least 6–8 months). In addition to this, the global vaccine production capacity is estimated at 350 million doses per year (Kieny et al., 2006); however, with a herd immunity threshold for most preventable diseases being in the range of 75–94% (CDC, 2007), the potential demand for vaccine doses to prevent a pandemic could be greater than 4.8 billion doses (assuming a global population of over 6.5 billion (United Nations, 2006)). Hence, new and innovative solutions must be found to deal with this untenable situation if vaccines are to play their role as the principal measure for preventing influenza pandemics. DNA based vaccines could assist to over come these problems. A working vaccine has previously been taken through the stages of initial development to final product in approximately 1 month (Bouchie, 2003). It must be noted that no commercial influenza DNA vaccine is currently available; however, Phase 1 human trials to protect against the influenza virus have been successful (Murphy, 2004). Further, cell-mediated immunity and humoral responses have been considered and reported for equine influenza virus, particlemediated DNA vaccination (Soboll et al., 2003). This study is of interest not only due to its success but also for its positive outcomes of DNA vaccination of a large animal, in this case a horse. As different DNA molecules have such similar physicochemical characteristics and production methods can be near-duplicated for different molecules, this time could conceivably be reduced to around 2 weeks. Hence, the production time issue could be solved. Being able to make enough doses in time is the last remaining issue and will hinge around the trade off between the capital investment required to have spare vaccine production capacity lying dormant for long periods of time versus the health benefits of having large numbers 347 of vaccine doses when required to combat a pandemic or epidemic. One future option, when DNA therapeutics really take off, is for a distributed global network of DNA manufacturers that routinely make DNA drugs (e.g. for expression of certain hormones) that can quickly change their production to DNA vaccines. The development of such a system will require government or philanthropic backing until such time that the sustained demand for DNA drugs reaches the multi-billion dollar level. This will be at least several decades away—what should happen in the mean time is open for discussion. 8. Conclusion Using DNA as a drug represents a novel alternative option for immunological disease control. With DNA technology, it is possible to replace antiquated production and storage methods of conventional therapeutic methods that have been unsuccessful in controlling certain diseases (Kaslow, 2004). By using DNA, it is possible to address several diseases or disease strains in one vaccine, which will meet growing need for multivalent combination vaccines leading to fewer injections (Brower, 1998). Thus, DNA will be an important component of the new generation of prophylactics, therapeutic drugs and vaccines. However, a much greater and wider body of clinical data and further improvements in bioprocess engineering and delivery are required before DNA may take a leading role in a new generation of blockbuster therapeutics. Acknowledgements Dr G.M. Forde and the BEL Team acknowledge the support of the Victorian Endowment for Science, Knowledge and Innovation and Monash University for their support of DNA related bioprocess engineering research. 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