Electrotransfer for drug delivery – from bench to bedside

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  • Professor Richard Heller, Professor and Director of the Frank Reidy Research Center for Bioelectrics at Old Dominion University has over 25 years’ research experience into the effects of electric pulses on biological systems. His pioneering research uses pulses of high-voltage electric fields to enable drug and gene delivery. His work centres on optimising these gene therapy tools with a view to developing exciting therapeutics for a wide range of diseases.

    Gene transfer or gene therapy is a form of treatment that involves inserting one or more genes (the basic biological unit of heredity) into a patient’s cells. Once inside the host cell, the new gene (or DNA) makes its way to the cell’s nucleus where the cell machinery then alters the levels of proteins coded by that gene. The altered levels of proteins can then correct or control disease. An exciting and active area of research, the potential for gene therapy is immense, holding huge promise for immunisation as well as a therapeutic tool for a range of diseases, including haemophilia, muscular dystrophy, immune deficiencies and types of cancer.

    Unlocking the power of gene therapy
    Gene therapy depends on safe and efficient gene delivery. Getting genes into the host cells is one of the most difficult aspects of gene therapy and poses a huge technical challenge to be overcome before it can be a viable approach to treating disease. Much research is devoted to finding ways to effectively introduce target genes into human cells. Even more tricky is getting the appropriate expression of the protein that is coded by the gene.

    Figure 1. Thermally Assisted Gene Electrotransfer Device. An optical fibre that was connected to an infrared laser and inserted into the device. The fibre is situated centrally between 4 electrodes. The fibre is also placed 1cm above a 3mm opening. This gap and opening allows for an increased spot size of the light as it exits into the space between the opening and the tissue which is set at 5mm. Modified and reprinted with permission from Current Gene Therapy, Vol. 16 issue 2 pages 83-89, 2014 (Figure 1).

    Overcoming these challenges, Professor Richard Heller, Frank Reidy Research Center for Bioelectrics at Old Dominion University, has pioneered the use of electrotransfer for drug and gene delivery. Originally used as a laboratory method to deliver DNA to experimental cells, Professor Heller and his research team have developed the electrotransfer tool for in vivo use, meaning that the genes are transferred directly into the cells inside the patient’s body.

    Professor Heller’s ground-breaking tool uses pulsed, high-voltage electric fields to target host cells allowing the uptake of molecules.

    Professor Heller’s ground-breaking tool uses pulsed, high-voltage electric fields to target host cells allowing the uptake of molecules. The electrical treatment causes a transient increase in the permeability of cell membranes, allowing drugs and genes to be taken up by the cell. Prof Heller’s early studies showed that the increase in permeability is temporary and has no effect on cell viability. Subsequent clinical studies have demonstrated it to be a safe and efficient therapeutic method. Gene therapy can be exploited to deliver drugs, DNA, proteins or even antibodies into cells.

    Professor Heller’s team realised that successful gene transfer depends on achieving a precise therapeutic protein dose. Although controlling the administered dose of the gene is achievable, controlling levels of the resultant expressed protein is far more challenging. Through his research, Professor Heller is developing new approaches to improve delivery and therefore impart more control on protein expression. His non-invasive electrode, the multielectrode array (MEA), efficiently delivers high levels of genes in the skin for up to 15 days after treatment. By exploring parameters including electric field strength, pulse duration, pulse number, electrode geometry and configuration, and also the delivery area, Heller’s team can control the onset, level, and duration of protein expression of the transgene. The researchers are also exploring the use of moderate heat controlled through an exogenous source to improve gene delivery in vivo. In this way, their sophisticated approach can be fine-tuned to control the levels and duration of expression of the new gene, allowing a tailored approach, specific for the patient.

    Figure 2. Blood Flow within the Heart. Coronary perfusion before and after treatment was determined using the SPY® Intraoperative Perfusion Assessment System (Novadaq Technologies, Inc, Bonita Springs, FL). Fluorescent microspheres are injected intravenously and fluorescent intensity measured with a camera. The SPY system enables the user to image, capture, and view dynamic fluorescence images of the myocardium. The level of perfusion has been shown to be directly related to changes in the intensity of fluorescence. The pre-occlusion image was taken prior to blocking the left anterior descending artery. The post-occlusion image was taken immediately after blocking the left anterior descending artery. The post-treatment image was taken two weeks after treatment with plasmid VEGF and gene electrotransfer.

    Prof Heller’s multidisciplinary collaborative work centres on the development of gene therapy tools for cancer and vascular diseases, as well as vaccine and immunotherapy protocols for cancer and infectious disease. His translational research has advanced to clinical trials for melanomas – aggressive skin cancers, representing the first successful in vivo electrogene delivery in humans. Since the first trial, the approach has been tested in other cancers as well.

    Pioneers of gene electrotransfer
    Prof Heller has demonstrated the power of his electrogene transfer tool for the treatment of ischaemia, both in peripheral tissues as well as in the heart. Caused by underlying problems with blood vessels (for example, arterial disease), ischaemia causes a restriction of blood supply to tissues with resultant damage to tissue and is a prevalent cause of morbidity and mortality in the western world. Prof Heller’s team successfully delivered genes coding for Fibroblast Growth Factor (FGF), a substance that promotes the growth of new blood vessels. Rats with ischaemic limbs were injected with the FGF gene followed by non-invasive electroporation. Following treatment, a significant increase in limb blood flow was seen in those rats who had received electroporation, compared to those who had received an injection of the FGF gene alone. The density of blood vessels was also significantly higher, suggesting that their treatment could be a potential non-invasive therapeutic approach to increase blood flow for ischaemia. In another study of ischaemia in the heart of pigs, Prof Heller’s gene electrotransfer tool successfully delivered Vascular Endothelial Growth Factor (VEGF) to the affected hearts and led to an increase in blood flow compared to control animals.

    Gene delivery of vaccines
    Prof Heller’s team evaluated the MEA tool to effectively deliver a DNA vaccine against B. anthracis, the bacteria responsible for causing anthrax. By adjusting the voltage, amount of DNA and number of treatments, the team optimised the tool to successfully administer the vaccine in mice in vivo. By changing the voltage in particular, the team found that mice had raised levels of protective antibody from the vaccination and generated a protective immune response, suggesting that MEA could be established as a non-invasive way to vaccinate against anthrax.

    A novel approach for malignant melanoma
    Much gene transfer research aims to push the expression of genes to the highest level. Prof Heller’s team, however, takes a different approach. By determining the optimum dose of the expressed gene, the researchers work out the delivery method to achieve this. This is not an easy task – although controlling the administered dose of the gene is relatively straightforward, controlling the levels of expressed protein is much harder. Prof Heller believes that the best clinical outcomes can be achieved by first optimising the expression profile. In this way, he has developed a successful protocol for the treatment of malignant melanoma.

    The deadliest form of skin cancer, occurring with increasing frequency, malignant melanoma will be diagnosed in an estimated 75,000 new patients in 2014. A major health concern, melanoma does not respond well to standard chemotherapy, and there is currently no effective therapy for advanced disease, which occurs in 20% of cases. Gene therapy offers therapeutic hope for this aggressive disease and unlike current therapies, has the potential to eliminate cancer cells without damaging normal, healthy tissue.

    Excitingly, Professor Heller’s landmark clinical research into electro gene immunotherapy represents the first time that electroporation has been used to deliver therapeutic DNA in humans. The team first tested the electroporation method in mice with melanoma. Mice were treated with the gene for interleukin-12 (IL-12), which stimulates the immune system to fight the cancer. A powerful immune system stimulant, previous studies injecting IL-12 protein caused toxic side effects and limited success. In contrast, Prof Heller’s strategy using his hand-held electroporation tool to safely deliver the IL-12 gene to melanoma tumour cells resulted in an 80% complete immune response in mouse models of melanoma. Importantly, the mice did not develop new tumours, suggesting that the therapy induced a systemic immune response against new cancers.

    Figure 3. GET of VEGF-A reduces the infarct size at treatment sites. Representative TTC staining at seven weeks shows viable muscle tissue in red and remodelled infarct tissue in white for GET (A), injection only (B), and sham control (C) treated hearts. Green stars denote treatment sites in the ischemic myocardium. Reprinted with permission from Gene Therapy, Vol. 23, Issue 8, pages 1-8, 2016 (Supplementary figure 3).

    Following successful preclinical studies, a phase I clinical trial was conducted in 24 patients with advanced metastatic melanoma. Patients were treated at seven dose levels. Treatments were found to be safe and effective at stimulating an immune response to the tumour. Following treatment, patients showed a dose-proportional increase in IL-12 protein levels, concomitant with damage to the tumour and the presence of protective immune cells. 10% of patients showed complete regression of advancing tumours and 42% of patients showed stabilisation of their disease. Since the conclusion of this Phase I study, a successful Phase II study was completed which confirmed the results observed in the original trial. Additional trials have been conducted for treatment of other cancers as well as combining the pIL-12 delivery with anti-PD-1 and anti-PDL1.

    Heller’s landmark clinical research into electro gene immunotherapy represents the first time that electroporation has been used to deliver therapeutic DNA in humans.

    Future research
    Prof Heller’s team are now building on their exciting results in malignant melanoma, paving the way for further clinical trials. The researchers are testing the idea that if IL-12 DNA is delivered under appropriate conditions (i.e. at the right dose and location), a change in the tumour microenvironment will occur that can be associated with an appropriate therapeutic response. Their goal is to characterise the response and identify potential markers that can indicate appropriate delivery and expression – can a specific pattern of response). Their hope is that with correct delivery and expression of DNA, not only can they achieve a favourable outcome on the primary tumour but also provoke an increased response at distant sites (i.e., on the spreading secondary tumours), through the activation of specialised immune cells.

    The development of ground-breaking protocols for gene therapy is exciting, holding promise to transform medicine and create options for patients who are living with difficult and currently untreatable diseases.

    Your research has significantly advanced the development of in vivo gene electrotransfer as a therapeutic tool. What originally sparked your interest in this area of research?

    In the 1990s, we had success delivering chemotherapeutic agents directly to tumours with electroporation. This was a localised response and the oncologists testing the therapy asked if we could enhance it to include effective treatment of distant metastases. The critical aspect was to develop a potentially effective, non-toxic and inexpensive option. We found that gene delivery with electrotransfer allowed sufficient control of the expressed protein levels, which produced the desired therapeutic result. This led to testing this concept for other potential therapies including coronary and peripheral ischemia, wound healing and DNA vaccine delivery.

    References

    • Bulysheva AA, Hargrave B, Burcus N, Lundberg CG, Murray L, Heller R. (2016). ‘Vascular endothelial growth factor-A gene electrotransfer promotes angiogenesis in a porcine model of cardiac ischemia’. Gene Ther, 23(8-9):649-656.
    • Donate A, Bulysheva A, Edelblute C, Jung D, Malik MA, Guo S, Burcus N, Schoenbach K, Heller R. (2016). ‘Thermal assisted in vivo gene electrotransfer’. Curr Gene Ther, 16(2):83-9.
    • Shirley SA, Lundberg CG, Li F, Burcus N, Heller R. (2015). ‘Controlled gene delivery can enhance therapeutic outcome for cancer immune therapy for melanoma’. Curr Gene Ther, 15(1):32-43.
    • Guo S, Donate A, Basu G, Lundberg C, Heller L, Heller R. (2011). ‘Electro-gene transfer to skin using a noninvasive multielectrode array’. J Control Release, 151(3):256-62.
    • Donate A, Heller R (2013). ‘Assessment of delivery parameters with the multi-electrode array for development of a DNA vaccine against Bacillus anthracis’. Bioelectrochemistry, 94:1-6.
    • Daud AI, DeConti RC, Andrews S, Urbas P, Riker AI, Sondak VK, Munster PN, Sullivan DM, Ugen KE, Messina JL, Heller R. (2008). J Clin Oncol, ‘Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma’. 26(36):5896-903.
    • Ferraro B, Cruz YL, Baldwin M, Coppola D, Heller R. (2010). ‘Increased perfusion and angiogenesis in a hindlimb ischemia model with plasmid FGF-2 delivered by noninvasive electroporation’. Gene Ther, 17(6):763-9.

  • Research Objectives
    A major focus of Professor Richard Heller’s current research is to develop in vivo delivery procedures for several non-viral gene therapy applications.

    Funding

    • NIH – NCI, NIBIB, NHLBI
    • DOD
    • Oncosec Medical, Inc

    Collaborators
    University of California San Francisco

    • Adil Daud, MD
    • Lawrence Fong, MD

    University of South Florida

    • Richard Gilbert, PhD
    • Mark J. Jaroszeski, PhD
    • Andrew Hoff

    Old Dominion University

    • Loree Heller, PhD
    • Siqi Guo, MD
    • Stephen Beebe, PhD
    • Barbara Hargrave, PhD
    • Shu Xiao, PhD
    • Anna Bulysheva, PhD

    Institute of Oncology, Ljubljana, Slovenia

    • Gregor Sersa, PhD

    Zealand University Hospital, University of Copenhagen, Denmark

    • Julie Gehl, MD

    Bio
    Richard Heller received his BS degree in Microbiology from Oregon State University, and a PhD from the University of South Florida, College of Medicine. Dr Heller has over 25 years of experience in evaluating the effects of electric pulses on biological systems. He is recognised as a pioneer on the use of electrotransfer for drug and gene delivery.

    Contact
    Professor Richard Heller
    Old Dominion University
    300 4211 Monarch Way
    Norfolk, VA 23529
    USA

    E: rheller@odu.edu
    T: +1 757 683 2690
    W:
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  • Electrotransfer for drug delivery – from bench to bedside

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