Publications by authors named "Richard J Connolly"

10 Publications

  • Page 1 of 1

Characterization of abscopal effects of intratumoral electroporation-mediated IL-12 gene therapy.

Gene Ther 2019 02 15;26(1-2):1-15. Epub 2018 Oct 15.

Fred Hutchinson Cancer Center, 1100 Fairview Avenue N, Seattle, WA, 98109, USA.

Intratumoral electroporation-mediated IL-12 gene therapy (IT-pIL12/EP) has been shown to be safe and effective in clinical trials, demonstrating systemic antitumor effects with local delivery of this potent cytokine. We recently optimized our IL-12 gene delivery platform to increase transgene expression and efficacy in preclinical models. Here we analyze the immunological changes induced with the new IT-pIL12/EP platform in both electroporated and distant, non-electroporated lesions. IT-pIL12/EP-treated tumors demonstrated rapid induction of IL-12-regulated pathways, as well as other cytokines and chemokines pathways, and upregulation of antigen presentation machinery. The distant tumors showed an increase in infiltrating lymphocytes and gene expression changes indicative of a de novo immune response in these untreated lesions. Flow cytometric analyses revealed a KLRG1 CD8 effector T-cell population uniquely present in mice treated with IT-pIL12/EP. Despite being highly activated, this population expressed diminished levels of PD-1 when re-exposed to antigen in the PD-L1-rich tumor. Other T-cell exhaustion markers appeared to be downregulated in concert, suggesting an orchestrated "armoring" of these effector T cells against T-cell checkpoints when primed in the presence of IL-12 in situ. These cells may represent an important mechanism by which local IL-12 gene therapy can induce a systemic antitumor immune response without the associated toxicity of systemic IL-12 exposure.
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http://dx.doi.org/10.1038/s41434-018-0044-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514882PMC
February 2019

Development of an adaptive electroporation system for intratumoral plasmid DNA delivery.

Bioelectrochemistry 2018 Aug 9;122:191-198. Epub 2018 Apr 9.

OncoSec Medical Incorporated, 5820 Nancy Ridge Drive, San Diego, CA, USA. Electronic address:

Intratumoral electroporation of plasmid DNA encoding the proinflammatory cytokine interleukin 12 promotes innate and adaptive immune responses correlating with anti-tumor effects. Clinical electroporation conditions are fixed parameters optimized in preclinical tumors, which consist of cells implanted into skin. These conditions have little translatability to clinically relevant tumors, as implanted models cannot capture the heterogeneity encountered in genetically engineered mouse models or clinical tumors. Variables affecting treatment outcome include tumor size, degree of vascularization, fibrosis, and necrosis, which can result in suboptimal gene transfer and variable therapeutic outcomes. To address this, a feedback controlled electroporation generator was developed, which is capable of assessing the electrochemical properties of tissue in real time. Determination of these properties is accomplished by impedance spectroscopy and equivalent circuit model parameter estimation. Model parameters that estimate electrical properties of cell membranes are used to adjust electroporation parameters for each applied pulse. Studies performed in syngeneic colon carcinoma tumors (MC38) and spontaneous mammary tumors (MMTV-PyVT) demonstrated feedback-based electroporation is capable of achieving maximum expression of reporter genes with significantly less variability and applied energy. These findings represent an advancement to the practice of gene electro-transfer, as reducing variability and retaining transfected cell viability is paramount to treatment success.
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http://dx.doi.org/10.1016/j.bioelechem.2018.04.005DOI Listing
August 2018

Direct Current Helium Plasma for Delivery of Plasmid DNA Encoding Erythropoietin to Murine Skin.

Plasma Med 2017 ;7(3):261-271

Dept of Chemical and Biomedical Engineering, University of South Florida, College of Engineering, Tampa, FL.

The use of electric fields to deliver DNA, called electroporation, has the potential to broadly impact vaccination and disease treatment. The evidence for this has emerged from a large number of recently completed and ongoing clinical trials. The methods for applying electric fields to tissues traditionally involve contact between metal electrodes and the tissue. In this study, we investigated the use of helium plasma as a noncontact method for electrically treating tissue in a manner that results in the uptake and expression of foreign DNA in murine skin. More specifically, our goal was to demonstrate that DNA encoding a model-secreted protein could be delivered, detected in the blood, and remain functional to produce its known biological effect. Murine erythropoietin (EPO) was the model-secreted protein. Results clearly demonstrated that an intradermal DNA injection followed by plasma treatment for 2 min resulted in elevated levels of EPO in the blood and corresponding hemoglobin increases that were statistically significant relative to DNA injection alone.
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http://dx.doi.org/10.1615/PlasmaMed.2017019506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404545PMC
January 2017

Optimization of a plasma facilitated DNA delivery method.

Bioelectrochemistry 2015 Jun 13;103:15-21. Epub 2014 Oct 13.

Department of Chemical and Biomedical Engineering, College of Engineering, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620, United States. Electronic address:

Plasma-based methods have recently emerged as a technique for augmenting plasmid DNA delivery to skin. This delivery modality relies on the deposition of ionized gas molecules on to targeted cells or tissue to establish an electric field. It is hypothesized that this electric field results in the dielectric breakdown of cell membranes, making cells permeable to exogenous molecules. This in vivo investigation sought to optimize the intradermal delivery of a luciferase expressing plasmid DNA by modulating the total exposure to the plasma source and the plasmid DNA dose. Varying the plasma exposure time from 2, 5, 10, and 20 min allowed the conditions resulting in the highest expression of luciferase to be found. These conditions correlated to the 10 minute exposure time for a plasma derived from either +8 kV or -8 kV, when the generator was operated 3 cm from the epidermal tissue surface with a helium flow rate of 15 L/min. Exposing the injected flank skin for 10 min resulted in a rise of 37.3-fold for a plasma created with +8 kV and 27.1-fold for a plasma created with -8 kV. When using this treatment time with 50, 100, or 200 μg of a luciferase expressing plasmid, it was found that 100 μg resulted in the highest peak luminescence.
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http://dx.doi.org/10.1016/j.bioelechem.2014.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346600PMC
June 2015

Electrogenetherapy of B16.F10 murine melanoma tumors with an interleukin-28 expressing DNA plasmid.

Hum Vaccin Immunother 2012 Nov 1;8(11):1722-8. Epub 2012 Nov 1.

Department of Molecular Medicine; Morsani College of Medicine; Tampa, FL USA.

Augmented delivery of cytokine-expressing DNA plasmids to subcutaneous tumors has been demonstrated to result in a level of enhanced anti-tumor activity. One delivery enhancement method which has been evaluated is in vivo electroporation (EP), a contact-dependent delivery technique where electric pulses are hypothesized to augment the transfer of DNA into cells and tissues through the induction of temporary cell membrane pores. Previous work by members of our group, as well as others, has demonstrated the anti-tumor effects of DNA plasmids expressing the cytokines IL-12 and IL-15. In this report the potential anti-tumor activity of a relatively newly-described cytokine, IL-28, was measured when administered intratumorally as a DNA expression plasmid (designated pIL28) to established murine (B16.F10) melanoma tumors. The administration of the IL-28 expressing plasmid was performed through enhanced delivery methods. One method was EP and the other a non-contact dependent technique using a helium plasma stream. IL-28 is a member of the type III interferon family of cytokines that has been characterized as possessing potent anti-viral activity. This cytokine has been demonstrated to function as an adjuvant in small animal model vaccination protocols and stimulates CD8+ CTL responses. In addition, stimulation of anti-tumor activity has been demonstrated in several studies using IL-28. Based on these activities, it was hypothesized that this cytokine could, when delivered through a DNA expression plasmid, mediate anti-tumor activity. The results of this study indicated that enhanced delivery of pIL-28 resulted in attenuation of tumor growth, compared with non-enhanced delivery. Of note, this is the first proof-of-concept experiment, of our knowledge, documenting the ability of a non-contact dependent helium plasma-based delivery method to mediate the enhancement of an anti-tumor effect by a cytokine-expressing DNA plasmid. This suggests the use of the helium plasma delivery method as an alternative or adjunctive method to EP for the effective delivery of agents that possess potential anti-tumor activity.
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http://dx.doi.org/10.4161/hv.22560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601148PMC
November 2012

Non-contact helium-based plasma for delivery of DNA vaccines. Enhancement of humoral and cellular immune responses.

Hum Vaccin Immunother 2012 Nov 16;8(11):1729-33. Epub 2012 Aug 16.

Center for Molecular Delivery, University of South Florida; Tampa, FL, USA.

Non-viral in vivo administration of plasmid DNA for vaccines and immunotherapeutics has been hampered by inefficient delivery. Methods to enhance delivery such as in vivo electroporation (EP) have demonstrated effectiveness in circumventing this difficulty. However, the contact-dependent nature of EP has resulting side effects in animals and humans. Noncontact delivery methods should, in principle, overcome some of these obstacles. This report describes a helium plasma-based delivery system that enhanced humoral and cellular antigen-specific immune responses in mice against an intradermally administered HIV gp120-expressing plasmid vaccine (pJRFLgp120). The most efficient plasma delivery parameters investigated resulted in the generation of geometric mean antibody-binding titers that were 19-fold higher than plasmid delivery alone. Plasma mediated delivery of pJRFLgp120 also resulted in a 17-fold increase in the number of interferon-gamma spot-forming cells, a measure of CD8+ cytotoxic T cells, compared with non-facilitated plasmid delivery. This is the first report demonstrating the ability of this contact-independent delivery method to enhance antigen-specific immune responses against a protein generated by a DNA vaccine.
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http://dx.doi.org/10.4161/hv.21624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601149PMC
November 2012

Enhancement of antigen specific humoral immune responses after delivery of a DNA plasmid based vaccine through a contact-independent helium plasma.

Vaccine 2011 Sep 31;29(39):6781-4. Epub 2010 Dec 31.

Department of Chemical and Biomedical Engineering, College of Engineering, University of South Florida, 4202 E. Fowler Avenue, ENB 118, Tampa, FL 33620, USA.

Non-viral in vivo delivery of DNA, encoding for specific proteins, has traditionally relied on chemical or physical forces applied directly to tissues. Physical methods typically involve contact between an applicator/electrode and tissue and often results in transient subject discomfort. To overcome these limitations of contact-dependent delivery, a helium plasma source was utilized to deposit ionized gasses to treatment/vaccination sites without direct contact between the applicator and the tissues. The study reported here evaluated the efficacy of this strategy as an effective method to administer DNA vaccines. Balb/C mice were vaccinated with a DNA plasmid expressing an HIVgp120 envelope glycoprotein either with or without co-administration of helium plasma or electroporation. The results indicated, for the first time, the potential efficacy of helium plasma delivery for the induction and enhancement of antigen specific immune responses following DNA vaccination.
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http://dx.doi.org/10.1016/j.vaccine.2010.12.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3156891PMC
September 2011

Characterization of plasma mediated molecular delivery to cells in vitro.

Int J Pharm 2010 Apr 18;389(1-2):53-7. Epub 2010 Jan 18.

Department of Chemical and Biomedical Engineering, University of South Florida, ENB 118, 4202 E. Fowler Avenue, Tampa, FL 33620, USA.

Ion-based strategies have recently emerged as a method to facilitate molecular delivery. These methods are attractive as they separate the applicator from the treatment site avoiding some issues encountered with other electrically driven methods. Current literature on plasma delivery has shown utility in vitro and in vivo for both drugs and genes. To advance this technology more information must become available on the mechanism responsible for delivery and the effects of ion exposure on eukaryotic cells. This in vitro investigation found that molecular delivery facilitated by a DC-based plasma follows a dose-response behavior, with optimum uptake of Sytox Green occurring in two cell lines after 600 s of exposure. In both cell lines exposure to the discharge caused no adverse effects in viability for exposure times up to 600 s. It was also found that membranes treated with ions remained permeabilized for several minutes following plasma treatment and that membrane resealing exhibited first order kinetics.
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http://dx.doi.org/10.1016/j.ijpharm.2010.01.016DOI Listing
April 2010

Plasma facilitated delivery of DNA to skin.

Biotechnol Bioeng 2009 Dec;104(5):1034-40

Department of Chemical and Biomedical Engineering, University of South Florida, ENB 118, Tampa, Florida 33620, USA.

Non-viral delivery of cell-impermeant drugs and DNA in vivo has traditionally relied upon either chemical or physical stress applied directly to target tissues. Physical methods typically use contact between an applicator, or electrode, and the target tissue and may involve patient discomfort. To overcome contact-dependent limitations of such delivery methodologies, an atmospheric helium plasma source was developed to deposit plasma products onto localized treatment sites. Experiments performed in murine skin showed that samples injected with plasmid DNA encoding luciferase and treated with plasma demonstrated increased levels of expression relative to skin samples that received injections of DNA alone. Increased response relative to injection alone was observed when either positive or negative voltage was used to generate the helium plasma. Quantitative results over a 26-day follow-up period showed that luciferase levels as high as 19-fold greater than the levels obtained by DNA injection alone could be achieved. These findings indicate that plasmas may compete with other physical delivery methodologies when skin is the target tissue.
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http://dx.doi.org/10.1002/bit.22451DOI Listing
December 2009
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