Cardiac Myosin Activation with Gene Therapy Produces Sustained Inotropic Effects and May Treat Heart Failure with Reduced Ejection Fraction.

Chronic inotropic therapy is effective for the treatment of heart failure with reduced ejection fraction, but has been limited by adverse long-term safety profiles, development of tolerance, and the need for chronic parenteral administration. A safe and convenient therapeutic agent that produces sustained inotropic effects could improve symptoms, functional capacity, and quality of life. Small amounts of 2-deoxy-adenosine triphosphate (dATP) activate cardiac myosin leading to enhanced contractility in normal and failing heart muscle. Cardiac myosin activation triggers faster myosin crossbridge cycling with greater force generation during each contraction. This paper describes the rationale and results of a translational medicine effort to increase dATP levels using a gene therapy strategy to deliver and upregulate ribonucleotide reductase (R1R2), the enzyme responsible for dATP synthesis, selectively in cardiomyocytes. In small and large animal models of heart failure, a single dose of this gene therapy has led to sustained inotropic effects with a benign safety profile. Further animal studies are appropriate with the goal of testing this agent in patients with heart failure.

Translation of Cardiac Myosin Activation with 2-deoxy-ATP to Treat Heart Failure via an Experimental Ribonucleotide Reductase-Based Gene Therapy.

Despite recent advances, chronic heart failure remains a significant and growing unmet medical need, reaching epidemic proportions carrying substantial morbidity, mortality, and costs. A safe and convenient therapeutic agent that produces sustained inotropic effects could ameliorate symptoms, and improve functional capacity and quality of life. We discovered small amounts of 2-deoxy-ATP (dATP) activate cardiac myosin leading to enhanced contractility in normal and failing heart muscle. Cardiac myosin activation triggers faster myosin crossbridge cycling with greater force generation during each contraction. We describe the rationale and results of a translational medicine effort to increase dATP levels using a gene therapy strategy that upregulates ribonucleotide reductase, the rate-limiting enzyme for dATP synthesis, selectively in cardiomyocytes. In small and large animal models of heart failure, a single dose of this gene therapy has led to sustained inotropic effects with no toxicity or safety concerns identified to-date. Further animal studies are being conducted with the goal of testing this agent in patients with heart failure.

Proangiogenic microtemplated fibrin scaffolds containing aprotinin promote improved wound healing responses.

Survival of tissue engineered constructs after implantation depends heavily on induction of a vascular response in host tissue, promoting a quick anastomosis of the cellular graft. Additionally, implanted constructs typically induce fibrous capsule formation, effectively preventing graft integration with host tissue. Previously we described the development of a high density microtemplated fibrin scaffold for cardiac tissue engineering applications with tunable degradation and mechanical properties which promoted seeded cell survival and organization in vitro (Thomson et al., Tissue Eng Part A, 2013). Scaffold degradation in vitro was controllable by addition of the serine protease inhibitor aprotinin and/or the fibrin cross-linker Factor XIII (FXIII). The goal of this study was to assess host tissue responses to these fibrin scaffold formulations by determining effects on scaffold degradation, angiogenic responses, and fibrous capsule formation in a subcutaneous implant model. Aprotinin significantly decreased scaffold degradation over 2 weeks of implantation. A significant increase in capillary infiltration of aprotinin implants was found after 1 and 2 weeks, with a significantly greater amount of capillaries reaching the interior of aprotinin scaffolds. Interestingly, after 2 weeks the aprotinin scaffolds had a significantly thinner, yet apparently more cellular fibrous capsule than unmodified scaffolds. These results indicate aprotinin not only inhibits fibrin scaffold degradation, but also induces significant responses in the host tissue. These included an angiogenic response resulting in increased vascularization of the scaffold material over a relatively short period of time. In addition, aprotinin release from scaffolds may reduce fibrous capsule formation, which could help promote improved integration of cell-seeded scaffolds with host tissue.

Prevascularized microtemplated fibrin scaffolds for cardiac tissue engineering applications.

Myocardial infarction (MI) causes significant cell loss and damage to myocardium. Cell-based therapies for treatment of MI aim to remuscularize the resultant scar tissue, but the majority of transplanted cells do not survive or integrate with the host tissue. Scaffolds can improve cell retention following construct implantation, but often do little to enhance host-graft integration and/or show limited biodegradation. Fibrin is an ideal biomaterial for cardiac tissue engineering as it is a natural, biodegradable polymer that can induce neovascularization, promote cell attachment, and has tunable mechanical properties. Here we describe a novel, high-density microtemplated fibrin scaffold seeded with a tri-cell mixture of cardiomyocytes, endothelial cells (ECs), and fibroblasts to mimic native cardiac tissue in structure and cellular composition to improve cell retention and promote integration with the host tissue. Scaffolds were designed with uniform architecture of parallel 60 µm microchannels surrounded by an interconnected microporous network of 27-µm-diameter pores and mechanical stiffness comparable to native cardiac tissues (70-90kPa). Scaffold degradation was controlled with the addition of Factor XIII (FXIII) and/or protease inhibitor (aprotinin). Unmodified scaffolds had a fast degradation profile both in vitro (19.9%±3.9% stiffness retention after 10 days) and in vivo. Scaffolds treated with FXIII showed an intermediate degradation profile in vitro (45.8%±5.9%), while scaffolds treated with aprotinin or both FXIII and aprotinin showed significantly slowed degradation in vitro (60.9%±5.2% and 76.4%±7.6%, respectively, p<0.05). Acellular aprotinin scaffold myocardial implants showed decreased collagen deposition after 7 days. Unmodified and aprotinin implants could not be located by 14 days, while 2 of 8 FXIII implants were found, but were significantly degraded. Constructs supported seeded cell survival and organization in vitro, promoting EC-lined lumen structure formation in construct channels and colocalization of viable ECs and cardiomyocytes. In addition, constructs promoted extracellular matrix deposition by seeded cells, as shown by collagen staining within construct channels and by significant increases in construct stiffness over 10 days in vitro (209%±32%, p<0.05). The data suggest our fibrin scaffolds are ideally designed to promote graft cell survival and organization, thus improving chances of promoting construct integration with the host tissue upon implantation.

Prophylactic anti-tumor effects in a B cell lymphoma model with DNA vaccines delivered on polyethylenimine (PEI) functionalized PLGA microparticles.

Idiotypic sequences, specific to the hypervariable regions of immunoglobulins expressed by malignant B cells offer a therapeutic target in B cell lymphoma. Efficient approaches have been described to clone a single chain fragment of the tumor immunoglobulin (Ig) comprising of heavy and light Ig chains (sFv) fused with proinflammatory chemokines. Tumor associated, poorly immunogenic self antigens encoded by plasmid DNA (pDNA) have been rendered immunogenic by chemokine fusion, thereby targeting to antigen presenting cells (APCs) which differentially express chemokine receptors. Here we present an injectable (parenteral) approach using synthetic polymer based cationic microparticle formulations for enhancing the potency of such chemokine/self antigen expressing plasmid construct. Branched and linear polyethyleneimine (PEI) were conjugated on poly (D, L lactide-co-glycolide) (PLGA) microparticles using carbodiimide chemistry followed by efficient loading of plasmid DNA. In addition to imparting significant buffering ability to these cationic microparticles, flow cytometry studies indicate that these DNA loaded microparticles significantly up regulate CD80 and MHC class II markers in phagocytic RAW264.7 cells, indicating intrinsic adjuvant effects. Intradermal injections in Balb/c mice with these formulations induced significant protection upon tumor challenge with 2.5 times the minimal lethal dose. Long term survival rates were significant (p < 0.05) in comparison with saline injected controls or blank microparticles. Further studies indicated that intramuscular delivery might provide better protection compared to intradermal injections and perform similar to gene gun mediated administration. We conclude, based on these promising in vivo results, that such surface-functionalized microparticles offer an attractive strategy to improve the potency of self antigen-based cancer DNA vaccines.