Heart disease statistics compiled by the American Heart Association in 2010 estimate 785,000 Americans, each year, will have a new coronary attack, another 470,000 will have a recurrent heart attack, and 195,000 will have a silent infarction, resulting in a coronary event every 25 seconds in the US. Myocardial cells affected by the infarction must endure hypoxic stress during the ischemic event and oxidative stress during reperfusion. Two decades of research suggests the induction of a heat-shock protein 70 (Hsp72) in myocardial cells counteracts these stresses and improves post-ischemic contractile recovery. To date, clinically relevant methods of inducing Hsp70 in these cells have yet to be devised. We developed the Fv fragment of a cell- penetrating antibody, mAb 3E10, as an intracellular transporter to deliver the Hsp70 into cells, and we have demonstrated cytoprotection against oxidative damage in vitro and in vivo. The objective here is to establish proof-of-principle in rabbits subjected to occlusion of the left coronary artery (LCA) followed by reperfusion of the heart and intravenous injection of Fv-Hsp70 or a sham control. Fv-Hsp70 treated rabbits should present a reduced infarct volume compared to the control. Our central hypothesis is that Fv-Hsp70 will be therapeutically effective in protecting against post-ischemic damage as a result of an infarction. The rationale for the proposed research is that the cell penetrating antibody, 3E10, is an intracellular transporter that can deliver Hsp70 directly into cells where it minimizes protein denaturation and aggregation caused by stress. The antibody binds extracellular DNA and nucleosides, targets that are quite accessible where there are damaged cells, and it penetrates still viable cells through an equilibrative nucleoside salvage pathway. 3E10 is unique in that it penetrates cells without apparent harm and has been administered to humans without evidence of toxicity. Fv- Hsp70 has already been created and shown to be an effective cytoprotectant in vivo, minimizing by 68% the infarct volume in brain tissue when administered to rats after a stroke. Our long-term goal is to determine the effectiveness of Fv-Hsp70 in improving myocardial recovery when administered by clinicians to a heart attack victim. We are requesting funding to achieve the following specific aims: 1) Manufacture and characterize multiple lots of Fv-Hsp70 and scFv 3E10 sham control for the infarction studies. 2) Evaluate the therapeutic efficacy of Fv-Hsp70 in vivo using SPECT imaging of the infarct volume with 99mTc-Annexin V. 3) Evaluate the therapeutic efficacy of Fv-Hsp70 ex vivo using tissue histology, as well as, creatine kinase and troponin I blood levels. The proposed research is significant because it develops a critical therapeutic for an unmet need. The proposed research is innovative because it utilizes a unique antibody-mediated, energy-independent intracellular delivery system for protein therapeutics. Inducing Hsp70 production in vivo can take time, whereas the impact of our product is the rapid delivery of Hsp70 into damaged cells to prevent cell death.
of the proposed project to public health Heart disease statistics compiled by the American Heart Association in 2010 estimate 785,000 Americans, each year, will have a new coronary attack, and another 470,000 will have a recurrent heart attack, resulting in a coronary event every 25 seconds in the US. The research proposal is relevant to public health because the development of myocardial cytoprotectants that minimize damage to heart tissue after a coronary event is an unmet medical need. This proposal is relevant to the overall mission of the SBIR program because a successful demonstration in Phase I of the Fv-Hsp70's myocardial cytoprotection will stimulate further funding in the private and public sectors, including a Phase II SBIR, for the product's ultimate commercialization as a front-line cardiac therapy.
