Intracellular targeting Hsp70 for myocardial cytoprotection after an infarction

细胞内靶向 Hsp70 用于梗塞后心肌细胞保护

• 批准号: 8780833
• 负责人: Missag Hagop Parseghian
• 金额: $ 22.33万
• 依托单位: RUBICON BIOTECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2015-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8780833
• 关键词: Address; Adverse effects; Affect; American; American Heart Association; Animal Model; Antibodies; Area; Autopsy; Binding; Biological Assay; Blood; Blood specimen; Cardiac; Cell Culture Techniques; Cell Death; Cell Survival; Cells; Cerebral Infarction; Cessation of life; Chimeric Proteins; Clinical Trials; Coronary; Coronary Occlusions; Creatine Kinase; Culture Media; Cyclic GMP; Cytoprotection; Cytoprotective Agent; DNA; Data; Development; Drug Formulations; Drug Kinetics; Dyes; Echocardiography; Effectiveness; Electrocardiogram; Enzyme-Linked Immunosorbent Assay; Evaluation; Evans blue stain; Event; FDA approved; Freezing; Funding; Goals; Heart; Heart Diseases; Heat-Shock Proteins 70; High Pressure Liquid Chromatography; Histology; Horseradish Peroxidase; Human; Hypoxia; In Vitro; Infarction; Injury; Intracellular Transport; Ion-Exchange Chromatography Procedure; Ischemia; Label; Mass Spectrum Analysis; Measures; Mediating; Medical; Methods; Mission; Modeling; Molecular; Monitor; Muscle Cells; Myocardial; Myocardial Infarction; Nucleosides; Oryctolagus cuniculus; Oxidative Stress; Pathway interactions; Performance; Phase; Phase III Clinical Trials; Private Sector; Process; Production; Protein Denaturation; Public Health; Public Sector; Radioimmunoconjugate; Rattus; Reagent; Recovery; Recurrence; Relaxation; Reperfusion Therapy; Research; Research Proposals; Risk; Sampling; Sarcolemma; Small Business Innovation Research Grant; Source; Staging; Staining method; Stains; Stress; Stroke; System; Testing; Therapeutic; Time; Tissues; Toxic effect; Treatment Efficacy; Troponin I; Ventricular Function; Work; annexin A5; attack victim; brain tissue; cell injury; clinically relevant; commercialization; extracellular; heart preservation; improved; in vivo; injured; innovation; intravenous injection; left coronary artery; nonhuman primate; oxidative damage; prevent; programs; protein aggregation; public health relevance; sample collection; scale up; single photon emission computed tomography; statistics; therapeutic protein

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：美国心脏协会在2010年在2010年汇编的心脏病统计数据估计，每年有785,000名美国人将发生新的冠状动脉攻击，另外470,000名将再次发作心脏病发作，195,000将有一个无声的梗塞，导致美国每25秒发生冠状动脉。受梗塞影响的心肌细胞必须在缺血事件期间忍受低氧应激，在再灌注过程中氧化应激。二十年的研究表明，在心肌细胞中诱导热蛋白70（HSP72）可以抵消这些应力并改善缺血后的收缩回收率。迄今为止，在这些细胞中诱导HSP70的临床相关方法尚未设计。我们开发了细胞穿透抗体MAB 3E10的FV片段，作为细胞内转运蛋白，以将Hsp70传递到细胞中，我们已经证明了在体外和体内抗氧化损伤的细胞保护作用。此处的目的是在受到左冠状动脉（LCA）的阻塞的兔子中建立原则证明，然后再灌注心脏，并静脉注射FV-HSP70或假对照。与对照相比，经过FV-HSP70治疗的兔子应呈现梗塞体积减少。我们的中心假设是，FV-HSP70将在治疗上有效防止由于梗塞而防止缺血后损害。拟议的研究的基本原理是细胞穿透抗体3E10是一种细胞内转运蛋白，可以将HSP70直接输送到细胞中，从而最大程度地减少蛋白质变性和由应激引起的聚集。抗体结合细胞外DNA和核苷，在损坏细胞的情况下易于访问，并且通过平衡的核苷拯救途径穿透了仍然活的细胞。 3E10是独一无二的，因为它可以穿透细胞而没有明显的伤害，并且已经对人类施用而没有毒性证据。 FV-HSP70已经被创建并证明是体内有效的细胞保护剂，当中风后给大鼠施用时，将脑组织中的梗死体积降至68％。我们的长期目标是确定FV-HSP70在临床医生对心脏病发作受害者管理时改善心肌恢复的有效性。我们要求资助以实现以下特定目的：1）制造和表征许多用于梗塞研究的FV-HSP70和SCFV 3E10假手术控制。 2）使用99MTC-ANNEXIN V使用SPECT对FV-HSP70在体内的治疗功效。3）评估FV-HSP70 EX VIVO的治疗功效，并使用组织组织学的治疗功效，以及肌酸酶激酶和曲霉素I血液水平。拟议的研究很重要，因为它为未满足的需求开发了关键的治疗方法。拟议的研究具有创新性，因为它利用了独特的抗体介导的蛋白质疗法的抗体介导的，无关的细胞内递送系统。在体内诱导HSP70的产生可能需要时间，而我们产品的影响是将Hsp70迅速递送到受损细胞中以防止细胞死亡。

项目成果

Cardioprotective Effects of HSP72 Administration on Ischemia-Reperfusion Injury.

• DOI: 10.1016/j.jacc.2017.07.762
• 发表时间: 2017-09-19
• 期刊: Journal of the American College of Cardiology
• 影响因子: 24
• 作者: Tanimoto T;Parseghian MH;Nakahara T;Kawai H;Narula N;Kim D;Nishimura R;Weisbart RH;Chan G;Richieri RA;Haider N;Chaudhry F;Reynolds GT;Billimek J;Blankenberg FG;Sengupta PP;Petrov AD;Akasaka T;Strauss HW;Narula J
• 通讯作者: Narula J