Engineered Cell Therapy for Sustained Local Myocardial Delivery of Nonsecreted Proteins

Abstract

Novel strategies for the treatment of congestive heart failure have taken the form of gene and cell therapy to induce angiogenesis, optimize calcium handling by cardiac myocytes, or regenerate damaged myocardial tissue. Arguably both gene-and cell-based therapies would be benefited by having the ability to locally deliver specific transcription factors and other usually nonsecreted proteins to cells in the surrounding myocardial tissue. The herpes simplex virus type 1 (HSV-1) tegument protein VP22 has been shown to mediate protein intercellular trafficking to mammalian cells and finally localize into the nucleus, which makes it a useful cargo-carrying functional protein in cell-based gene therapy. While VP22 has been studied as a means to modulate tumor growth, little is known about the distribution and transport kinetics of VP22 in the heart and its potential application in combination with autologous cell transplantation for the delivery of proteins to myocardial tissue. The aim of this study was to evaluate the efficacy of VP22 fusion protein intercellular trafficking combined with autologous cell transplantation in the heart. In an in vitro study untransfected rat heart cells were cocultured with stably transfected rat cardiac fibroblasts (RCF) with fusion constructs of VP22. The control experiment was untransfected rat heart cells co-plated with RCF stably transfected with enhanced green fluorescence protein (eGFP). The Lewis rat model was selected for in vivo study. In the in vitro studies there was a 14-fold increase in the number of GFP-positive cells 48 h after initiating coculture with VP22-eGFP RCF compared to eGFP RCF. In the rat model, transplantation of VP22-eGFP expressing RCF led to VP22-eGFP fusion protein delivery to an area of myocardial tissue that was 20-fold greater than that observed when eGFP RCF were transplanted. This area appeared to reach a steady state between 7 and 10 days after transplantation. The VP22-eGFP area consisted of eGFP-positive endothelium, smooth muscle cells, and cardiac myocytes with delivery to an area of approximately 1 mm 2 of myocardial tissue. Our data suggest a viable strategy for the delivery of proteins that are not naturally secreted or internalized, and provide the first insight into the feasibility and effectiveness of cell-penetrating proteins combined with cell transplantation in the heart.

Genetic Enhancement of Stem Cell Engraftment, Survival, and Efficacy

“…101 Another approach that is being developed to direct cardiac differentiation of adult stem cells is the delivery of chimeric proteins encoding cell penetrating peptides and cardiac specific transcription factors. 24,102 Cell penetrating peptides (CPP) cause nonsecreted proteins to be secreted and to be internalized by surrounding cells. We have demonstrated that the transplantation into the myocardium of cells genetically enhanced to express a CPP-GFP protein results in GFP expression in native cardiac myocytes.…”

“…We have demonstrated that the transplantation into the myocardium of cells genetically enhanced to express a CPP-GFP protein results in GFP expression in native cardiac myocytes. 102 To determine whether we could deliver functional transcription factors to the myocardium, we developed a CPP-GATA4 construct and transplanted cardiac fibroblasts that were stably transfected with the CPP-GATA4 construct 1 month after myocardial infarction in the Lewis rat. The infarct border zone of animals that received CPP-GATA4 demonstrated increased cardiac myosin and Bcl-2 expression.…”

The Microenvironment of Freeze-Injured Mouse Urinary Bladders Enables Successful Tissue Engineering

“…3,4 Before clinical applications of these cells is possible, researchers and clinicians must overcome several problems including improved survival rate for the implanted cells, differentiation into the target cell type, and structural support that enables the reconstruction of the recipient tissues. [5][6][7][8] To overcome the problems, the utilization of scaffolds, 9,10 growth factors, 11 and combinations of these materials 12,13 has been investigated. The survival, differentiation, and reorganization of the implanted cells are affected by the microenvironment within the recipient tissues [14][15][16][17] ; however, our understanding of these microenvironmental variables is currently insufficient to provide for clinically effective and reliable resources for regenerative medicine.…”

Effect of Cell-Based Intercellular Delivery of Transcription Factor GATA4 on Ischemic Cardiomyopathy

“…We recently demonstrated the utility of this strategy to deliver the nonsecreted marker protein green fluorescent protein (GFP) to myocardial tissue. 12 In this previous study, we demonstrated that VP22 chimeric proteins are delivered to Ϸ1 mm 2 of myocardium in a direction radial to the needle track. In the present study, we quantified the effects of sustained GATA4:VP22 release into the infarct border zone in a rat model of ischemic cardiomyopathy induced by left anterior descending (LAD) ligation 1 month before cell delivery.…”