Hypoxia-inducible factor-1 alpha (HIF-1 alpha) gene therapy holds great promise for the treatment of myocardial ischemia. Both preclinical and clinical evaluations of this therapy are underway and can benefit from a vector strategy that allows noninvasive assessment of HIF-1 alpha expression as an objective measure of gene delivery. We have developed a novel bidirectional plasmid vector (pcTnT-HIF-1 alpha-VP2-TSTA-fluc), which employs the cardiac troponin T (cTnT) promoter in conjunction with a two-step transcriptional amplification (TSTA) system to drive the linked expression of a recombinant HIF-1 alpha gene (HIF-1 alpha-VP2) and the firefly luciferase gene (fluc). The firefly luciferase (FLuc) activity serves as a surrogate for HIF-1 alpha-VP2 expression, and can be noninvasively assessed in mice using bioluminescence imaging after vector delivery. Transfection of cultured HL-1 cardiomyocytes with pcTnT-HIF-1 alpha-VP2-TSTA-fluc led to a strong correlation between FLuc and HIF-1 alpha-dependent vascular endothelial growth factor expression (r(2) = 0.88). Intramyocardial delivery of pcTnT-HIF-1 alpha-VP2-TSTA-fluc into infarcted mouse myocardium led to persistent HIF-1 alpha-VP2 expression for 4 weeks, even though it improved neither CD31 + microvessel density nor echocardiographically determined left ventricular systolic function. These results lend support to recent findings of suboptimal efficacy associated with plasmid-mediated HIF-1 alpha therapy. The imaging techniques developed herein should be useful for further optimizing HIF-1 alpha-VP2 therapy in preclinical models of myocardial ischemia.

PURPOSE:We tested a novel imaging strategy, in which both the survival of transplanted myoblasts and their therapeutic transgene expression, a recombinant hypoxia-inducible factor-1α (HIF-1α-VP2), can be monitored using firefly luciferase (fluc) and Renilla luciferase (hrl) bioluminescence reporter genes, respectively. PROCEDURES:The plasmid pUbi-hrl-pUbi-HIF-1α-VP2, which expresses both hrl and HIF-1α-VP2 using two ubiquitin promoters, was characterized in vitro. C2c12 myoblasts stably expressing fluc and transiently transfected with pUbi-hrl-pUbi-HIF-1α-VP2 were injected into the mouse hindlimb. Both hrl and fluc expression were monitored using bioluminescence imaging (BLI). RESULTS:Strong correlations existed between the expression of hRL and each of HIF-1α-VP2, VEGF, and PlGF (r(2) > 0.83, r(2) > 0.82, and r(2) > 0.97, respectively). In vivo, both transplanted cells and HIF-1α-VP2 transgene expression were successfully imaged using BLI. CONCLUSIONS:An objective evaluation of myoblast-mediated gene transfer in living mice can be performed by monitoring both the survival and the transgene expression of transplanted myoblasts using the techniques developed herein.

OBJECTIVE — b -Cells that express an imaging reporter have pro- vided powerful tools for studying b -cell development, islet transplantation, and b -cell autoimmunity. To further expedite diabetes research, we generated transgenic C57Bl/6 MIP-TF mice that express a trifusion protein of three imaging reporters (luciferase/ EGFP/HSV1-sr39 thymidine kinase) in their b -cells. This should enable the noninvasive imaging of b -cells by charge-coupled de- vice (CCD) and micro positron emission tomography (PET) and the identi fi cation of b -cells at the cellular level by fl uorescent microscopy. RESULTS — MIP-TF mouse b -cells were readily identi fi ed in pan- creatic tissue sections using fl uorescent microscopy. We show for the fi rst time that MIP-TF b -cells can be noninvasively imaged using microPET. There was a correlation between CCD and microPET signals from the pancreas region of individual mice. After low-dose streptozotocin administration to induce type 1 diabetes, we observed a progressive reduction in bioluminescence from the pancreas region before the appearance of hyperglycemia. Although there have been reports of hyperglycemia inducing pro- insulin expression in extrapancreatic tissues, we did not observe bioluminescent signals from extrapancreatic tissues of diabetic MIP-TF mice. Because MIP-TF mouse b -cells express a viral thymidine kinase, ganciclovir treatment induced hyperglycemia, pro- viding b 1 diabetes 2

OBJECTIVE β-Cells that express an imaging reporter have provided powerful tools for studying β-cell development, islet transplantation, and β-cell autoimmunity. To further expedite diabetes research, we generated transgenic C57BL/6 “MIP-TF” mice that have a mouse insulin promoter (MIP) driving the expression of a trifusion (TF) protein of three imaging reporters (luciferase/enhanced green fluorescent protein/HSV1-sr39 thymidine kinase) in their β-cells. This should enable the noninvasive imaging of β-cells by charge-coupled device (CCD) and micro-positron emission tomography (PET), as well as the identification of β-cells at the cellular level by fluorescent microscopy. RESEARCH DESIGN AND METHODS MIP-TF mouse β-cells were multimodality imaged in models of type 1 and type 2 diabetes. RESULTS MIP-TF mouse β-cells were readily identified in pancreatic tissue sections using fluorescent microscopy. We show that MIP-TF β-cells can be noninvasively imaged using microPET. There was a correlation between CCD and microPET signals from the pancreas region of individual mice. After low-dose streptozotocin administration to induce type 1 diabetes, we observed a progressive reduction in bioluminescence from the pancreas region before the appearance of hyperglycemia. Although there have been reports of hyperglycemia inducing proinsulin expression in extrapancreatic tissues, we did not observe bioluminescent signals from extrapancreatic tissues of diabetic MIP-TF mice. Because MIP-TF mouse β-cells express a viral thymidine kinase, ganciclovir treatment induced hyperglycemia, providing a new experimental model of type 1 diabetes. Mice fed a high-fat diet to model early type 2 diabetes displayed a progressive increase in their pancreatic bioluminescent signals, which were positively correlated with area under the curve–intraperitoneal glucose tolerance test (AUC-IPGTT). CONCLUSIONS MIP-TF mice provide a new tool for monitoring β-cells from the single cell level to noninvasive assessments of β-cells in models of type 1 diabetes and type 2 diabetes.