Surgical and catheter delivery of autologous myoblasts in patients with congestive heart failure.

Autologous skeletal myoblast (ASM) transplantation is being explored as a possible therapy for patients who have suffered a myocardial infarction. Our initial experience with direct injection during coronary artery bypass grafting demonstrated that this method of delivery was both feasible and safe. In addition, proof of concept of the engraftment and survival of ASMs was shown. However, since many patients who have survived a myocardial infarction are not candidates for surgery, a less invasive delivery method is preferred. We implemented a series of translational research steps to bring catheter-based technology to a clinical application. This included assessing the biocompatibility of the ASM and a novel needle injection catheter using a 3-dimensional endoventricular navigation system, the bioretention and biodistribution of ASMs in a porcine model of myocardial infarction, and the safety and efficacy of ASM transplantation for cardiac function in the porcine model. After catheter functionality had been demonstrated, electromechanical mapping was used to assess the viability in the region of ASM transplantation, and echocardiography, electrocardiogram, and angiography tests were used to assess cardiac function 2 months after ASM transplantation. The results from these preclinical studies were used as a foundation for application of these concepts to a human clinical trial. Here we review the results from our preclinical experiments and surgical delivery clinical trial, and describe the recent clinical studies undertaken to assess the safety and feasibility of catheter-based ASM transplantation into human subjects.

Safety and feasibility of percutaneous autologous skeletal myoblast transplantation in the coil-infarcted swine myocardium.

INTRODUCTION: Autologous skeletal myoblast transplantation (ASMT) for myocardial regeneration is a promising new treatment for patients with congestive heart failure secondary to myocardial infarction (MI). However, non-surgical delivery could broaden the utility of this approach. The present study was designed to evaluate the safety and feasibility of transplanting autologous skeletal myoblast (ASM) via endovascular delivery into the infarcted swine myocardium. METHODS: Seven female Yorkshire swine successfully underwent induced left ventricular MI. ASM biopsies were obtained from the hind limb of each animal and myoblasts were expanded in vitro. In a pilot experiment, ASM were labeled with iridium and short-term retention and biodistribution was determined 2 h after ASM delivery via the MyoStar needle-injection catheter inserted through the femoral artery. At 30 days post-infarction, the remaining animals were divided into three groups containing 2 animals each for percutaneous catheter delivery into the infarcted zone: group 1 control animals were injected with media only, group 2 and 3 animals were injected with approximately 300 x 10(6) and 600 x 10(6) ASM, respectively. Sixty days post-transplantation, the swine hearts were harvested. RESULTS: During the 60-day period between transplantation and harvest, no adverse events were recorded, and continuous rhythm monitoring revealed no arrhythmias. In the small sampling size, myocardial function assessments revealed a trend toward improvement in the treatment groups with respect to ejection fraction, viability, and cardiac index. However, histology of treated swine hearts identified no skeletal muscle cells. DISCUSSION: Percutaneous ASMT into an infarcted swine myocardium is feasible and safe, and may contribute to overall improved heart function.

A percutaneous swine model of myocardial infarction.

INTRODUCTION: The aim of this study was to develop a percutaneous, low risk, and reproducible technique of MI that simulates human disease. METHODS: MI was induced in 44 swine (32.8+/-7.2 kg) by percutaneous embolization coil deployment in the left anterior descending coronary artery. Hemodynamic measurements, left heart catheterization, and echocardiography were performed pre, post, and 30 days after MI. 3D NOGA viability mapping was performed at baseline and 30 days. Excised hearts were examined histologically. RESULTS: Pre-MI mortality was 6.8% and 24 h mortality was 13.6%. All pigs that survived 24 h after MI remained alive at 30 days. The mean left ventricular ejection fraction decreased from 58.4% to 42.1% (p<0.001) at 30 days. The average thrombolysis in myocardial infarction score was 3, 0, and 1.5 at baseline, post-MI, and 30 days, respectively. At 30 days, the end diastolic diameter, end diastolic volume, end systolic volume, and wall motion index increased from 3.76 to 3.89 cm, 32.5 to 50.0 ml, 14.9 to 27.0 ml, and 1.01 to 1.38, respectively (all p<0.05), while the ejection fraction decreased from 56.5% to 49.4% (p<0.01). Additionally, at 30 days, statistically significant reductions in both unipolar and bipolar voltage in the mid and apical regions of the left ventricle were observed. Postmortem pathology showed a transmural scar in the apical anteroseptal regions with fibrosis in the MI region, which accounted for 14.8% and 14.2% of the total left and right ventricular myocardial area and volume, respectively. DISCUSSION: This model of MI is reliable, reproducible, has a pathophysiology similar to humans, and a lower mortality and ventricular fibrillation rates compared to other models. This model may be used to evaluate the effects of pharmacologics, gene therapy, and stem cell transplantation for the treatment of cardiovascular disease as well as studying mechanisms of cardiac remodeling.

Safety and feasibility of autologous myoblast transplantation in patients with ischemic cardiomyopathy: four-year follow-up.

BACKGROUND: Successful autologous skeletal myoblast transplantation into infarcted myocardium in a variety of animal models has demonstrated improvement in cardiac function. We evaluated the safety and feasibility of transplanting autologous myoblasts into infarcted myocardium of patients undergoing concurrent coronary artery bypass grafting (CABG) or left ventricular assist device (LVAD) implantation. In addition, we sought to gain preliminary information on graft survival and any associated changes in cardiac function. METHODS AND RESULTS: Thirty patients with a history of ischemic cardiomyopathy participated in a phase I, nonrandomized, multicenter pilot study of autologous skeletal myoblast transplantation concurrent with CABG or LVAD implantation. Twenty-four patients with a history of previous myocardial infarction and a left ventricular ejection fraction <40% were enrolled in the CABG arm. In a second arm, 6 patients underwent LVAD implantation as a bridge to heart transplantation, and patients donated their explanted native hearts for testing at the time of heart transplantation. Myoblasts were successfully transplanted in all patients without any acute injection-related complications or significant long-term, unexpected adverse events. Follow-up positron emission tomography scans showed new areas of glucose uptake within the infarct scar in CABG patients. Echocardiography measured an average change in left ventricular ejection fraction from 28% to 35% at 1 year and of 36% at 2 years. Histological evaluation in 4 of 6 patients who underwent heart transplantation documented survival and engraftment of the skeletal myoblasts within the infarcted myocardium. CONCLUSIONS: These results demonstrate the survival, feasibility, and safety of autologous myoblast transplantation and suggest that this modality offers a potential therapeutic treatment for end-stage heart disease.

Feasibility and safety of autologous myoblast transplantation in patients with ischemic cardiomyopathy.

Successful autologous skeletal myoblast transplantation into infarcted myocardium in a variety of animal models has demonstrated improvement in cardiac function. We evaluated the safety and feasibility of transplanting autologous myoblasts into infarcted myocardium of patients undergoing concurrent coronary artery bypass grafting (CABG) or left ventricular assist device implantation (LVAD). In addition, we sought to gain preliminary information on graft survival and any potential improvement of cardiac function. Eighteen patients with a history of ischemic cardiomyopathy participated in a phase I, nonrandomized, multicenter pilot study of autologous skeletal myoblast transplantation concurrent with CABG or LVAD implantation. Twelve patients with a history of previous myocardial infarction (MI) and a left ventricular ejection of less than 30% were enrolled in the CABG arm. In a second arm, six patients underwent LVAD implantation as a bridge to heart transplantation and were required to donate their heart for testing at the time of heart transplant. Myoblasts were successfully transplanted in all patients without any acute injection-related complications or significant long-term unexpected adverse events. Follow-up PET scans showed new areas of viability within the infarct scar in CABG patients. Echocardiography measured an average improvement in left ventricular ejection fraction (LVEF) from 25% to 34%. Histological evaluation in four out of five patients who underwent heart transplantation documented survival and engraftment of the skeletal myoblasts within the infarcted myocardium. These interim results demonstrate survival, feasibility, and safety of autologous myoblast transplantation and suggest that this modality may offer a potential therapeutic treatment for end-stage heart disease.

Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus.

Direct insertion of amino acid sequences into the adeno-associated virus type 2 (AAV) capsid open reading frame (cap ORF) is one strategy currently being developed for retargeting this prototypical gene therapy vector. While this approach has successfully resulted in the formation of AAV particles that have expanded or retargeted viral tropism, the inserted sequences have been relatively short, linear receptor binding ligands. Since many receptor-ligand interactions involve nonlinear, conformation-dependent binding domains, we investigated the insertion of full-length peptides into the AAV cap ORF. To minimize disruption of critical VP3 structural domains, we confined the insertions to residue 138 within the VP1-VP2 overlap, which has been shown to be on the surface of the particle following insertion of smaller epitopes. The insertion of coding sequences for the 8-kDa chemokine binding domain of rat fractalkine (CX3CL1), the 18-kDa human hormone leptin, and the 30-kDa green fluorescent protein (GFP) after residue 138 failed to lead to formation of particles due to the loss of VP3 expression. To test the ability to complement these insertions with the missing capsid proteins in trans, we designed a system for producing AAV vectors in which expression of one capsid protein is isolated and combined with the remaining two capsid proteins expressed separately. Such an approach allows for genetic modification of a specific capsid protein across its entire coding sequence leaving the remaining capsid proteins unaffected. An examination of particle formation from the individual components of the system revealed that genome-containing particles formed as long as the VP3 capsid protein was present and demonstrated that the VP2 capsid protein is nonessential for viral infectivity. Viable particles composed of all three capsid proteins were obtained from the capsid complementation groups regardless of which capsid proteins were supplied separately in trans. Significant overexpression of VP2 resulted in the formation of particles with altered capsid protein stoichiometry. The key finding was that by using this system we successfully obtained nearly wild-type levels of recombinant AAV-like particles with large ligands inserted after residue 138 in VP1 and VP2 or in VP2 exclusively. While insertions at residue 138 in VP1 significantly decreased infectivity, insertions at residue 138 that were exclusively in VP2 had a minimal effect on viral assembly or infectivity. Finally, insertion of GFP into VP1 and VP2 resulted in a particle whose trafficking could be temporally monitored by using confocal microscopy. Thus, we have demonstrated a method that can be used to insert large (up to 30-kDa) peptide ligands into the AAV particle. This system allows greater flexibility than current approaches in genetically manipulating the composition of the AAV particle and, in particular, may allow vector retargeting to alternative receptors requiring interaction with full-length conformation-dependent peptide ligands.

Local endovascular delivery, gene therapy, and cell transplantation for peripheral arterial disease.

Advances in catheter technology, gene identification, and cell biology may provide novel treatment options for patients with peripheral arterial disease (PAD) who are not candidates for standard revascularization procedures. Animal studies and recent results in human beings suggest that transfer of growth factors or regulatory genes and transplantation of progenitor cells may provide novel therapy options by inducing therapeutic angiogenesis or by inhibiting restenosis. This review will discuss the development of a variety of catheters for localized endovascular delivery, as well as the various cellular and genetic strategies that exist to restore blood flow to ischemic tissue and to reduce neointimal hyperplasia.

Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding.

The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.