Therapeutic angiomyogenesis using human non-viral transduced VEGF 165-myoblasts *

This article reviews the scientific development of angiomyogenesis using VEGF165-myoblasts, a patented biotechnology platform in regenerative medicine associated with Human Myoblast Genome Therapy (HMGT), also known as Myoblast Transfer Therapy (MTT). VEGF165-myoblasts are the leading biologics for angiomyogenesis. This review also compares the safety and efficacy of VEGF165-myoblasts transduced using adenoviral vectors, nanoparticles or liposomes, in anticipation of their application in clinical trials in the near future. VEGF165-myoblasts are differentiated myogenic cells capable of extensive division, natural cell fusion, nucleus transfer, cell therapy and genome therapy. Following transplantation they survive, develop and function to revitalize degenerative myocardium in heart failure and ischemic cardiomyopathy animal studies. VEGF165-myoblasts are second generation products of HMGT/MTT which replenishes live cells and genetically repairs degenerating myofibers in Type II diabetes, muscular dystrophies, aging dysfunction and disfigurement. Myoblasts have also been used to enhance skin and muscle appearance in cosmetology. We envision that VEGF165-myoblasts will provide better outcome than their non-tranduced counterparts. Myoblasts are not stem cells. Their competitive advantages over stem cells are presented.


INTRODUCTION
The human body consists of living cells supplied 100% with capillaries.More than 55% by volume of these cells are myogenic.Muscle degeneration is the common pathway of many fatal and debilitating diseases such as muscular dystrophies, heart failure, ischemic cardiomyopathy, Type II diabetes and aging disfigurement.Therapeutic angiomyogenesis, the concomitant regeneration of muscles and capillaries using biologics is the leading approach to combat these most eminent killers of mankind.This has already been achieved in animals with the unique platform technology of VEGF 165 -myoblast transfer.

BIOLOGICS: CELLULAR VS. MOLECULAR
The cell is the origin of all life.Contained within its nucleus are more than 30,000 genes that determine cell normality and cell characteristics [1].The genes are composed of deoxyribonucleic acid (DNA) that is spatially and temporally switched on and off during development to produce more than 100,000 different transcripts of ribonucleic acid (RNA).The transcriptional events occur inside the nucleus and require the nuclear matrix and/or the chromatin to operate efficiently.These regulatory events are poorly understood but invariably involve polygenic interactions.
Whereas the basic sequence of the human genome has been determined, exactly how the genome functions will take many decades of further research.Scientists do not know the spatial and temporal interactions of the RNA transcripts and know little of their modes of action.Numerous methods have yet to be developed to determine the diverse functions of some 30,000 genes and more techniques have to be refined to effect gene regulation and expression.It is through this knowledge that pharmacogenetics may one day provide rational approaches in therapeutics.Today, no genetic treatment of human disease has been developed based on the Human Genome Project.The analysis of DNA/RNA variations and gene expression are used mainly in diagnostics, while gene therapy success through single gene manipulation has been rare.

HMGT/MTT: A COMBINED CELL THERAPY AND GENOME THERAPY
An alternative perspective is that a genetically abnormal cell degenerates due to the lack of the normal genome.In hereditary degenerative diseases such as muscular dystrophies, the much-needed normal genome can be incorporated into the dystrophic muscle fibers.This is achieved by taking a muscle biopsy from normal male donors, culturing pure mono-lineaged myogenic cells called "myoblasts" and injecting the normal myoblasts into dystrophic muscles.This cell transplant procedure is called myoblast transfer therapy (MTT) or human myoblast genome therapy (HMGT).Myoblast transfer is a treatment of important potential value [2].
Through natural cell fusion, which is inherent in myogenesis and muscle regeneration, the donor myoblasts insert their normal nuclei that contain the human genome into the dystrophic muscle fibers, forming multinucleated heterokaryons to effect genetic complementation repair [3].The donor nuclei operate to transcribe the missing RNA.Only transfer of the normal nuclei, carrying the genomic software and the chromosomal hardware, will allow the orderly provision of various co-factors necessary for the regulation and the expression of the transgene [4].
Natural transcription of the normal genome within the donor nuclei following MTT ensures orderly replacement of any protein deficiency resulting from single gene defect such as dystrophin for Duchenne muscular dystrophy (DMD), or from haphazard polygenic interactions such as GLUT4/IRAP for Type II diabetes.This differs significantly from single gene transduction, effected through viral or non-viral vectors, in that the transgene may find no transcriptional factors/co-factors in the adult environment for its regulation and expression.Many of these co-factors are the products of other genes that are only operative in early development.In addition, myoblasts are normal human cells and do not have complications of the Gene Therapy Medicinal Products (Table 1).
HMGT/MTT is a patented platform technology of cell transplantation, nuclear transfer, genome therapy and tissue engineering.It is the only human genome therapy in existence, and will remain so until another modality is discovered to deliver the human genome into the defective cells of a genetically ill patient.Myoblast is the only somatic cell type that has the ability of natural cell fusion.MTT is uniquely suited to treat hereditary muscle degeneration and weakness through nuclear transfer or genome transfer.In addition, when donor myoblasts fuse among themselves after MTT, they form new muscle fibers to repopulate the degenerative organ, depositing contractile filaments to augment its function.Thus, as a cell therapy, MTT find application not only to all forms of skeletal muscle degeneration, but to heart muscle degeneration, body-building, anti-ageing and soft tissue enhancement [5].MTT replenishes live cells through cell therapy, and repairs degenerating cells through genome therapy.

HMGT/MTT FOR MUSCULAR DYSTROPIES
First conducted in February 1990 and published on 14 July 1990, MTT is the world's first human gene therapy [3].This contention is not without contest [26][27][28][29], and only the record can set it straight for such an important claim.
HMGT/ MTT procedures have been conducted on patients suffering from Duchenne, Becker or Limb-Girdle muscular dystrophies.Beginning with 8 million myoblasts into a small foot muscle, Law's team proceeded to test 5 billion cells into 22 leg muscles, 25 billion cells into 64 body muscles, and then 50 billion cells into 82 muscles.With 280 procedures having been conducted to date, the complete safety of the HMGT/MTT procedure has been proven [3,5,[30][31][32][33][34][35].
There have been no adverse reactions or side effects.There has not been a single death or coma or failure in heart, lung, kidney or liver function related to the MTT procedures.Expected adverse reactions include mild fever (<101˚F), pain, nausea and/or erythema lasting up to three Therapeutic gene expression of dystrophin is efficient and stable, lasting up to six years after MTT [36].Phase II/III clinical trials had provided significant safety and efficacy data (Table 2) that the US Food and Drug Administration (FDA) approved direct cost recovery.

HEART CELL THERAPY (HCT)
Heart muscle degeneration is the leading cause of debilitation and death in humans.It results in loss of live cardiomyocytes, contractile filaments, contractility, heart function and healthy circulation.The damaged heart responds by cell division of cardiomyocytes.Cardiomyocytes do not multiply significantly because the human telomeric DNA repeats in these terminally differentiated cells are minimal.Without significant mitotic activity, surviving cardiomyocytes cannot provide enough new cells to deposit the contractile filaments necessary to sustain normal heart contractility.
Bioengineering the regenerative heart has been shown to provide a potential treatment for cardiovascular diseases.Through endomyocardial injections of cultured skeletal myoblasts, the latter spontaneously transfer their nuclei into cardiomyocytes to impart myogenic regeneration.Injected myoblasts trans-differentiate to become cardiomyocytes.Donor myoblasts also fuse among themselves to form new myofibers, depositing contractile filaments to improve heart contractility.These myofibers contain satellite cells with regenerative vigor to combat heart muscle degeneration.
Three myogenesis mechanisms were elucidated as proof of concept with 50 human/porcine xenografts using cyclosporine as immunosuppressant [37][38][39].Some myoblasts trans-differentiated to become cardiomyocytes.Others transferred their nuclei into host cardiomyocytes through natural cell fusion.As yet others formed skeletal myofibers with satellite cells.De novo production of contractile filaments augmented heart contractility [40,41].Whereas the newly formed myofibers harbor satellite cells and impart regenerative capacity to the heart muscle, the genetic transformation of cardiomyocytes in vivo to become regenerative heterokaryons through myoblast genome transfer [37] constitutes the ultimate heart repair.The regenerative heart [38] also contains transdifferentiated cardiomyocytes of myoblastic origin.In all three scenarios, new contractile filaments are deposited to improve heart contractility.This latter can be translated into the improvement in the quality of life of heart patients and in the prevention of heart attacks.
The first human myoblast transfer into the heart re- vealed that it was safe to administer one billion myoblasts at 100 × 10 6 /ml through the Myostar catheter of the NOGA system (Biosense Webster Inc.) using 20 injections at different locations inside the left ventricle of a swine [42,43].It was determined that 0.3 ml to 0.5 ml would be the optimal volume per injection.EKG was normal throughout the study without arrhythmia.Heart Cell Therapy (HCT) [44], as this is called, is administered with the vision that the myoblasts will survive, develop and function as aliens in the heart, and their nuclei as aliens within cardiomyocytes and myofibers.The myocardial aliens are newly formed skeletal myofibers that contribute to cardiac output through production of contractile filaments.They are donor in origin and as skeletal myofibers will have satellite cells and regenerative capability.The cardiomyocyte aliens are donor myoblast nuclei carrying chromosomes with long telomeric DNA subunits that are essential for mitosis.Upon injury of the trans-differentiated or heterokaryotic cardiomyocyte, the myoblast regenerative genome will be activated to produce foreign contractile filaments such as myosin and actin.
There was a transient elevation of the porcine antihuman-myoblast antibodies at one week after the xenograft [45][46][47].The antibody level subsided at the second week after HMGT, indicating that no more than two weeks of cyclosporine immunosuppression would be necessary for human/pig xenografts or for human allografts.
Myoblasts can be transduced to secrete VEGF 165 or other angiogenic factors to promote survival, development and functioning after HCT [7].In addition, the use of controlled cell-fusion technologies enables cell fusion between myoblasts and cardiomyocytes, producing heterokaryotic cardiomyocytes that are capable of extensive mitosis [8,12].
When compared with a heart transplant, HCT eliminates the use of lifelong immune-suppressants, which is the major cause of infection and death of heart transplant patients.HCT is much less invasive, and tissue availability is not an issue.At a fraction of the cost of a heart transplant, it also promises a reduction in health costs.

THERAPEUTIC ANGIOMYOGENESIS
Therapeutic angiogenesis alleviates tissue ischemia through induction of the intrinsic process of angiogenesis [48].Vascular endothelial growth factor 165 (VEGF 165 ) is the major angiogenic factor involved in physiological as well as pathological angiogenesis [49].The neovascularization is mainly achieved by endothelial cell proliferation triggered through VEGF 165 receptors, especially the VEGF 165 receptor-2 [50].Angiogenesis occurs by sprouting of the former vasculature using previously differentiated cells [51].Therapeutic angiogenesis exploits the natural process for enhanced neovascularization [52].Transplantation of genetically modified myoblasts provides a reservoir to produce biologically active angiogenic factors in a localized and sustained pattern.The technology not only induces new capillary formation, but also allows the transduced myoblasts to differentiate into cardiomyocytes to restore injured myocardium, a process called angiomyogenesis [39,47,48].
In the angiomyogenesis study, human myoblasts were transduced with viral vectors, nano-particles or CD liposomes carrying human VEGF 165 genes.[47,48,[53][54][55].The cells were characterized for VEGF 165 transduction and expression efficiency by immunostaining, enzymelinked immunosorbent assay (ELISA), immunoblotting and RT-PCR.A porcine heart model of infarction was created in female swine by left circumflex artery ligation.The animals were grouped either as control or myoblastimplanted.Angiography was performed to ensure complete occlusion of the blood vessel.Infarction was confirmed with MIBI-Tc99mTc-tetrofosmin SPECT scanning.Four weeks later, 5ml basal DMEM without or with 3 × 10 8 human myoblasts carrying VEGF 165 and Lac-Z genes were implanted into the infarcted areas of the porcine left ventricles.The animals were maintained on cyclosporine (5 mg/kg body weight) for six weeks post-operatively.Hearts were then explanted and processed for immunocytochemical studies.Increased blood vessel density and heart function were the result of these transduced myoblasts treatment [39,48,[53][54][55].

SECOND GENERATION PRODUCTS
Human myoblasts transduced with VEGF 165 using Polyethylenimine-25 nanoparticles or CD liposomes are second generation products for heart angiomyogenesis (Table 3) [54,55].They are promising biologics in treating ischemic cardiomyopathy.Implantation of these myoblasts is safe and efficacious in inducing angiomyogenesis and recovery of left ventricular function of infarcted rat hearts.Approximately five times more capillaries blood capillaries and muscle were found in the VEGF 165 -myoblast injected myocardia as compared to the controls injected with carrier solution (Figure 1).
As determined by ELISA, non-transduced myoblasts secreted VEGF 165 in vitro (300 ± 50 pg/mL) that is enhanced many folds (37 ± 3 ng/mL) in VEGF 165 -myoblasts transduced with a replication incompetent adenoviral vector.Immunostaining showed >95% VEGF 165 positive myoblasts following transduction.Concentration of VEGF 165 released in the culture medium peaked (37 ± 3 ng/mL) at 8 days post-transduction.Cell proliferation assay on human umbilical vein endothelial cells using supernatant from VEGF 165 transduced myoblasts revealed extensive proliferation of cells which was suppressed in the presence of anti-human VEGF 165 antibody in culture medium and was further confirmed by thymidine incorporation assay [53,54].

PEI-VEGF 165 MYOBLASTS
Non-viral vector gene delivery approach provides a safer alternative to overcome the untoward effects of viral vectors.Use of plasmid DNA either alone or in association with cationic liposomes/polymers were assessed with encouraging results [63,64].Similarly, the use of polymer based nanoparticles conferred several advantages including ease of preparation, purification and chemical modification as well as their enormous stability [64,65].Polyethylenimine (PEI) has been widely used for nonviral transduction of cells [65].PEI is cationic in nature and has strong DNA compaction capacity, effective DNA protection and with an intrinsic endosomolytic activity [65].All these properties of PEI contribute to its transfection efficacy.PEI (molecular weight 25 kDa: PEI-25) nanoparticles were designed and transduction conditions optimized to transduce the VEGF 165 gene into human myoblasts with minimum cytotoxic effects.
The feasibility and efficacy of implanting PEI-VEGF 165 myoblasts for angiomyogenic treatment of rat infarcted myocardium were confirmed.Experimental design consisted of rat heart model of acute myocardial infarction receiving injections in group-1/DMEM, group-2/nontransduced myoblasts and group-3/PEI-VEGF 165 -myoblasts.Rats were immuno-suppressed with cyclosporine injection from 3 days before and until 4 weeks after cell transplantation.
Based on optimized transfection conditions, PEI-VEGF 165 -myoblasts expressed VEGF 165 for 18 days with >90% cell viability in vitro.Apoptotic index was reduced in group-2 and group-3 as compared with group-1.Blood vessel density (×400) by immunostaining for PECAM-1 in group-3 was significantly higher (P = 0.043 for both) as compared with group-1 and group-2 at 4 weeks.Regional blood flow (ml/min/g) in the left ventricular anterior wall was higher in group-3 (P = 0.043 for both) as compared with group-1 and group-2.Improved ejection fraction was achieved in group-3 (58.44 ± 4.92%) as compared with group-1 (P = 0.004).It was concluded that PEI-VEGF 165 myoblasts served as an efficient alternative for angiomyogenesis in cardiac repair.We anticipate that the use of PEI nanoparticles for VEGF 165 transduction of human myoblasts is a safe and efficient approach for repair of the infarcted heart.Nanoparticle gene transduction will envision a new approach for gene therapy in human cardiovascular research [54].
Experimental design consisted of rat heart model of acute myocardial infarction receiving injections in group-1/DMEM, group-2/non-transduced myoblasts and group-3/CD-VEGF 165 -myoblasts.Rats were immunosuppressed with daily cyclosporine injections beginning at 3 days before and ending at 4 weeks after CD-VEGF 165 -myoblast transfer.Animal studies revealed reduced apoptosis and improved increased blood flow in group-3 as compared to group-1 and 2. Ejection fraction was best recovered in group-3 animals [55].
Although VEGF 165 gene transduction efficiency was low with CD-VEGF 165 -myoblasts, their gene expression efficiency was sufficient to induce neovascularization, improving blood flow and contractility in infracted rat myocardium.Even a low liposome transduction efficiency with minimal therapeutic protein production can be compensated with a large quantity of myoblast transferred.A substantial amount of the therapeutic gene can be harbored.The transduced muscle acts as a stable bioreactor to deliver a long-term supply of therapeutic protein at basal level.

MYOBLASTS ARE NOT STEM CELLS
By definition, a stem cell is a non-differentiated cell that can differentiate into at least two different cell types.Stem cells are pluripotent and can differentiate into multiple lineages of cell types such as cardiomyocytes, myoblasts, fibroblasts, adipocytes, osteoblasts, and chondrocytes.Myoblasts are not stem cells because they are already differentiated and can only develop to become one cell type which is muscle.
Today, the field of cell therapy is blemished by controversial stem-cell treatments, whereas gene therapy is tainted by mishaps of 'viral vector' technology.HMGT/ MTT does not involve the use of controversial stem cells or the use of dangerous viral agents that have been the cause of death in various clinical trials [59][60][61].MTT/ HMGT has been proven safe on approximately 280 human muscular dystrophy procedures and 300 human heart procedures since year 1990 [67].There are much competitive advantages of HMGT/MTT over stem cell therapy for somatic tissues (Table 4).
Making patient safety the centerpiece of medical liability reform in USA [68], one must realize that the major deficiency of the stem cell technology is scientific.Scientists do not know the transcriptional pathways that trigger stem cells to develop along specific lineages, e.g., to differentiate only into heart muscle cells and not into other cell types.Such knowledge is not likely to be available in the near future.Until then, stem-cell transplants into the heart for example may result in bony, cartilageous, fatty and fibrotic elements that are detrimental to heart function [67].
No effect of intracoronary injection of autologous mononuclear bone marrow stem cells on global left ventricular function was found [69].Whereas other pilot trials suggested that the intracoronary infusion of autologous progenitor cells might have improved left ventricular function after acute myocardial infarction, the statistics of these studies and thus the study conclusions could not hold because the standard deviations were greater than the means of the left ventricular ejection fractions reported [70,71].There was no evidence of donor cell engraftment.One must critically question how the simple act of intracoronary infusion of cells could lead to engraftment.

VEGF 165 -MYOBLAST TARGET SPEC-TRUM
It is anticipated that therapeutic angiomyogenesis using PEI-VEGF 165 -myoblasts or CD-VEGF 165 -myoblasts will find applications in the development of treatments for human heart failure, myocardial infarction in ischemic cardiomyopathy, ischemic limb and diabetic cardiomyopathy in Type II diabetes, various forms of muscular dystrophies and male/female impotency.In addition, newly regenerated capillaries and muscle cells will provide booster tissues for the construction of thicker and redder lips, prominent noses, cheeks and jaws, pink face, and revitalized hair base.Layers of myogenic cells densely populated with capillaries will provide a fertile ground to seed new hair follicle cells on the bald head or other parts of the body to grow hairs of desirable color, density and consistency.

CONCLUSION
Animal experimental data have culminated that pure VEGF 165 -myoblasts, when injected intramyocardially, are potential therapeutic transgene vehicles for concomitant angiogenesis and myogenesis to treat heart failure and ischemic cardiomyopathy.Therapeutic angiomyogenesis has potential application to a host of fatal and debilitating diseases and conditions.In anticipation of its transitional application into clinical trials in the near future, we envision that non-viral transduced VEGF 165myoblasts will provide better outcome than their nontranduced counterparts.

Table 1 .
Complications of viral and non-viral vectors and plasmids.
to prevent rejection of the foreign myoblasts because days.Two months of immunosuppression is sufficient mature muscle fibers do not express MHC-1 surface antigens.

Table 2 .
Efficacy demonstrated in myoblast-injected DMD muscles in US-FDA approved 50-B MTT Phase II/III clinical trials.

Table 4 .
The competitive advantages of myoblasts over stem cells.