Enhanced Efficiency Of Myoblast Mediated Gene Delivery To Muscle Fibers At The Presence Of Myogenic Specific Fusion Factors For The Production Of Target Proteins

An important recent finding is that skeletal muscle fibers can be used as recombinant protein factories to produce and secrete proteins that could act either locally in skeletal muscle for the treatment of muscular disorders or distally for other systemic disorders. The in-depth knowledge accumulated over the years on this tissue provides insights for developing strategies of gene delivery and for assessing and controlling therapeutic protein expression. The following advantages make skeletal muscle an optimal target tissue for genetic manipulation in various gene therapy strategies. Firstly, skeletal muscle could be looked on as the largest secretoty organ with easy access;secondly, skeletal muscle has an abundant blood vascular supply, thus providing an efficient transport system for the carriage of secreted protein into the circulation;thirdly, myofibers are terminally differentiated long-lived, providing a stable environment for long-term expression ofrecombinant transgenes. Even when muscle fibers are damaged, only short segments of individual fibers undergo degeneration, and the myonuclei of surviving segments remain viable;finally, skeletal muscle also has regenerative capacity due to the presence of satellite cells. These cells, while quiescent in normal muscle, can be activated in response to muscle damage. These cells undergo proliferating, migrating and fusing with one another to replace lost fibers, resulting in a regeneration of the lesion. Moreover, satellite cells can be purified from muscles and cultured without losing their ability to differentiate into muscle fibers.The highlighted advantages of skeletal muscle make it ideal target for recombination of proteins, thus very useful in gene therapy. But the limitation is the low efficiency of incorporation although many efforts have been made on it.Many kinds of vectors have been tried as vehicles to transfer target genes into host cells. Liposomes have the advantage of no toxicity, but it can not get through the nuclei membrane, and thus transient and minimal expression of transgenes. Retroviral vectors can transduce dividing cells by integrating into host chromosomal DNA, and is also non-toxic. But they integrate randomly and may cause mutation or cell death. Although adeno-viruses and adeno-associated viruses can be efficiently taken up by non-dividing cells in vivo and do not integrate with host DNA and therefore no mutation risks caused, their insert capacity is restricted to 5kb. Myoblasts are the only cells that divide extensively, migrate and fuse naturally to form myotubes. Once fused with host cells, all of the normal or genetically engineered DNA would be part of the multinucleated myofibers of the host to affect genetic repair. Being endogenous cells, myocytes do not produce those adverse effects of viral vectors, and has not the problematic immuno-rejection if transplanted with autologous cells.Myoblast-mediated gene transfer, despite unsatisfactory research and clinical trial results in the treatment of Duchenne muscular dystrophy, is still a potential method for the rectification of primary muscle myopathies and some systemic disorders, provided the major set-back of the extreme low efficiency of incorporating donor myoblasts into host muscle fibers can be significantly improved.Some results demonstrated that extracts from crushed muscle contained satellite cell mitogens. Preliminary experiments indicate that the crushed muscle extracts contained over twice as much soluble protein as found in the uncrushed extracts. This may indicate that there were some pre-existing myoblast specific fusion factor(s) (MSF) and were released by the injury. Based on the above phenomenon, it is postulated that some factors would be released at the injected or damaged site to enhance the myoblast proliferation and incorporation. Here in this study, BMP-4 was used as marker gene. Delivery of the BMP gene as a transgene for the target cells remains a potential strategy. When incorporated into host cells and expressed properly, the myogenic cell line is expected to express proteins associated with bone formation, and transdifferentiated into osteo-lineage, thus give easy and significant evidence of the successful incorporation of the donor myoblasts with the recipient.Therefore, the present study aimed at circumventing the major problem in myoblast mediated gene therapy with BMP-4 as marker gene by using aqueous crushed muscle extract (CME) to enhance the incorporation rate. It is believed that successful application of these methods could lead to effective therapies for a variety of genetic and acquired diseases, many of which are non-treatable today.Refolding and one-step purification ofBMP-4 expressed in Escherichia coli as inclusion bodies - In this part, E. coli were engineered to produce recombinant human bone morphogenetic protein-4 (rhBMP-4) in an inclusion form using a temperature-inducible expression system. After washing and solubilizing the inclusion bodies, the rhBMP-4 was refolded and dimerized at concentrations up to 100 mg/l by means of a simple dilution method. A one-step purification procedure based on affinity chromatography was used to isolate the rhBMP-4 dimer. The effects of the purified rhBMP-4 dimer were tested by using C2C12 cell culture system. The results showed that the myoblast cultured in the presence of the purified rhBMP-4 dimer, subject to both proliferation or differentiation stages did not form myotubes and neither expressed appreciable levels of myosin heavy chain (MHC). Instead, they formed osteoblast-like morphology with significantly increased alkaline phosphatase (ALP) activity and significantly increased osteocalcin production. It could be concluded from this part that the established method of a strong temperature-inducible expression vector in combination with a high-cell-density cultivation (HCDC) procedure, proceeding a simplified and high yielding method for renaturation of rhBMP-4 and then a one-step purification using affinity chromatography develop a highly productive and simplified process for rhBMP-4 production. Functional assay showed significant ALP activity and significant osteocalcin production in the myogenic cell line at the presence of rhBMP-4.The revised cell cycle commitment of the myoblast induced by Bone Morphogenetic Protein-4: - To better understand the activity of implantedrecombinant human mature domain of bone morphogenetic protein-4 (rhBMP-4), which should induce ectopic bone formation if functional, we investigated in this part whether the presence of rhBMP-4 in myoblast culture medium or a gene transfer of full length BMP-4 into C2C12 myoblasts could convert the committed differentiation pathway of myoblast into that of osteoblast lineage. Recombinant mature domain of human BMP-4 (rhBMP-4) was produced in E. coli, purified and refolded into active form, as described in the above part. The effects of BMP-4 on revising cell cycle commitment of myoblast were studied by using C2C12 cell culture system with the treatment of rhBMP-4 or by the engineered expression of BMP-4 in the cytoplasm of C2C12. The results showed that the myoblast cultured in the presence of rhBMP-4, subject to both proliferation or differentiation stages did not form myotubes and neither expressed appreciable levels of MHC. Instead, they formed osteoblast-like morphology with significantly increased alkaline phosphatase (ALP) activity and significantly increased osteocalcin production. The undifferentiated C2C12 cells with/without pCI-neo plasmid showed no BMP-4 expression and displayed the characteristic stellate morphology with no significant expression of ALP activity and undetectable osteocalcin production. These cells, when subject to differentiation stage, fused with each other forming characteristic thin, elongated multinucleated myotubes. The myoblasts transfected with pCI/BMP-4 recombinant plasmid showed abundant BMP-4 expression both in their proliferating and differentiating stages and the formation of multinucleated myotubes were inhibited, but an osteoblastic like morphology was observed. The C2C12 cells, which had been converted to differentiate into osteoblastic lineage cells by rhBMP-4 pretreatment, were capable of reversing back to myoblastic cells again when the BMP-4 was removed from their culture medium with decreasedALP activity and decreased osteocalcin production. In contrast, the cells harboring pCI/BMP-4 gene showed no-sign of reversing back when the culture medium was freshly changed. The conclusion is that the recombinant BMP-4 refolded from inclusion bodies has proper biological activities. And the introduction of BMP-4 gene into C2C12 cells could convert their committed myogenic differentiation pathway into that of osteoblast lineage, and the conversion of differentiation pathway is heritable. In contrast, the presence of rhBMP-4 in the culture medium could also convert the differentiation pathway of C2C12 cells;however, the conversion of phenotype is not heritable when rhBMP-4 is withdrawn. It was also indicated that BMP-4, as marker gene, can provide significant and easily detected markers once the marked cells were converted to other lineage.Enhanced Efficiency of Myoblast Mediated Gene Delivery to Muscle fibers at the presence of myogenic specific fusion factors - The biggestchallenge to gene therapy is how to efficiently introduce and express the desired therapeutic gene and how properly to fold the nascent polypeptides and/or secret them into extracellular space or blood stream, if required, in a sufficient number of cells to achieve clinical efficacy. In the present study, we have identified a partially purified aqueous extract of rat muscle, which was supposed to contain myoblast specific fusion factor(s) (MSF), that could significantly enhance the fusion of genetically engineered myoblasts with intact muscle fibers of the host rats, and therefore, the nuclei or the introduced genetic construct of the donor myoblasts would incorporate into the hybrid muscle fibers. Once incorporated, the introduced nuclei or the genetic construct could direct the machinery of the hybrid cells toexpress the desired protein(s). C2C12 myobalsts, which was manipulated to contain a plasmid carrying a marker (BMP-4) protein coding sequence, were used as a foreign gene delivery vehicle. BrdU labeling of the manipulated myoblasts allowed tracing of the fate of donor myoblasts. Immunohistochemistry using anti-BMP-4 antibody demonstrated the expression of the introduced gene construct using cellular machinery of the host cells. Assays for production of bone formation makers- osteocalcin and ALP would give the indications that whether the produced BMP-4 in the hybrid cells could fold and secret to extracellular space correctly, and whether undergo proper post-translational modification, and therefore, is active. By using these methods, we demonstrated that in the presence of MSF, numerous BrdU positive nuclei could be seen and the expression of BMP-4 could also be observed in the hybrid fibers of the recipient. While in the control group using BSA to replace MSF, only a few BrdU positive signal was detected. The expression of osteocalcin and the elevated ALP activity detected in the hybrid muscle fibers at the presence of MSF indicated the proper folding, secretion and post-translational modification of the expressed foreign protein. It could be concluded that this strategy of enhanced myoblast mediated gene transfer at the presence of MSF would break the major barrier in current practice of normal or engineered myoblast transplantation in the management of genetic muscle diseases or systemic genetic disorders.