Continuous modeling of metabolic networks with gene regulation in yeast and in vivo determination of rate parameters.

A continuous model of a metabolic network including gene regulation to simulate metabolic fluxes during batch cultivation of yeast Saccharomyces cerevisiae was developed. The metabolic network includes reactions of glycolysis, gluconeogenesis, glycerol and ethanol synthesis and consumption, the tricarboxylic acid cycle, and protein synthesis. Carbon sources considered were glucose and then ethanol synthesized during growth on glucose. The metabolic network has 39 fluxes, which represent the action of 50 enzymes and 64 genes and it is coupled with a gene regulation network which defines enzyme synthesis (activities) and incorporates regulation by glucose (enzyme induction and repression), modeled using ordinary differential equations. The model includes enzyme kinetics, equations that follow both mass-action law and transport as well as inducible, repressible, and constitutive enzymes of metabolism. The model was able to simulate a fermentation of S. cerevisiae during the exponential growth phase on glucose and the exponential growth phase on ethanol using only one set of kinetic parameters. All fluxes in the continuous model followed the behavior shown by the metabolic flux analysis (MFA) obtained from experimental results. The differences obtained between the fluxes given by the model and the fluxes determined by the MFA do not exceed 25% in 75% of the cases during exponential growth on glucose, and 20% in 90% of the cases during exponential growth on ethanol. Furthermore, the adjustment of the fermentation profiles of biomass, glucose, and ethanol were 95%, 95%, and 79%, respectively. With these results the simulation was considered successful. A comparison between the simulation of the continuous model and the experimental data of the diauxic yeast fermentation for glucose, biomass, and ethanol, shows an extremely good match using the parameters found. The small discrepancies between the fluxes obtained through MFA and those predicted by the differential equations, as well as the good match between the profiles of glucose, biomass, and ethanol, and our simulation, show that this simple model, that does not rely on complex kinetic expressions, is able to capture the global behavior of the experimental data. Also, the determination of parameters using a straightforward minimization technique using data at only two points in time was sufficient to produce a relatively accurate model. Thus, even with a small amount of experimental data (rates and not concentrations) it was possible to estimate the parameters minimizing a simple objective function. The method proposed allows the obtention of reasonable parameters and concentrations in a system with a much larger number of unknowns than equations. Hence a contribution of this study is to present a convenient way to find in vivo rate parameters to model metabolic and genetic networks under different conditions.

Gene therapy: a strategy for the treatment of inherited muscle diseases?

The emergence of new vectors of viral origin (recombinant adeno-associated viruses, second and third generation adenoviruses) and a new potential source of cells for transplantation (muscle-derived stem cells) are broadening the panel of therapeutic options for myopathies. Although the perfect gene-transfer method(s) have not yet been found, recent findings will certainly constitute a strong knowledge base for future clinical trials.

Functional EGFP-dystrophin fusion proteins for gene therapy vector development.

Transfection and transduction studies involving the use of the full-length dystrophin (11 kb) or the truncated mini-gene (6 kb) cDNAs are hampered by the large size of the resulting viral or non-viral expression vectors. This usually results in very low yields of transgene-expressing cells. Moreover, the detection of the few transgene-expressing cells is often tedious and costly. For these reasons, expression vectors containing the enhanced green fluorescent protein (EGFP) fused with the N-termini of mini- and full-length human dystrophin were constructed. These constructs were tested by transfection of Phoenix cells with Effectene, resulting after 48 h in a green fluorescent signal in 20% of cells. Analysis of the cell extracts by immunoblotting with the use of a monoclonal antibody specific to the dystrophin C-terminus confirmed the expression of EGFP-mini- (240 kDa) and EGFP-full-length human dystrophin (450 kDa) fusion proteins. Moreover, following the in vivo electroporation of the plasmids containing the EGFP-mini- and full-length dystrophin in mouse muscles, both fluorescent proteins were observed in cryostat sections in their normal location under the plasma membrane. This indicates that the fusion of EGFP to dystrophin or mini-dystrophin did not interfere with the normal localization of the protein. In conclusion, the fusion of EGFP provides a good tool for the search of the best methods to introduce mini- or full-length dystrophin cDNA in the cells (in vitro) or muscle fibers (in vivo) for the establishment of a treatment by gene therapy of Duchenne muscular dystrophy patients.

Systemic production of human granulocyte colony-stimulating factor in nonhuman primates by transplantation of genetically modified myoblasts.

Clinical use of human granulocyte-colony stimulating factor (hG-CSF) to treat various diseases involving neutropenia has been previously shown to (1) successfully increase circulating neutrophils, (2) reduce condition-related infections, and (3) cause few side effects in patients. To alleviate the symptoms of neutropenia, the patient must receive frequent injections of recombinant hG-CSF. Permanent ways to deliver stable levels of the molecule to the patient are being investigated. Among them, the transplantation of hG-CSF-secreting cells has been proposed and performed successfully in rodents, using fibroblast cell lines and primary muscle cells. We thus investigated whether similar results could be obtained by intramuscular myoblast transplantation in a large animal model. When 1-3 x 10(8) myoblasts were injected into three Macaca mulatta, hG-CSF was detected at high levels (300-900 pg/ml), which in turn led to a four- to fivefold increase in circulating neutrophils. However, both the concentrations of hG-CSF and neutrophil levels were found to decrease over time. Nonetheless, neutrophils were found at higher levels from the fourth week until the end the experiment (up to 29 weeks) in G-CSF monkeys compared with control animals. These results show that transplantation of hG-CSF-secreting myoblasts may indeed be a therapeutic option for the treatment of neutropenic patients.

Transplantation of normal and DMD myoblasts expressing the telomerase gene in SCID mice.

The limited proliferative capacity of dystrophic human myoblasts severely limits their ability to be genetically modified and used for myoblast transplantation. The forced expression of the catalytic subunit of telomerase can prevent telomere erosion and can immortalize different cell types. We thus tested the ability of telomerase to immortalize myoblasts and analyzed the effect of telomerase expression on the success of myoblast transplantation. Telomerase expression did not significantly extend the human myoblast life span. The telomerase expressing myoblasts were nonetheless competent to participate in myofiber formation after infection with the retroviral vector. Although the new fibers obtained are less numerous than after the transplantation of normal myoblasts, these results demonstrate that the forced expression of telomerase does not block the ability of normal or dystrophic myoblasts to differentiate in vivo. It will be now necessary to determine the factors that prevent telomerase from extending the life span of human myoblasts before the potential of this intervention can be fully examined.

Transplantation of dermal fibroblasts expressing MyoD1 in mouse muscles.

Transplantation of normal myoblasts into dystrophic muscles is a potential treatment for Duchenne muscular dystrophy (DMD). However, the success of such grafts is limited by the immune system responses. To avoid rejection problems, autologous transplantation of the patient's corrected myoblasts has been proposed. Regretfully, the low proliferative capacity of DMD myoblasts in culture (due to their premature senescence) limits such procedure. On the other hand, modification of dermal fibroblasts leading to the myogenic pathway using a master regulatory gene for myogenesis is an interesting alternative approach. In this study, the retrovirally encoded MyoD1 cDNA was introduced in dermal fibroblasts of TnI LacZ mice to provoke their conversion into myoblast-like cells. In vitro and in vivo assays were done and the results were compared to those obtained with uninfected fibroblasts and myoblasts. Some MyoD1-expressing fibroblasts were able to fuse and to express beta-galactosidase (under the transcriptional control of the Troponin I promoter), dystrophin and desmin in vitro. Thirty days following implantation of these modified fibroblasts in muscles of mdx mice, an average of 7 beta-Gal+/Dys-muscle fibers were observed. No beta-Gal+ fibers were observed after the transplantation of uninfected fibroblasts. Our results indicate that the successful implantation of myoblasts obtained from genetically modified fibroblasts is indeed feasible. However, the in vitro conversion rate and the in vivo fusion of genetically modified fibroblasts must be largely increased to consider this approach as a potential therapy for DMD and other myopathies.

Successful transplantation of genetically corrected DMD myoblasts following ex vivo transduction with the dystrophin minigene.

Myoblast transplantation and gene therapy are two promising therapeutical approaches for the treatment of Duchenne Muscular Dystrophy (DMD). So far, both strategies have met many hurdles, mainly because of immune reactions. In this study, we investigated a third and novel strategy based on the combination of these two basic ones, i.e., transplantation of genetically modified myoblasts. We first derived a primary culture from a muscle biopsy of a young DMD patient (3 years old). Adenoviral-mediated dystrophin gene transfer into these DMD cultures and expression of the dystrophin transgene were achieved in vitro. The transduced cultures were then transplanted the same day in immunodeficient SCID mouse muscles. Three weeks following the graft, many human dystrophin-positive fibers were observed throughout sections of the injected muscles. However, many fibers expressed human MHC antigens without expressing human dystrophin due to the low percentage of infected primary muscle cells in vitro (even when a high MOI [400] was used) and to a reduction and even to a complete loss of transgene copy number during myoblast replication. From our results, we conclude that, although not at a high proportion, (1) DMD primary myoblast cultures are infectable by adenoviruses; (2) they can be efficiently transplanted back in a muscle, leading to normal fusion of infected myoblasts with the host fibers; and (3) they can correct the dystrophin deficiency in the host fibers by the expression of a mini-dystrophin transgene.

Expression of human dystrophin following the transplantation of genetically modified mdx myoblasts.

Transplantation of genetically modified autologous myoblasts has been proposed as a possible solution to avoid long-term use of immunosuppressive drugs. To determine the conditions to be used in this kind of approach for possible treatment of dystrophin deficiency, mdx myoblasts were infected at different multiplicities of infection (MOI or 0.01-1000) with an adenoviral vector containing a CMV promoter/enhancer driven 6.3 kb human dystrophin cDNA (minigene) and tested in vitro for transgene expression. In these cultures, dystrophin mRNA was found to be proportionate with increasing MOI. Primary myoblast cultures derived from transgenic mdx mice expressing beta-Gal under a muscle-specific promoter and showing high expression of the human mini-dystrophin transgene introduced by the adenoviral vector were grafted into anterior tibialis muscles of SCID mice. Ten and 24 days after transplantation, numerous muscle fibers expressing both human dystrophin and beta-Gal were detected throughout the mouse muscles by immunohistochemistry using an antibody specific for human dystrophin. The presence of the human mini-dystrophin mRNA was also detected by RT-PCR. These results demonstrate that three essential conditions in autologous myoblast transplantation can be achieved: (1) in vivo survival of at least some of the transduced myoblasts; (2) efficient fusion of these cells with the host muscle fibers; and (3) the high expression of the dystrophin transgene in situ. Furthermore, this article provides a novel RT-PCR-based technique to quantify the human dystrophin minigene expression in vitro and in vivo.

Inflammatory damage following first-generation replication-defective adenovirus controlled by anti-LFA-1.

First-generation replication-defective adenoviruses have been reported to lead to transient reporter gene expression due to a specific immune reaction involving T and B lymphocytes. Some recent reports have also demonstrated the presence of a nonspecific inflammatory reaction involving macrophages and neutrophils after both intramuscular injections and viral vectors transduction. To further investigate this nonspecific inflammatory reaction, deltaE1/E3a adenoviruses were injected intramuscularly in immunocompetent mice. Some of these mice were treated with anti-LFA-1. The adenovirus-injected muscles showed abundant CD4+, CD8+, LFA-1+, and Mac-1+ cell infiltration 3 days after the deltaE1/E3a injection. The anti-LFA-1 monoclonal antibody was able to block the nonspecific inflammatory damage due mostly to neutrophils and macrophages. The anti-LFA-1 did not produce this effect by reducing the muscle infiltration by LFA-1+ cells. It may instead have blocked the direct interaction between LFA-1 and ICAM-1 thus preventing the damage produced by the respiratory burst of neutrophils. Blocking the resulting damage of this inflammatory reaction with anti-LFA-1 in animals also treated with FK506, a powerful immunosuppressant for gene therapy, largely increased the long-term transgene expression compared with mice only treated with FK506.