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WO1999030730A1 - Procedes et compositions permettant d'ameliorer la reussite de la transplantation cellulaire chez un receveur - Google Patents

Procedes et compositions permettant d'ameliorer la reussite de la transplantation cellulaire chez un receveur Download PDF

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WO1999030730A1
WO1999030730A1 PCT/CA1998/001176 CA9801176W WO9930730A1 WO 1999030730 A1 WO1999030730 A1 WO 1999030730A1 CA 9801176 W CA9801176 W CA 9801176W WO 9930730 A1 WO9930730 A1 WO 9930730A1
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composition
cells
myoblasts
muscle
transplantation
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PCT/CA1998/001176
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Jacques P. Tremblay
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Universite Laval
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Priority to AU18649/99A priority Critical patent/AU1864999A/en
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24023—Matrilysin (3.4.24.23)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells

Definitions

  • This invention relates to a method of restoring a functional protein in a tissue by transplanting donor cells or genetically modified donor or host cells, the method comprising one or more of the following the steps : a) reducing the early mortality by way of an anti-inflammatory therapy, b) increasing the diffusion of surviving donor cells into the host tissue, c) injecting a greater amount of donor cells already grown and multiplied in vitro and injecting cells which have been stimulated to proliferate or preconditioned for survival, d) increasing the fusion between the donor and host cells, and e) undertaking a suitable immuno-suppressive therapy.
  • Cell transplantation is a potential treatment for several diseases.
  • Myoblast transplantation may indeed be used to treat Duchenne Muscular Dystrophy (Partridge 1991), heart insufficiency, nanism (Dhawan et al. 1991), hemophilia (Dai et al. 1992) and Parkinson (Jiao et al. 1993); neuron transplantation may be used to treat Huntington disease (Emerich, 1995) and Parkinson disease (Borlongan and Sandberg, 1995); islet may be used for diabetic (Hering et al. 1993), hepatocytes for liver diseases (Raper and Wilson, 1993). The success of these cell transplantations has been so far rather limited.
  • the principal problems to overcome during cell transplantation are the following: a) a host specific immune reaction towards donor cells; b) the number of cells expressing the non-defective protein; c) the diffusion of donor cells in the recipient tissue; d) the amount of non-defective protein expressed in the cells participating to tissue function recovery; and e) the high donor cell death rate.
  • Nonspecific immunosuppression has been found successful for improving and maintaining the success of transplantation.
  • Immunosuppressors like FK506, rapamycin, cyclosporine A and cyclophosphamide did improve the survival time of the transplant.
  • cyclosporine A, FK506 and rapamycin have been found the most potent. Since immunosuppressors provoke undesirable side effects, it is highly desirable to match histocompatible subjects and to use a more specific immunotherapy, when needed or to genetically modify the host's own cells. With adequate immunosuppression, it may be possible to transplant cells originating from a different species, for example normal or genetically modified pig myoblasts could be transplanted into human muscles to restore a defective protein.
  • non-defective protein e.g., fused donor cells or hybrid donor-recipient muscle fibers
  • a non-defective protein e.g., fused donor cells or hybrid donor-recipient muscle fibers
  • other strategies have been used to increase the production of non-defective proteins. It has been found that pre-treating recipient mdx mice (an animal model for Duchenne muscular dystrophy) with notexin and radiation, which has for effect to destroy the capability of the recipient muscle cells to proliferate, gives a much better chance to the donor cells to proliferate and to reconstruct a functional tissue.
  • the extra-cellular matrix represents a barrier to the diffusion of transplanted cells, and metalloproteases have been proved to be tool to moderately favor the diffusion of donor cells into the recipient tissue.
  • the most spectacular results from metalloproteases are found in the increase of the rate of fusion between donor and host cells which participate in the recovery of a normal muscular function.
  • An alternative way to resolve the limited diffusion of myoblasts is to do very closely spaced myoblast injections (1 to 1.5 mm apart).
  • Such close injection trajectories may be done by using a robotic device controlled by a computer receiving information from an imaging system such as NMR (Nuclear Magnetic Resonance), echography system or low level X ray system such as those used for cathetherism.
  • an imaging system such as NMR (Nuclear Magnetic Resonance), echography system or low level X ray system such as those used for cathetherism.
  • NMR Nuclear Magnetic Resonance
  • echography system low level X ray system
  • low level X ray system such as those used for cathetherism
  • transplanted cells Although a high percentage of cells which survive after one week of transplantation expresses a non-defective protein (about 90% expressing dystrophin in an mdx mouse), the level of expression of a functional protein (dystrophin) per cell remains quite variable. There is therefore room for improving the level of expression of a non-defective protein in transplanted or fused cells specially when the transplanted cells are the host own cells genetically modified to express a protein lacking or defective in this patient, e) The high death of transplanted cells:
  • Inflammation may be one of the major factors involved in the important early mortality, e.g., before the building of a cellular or humoral response. Inflammation may be also partly responsible for the poor success of muscle regeneration observed after the transplantation of myoblasts between identical twins.
  • anti-LFA-1 or anti-ICAM-1 achieved an efficient decrease in the early cell mortality rate, there is still room for improving the success of survival, and for improving the overall success of cell transplantation.
  • the present invention covers significant improvements for each event involved in the transplantation success or graft survival. These improvements, separately or combined with each other, greatly ameliorate the recovery of a tissue towards a normal function.
  • the first object of the invention is to reduce the early death of the transplanted cells.
  • anti-LFA-1 or anti-ICAM-1 antibodies other anti-inflammatory agents are proposed, alone or in combination.
  • One or more of these anti- inflammatory agents may be combined with the anti-LFA-1 or anti-ICAM-1 antibody, to prevent the early death of transplanted cells.
  • the anti-inflammatory agent may be one or more agents selected from the group consisting of TGF-beta1 , ligand(s) against CD18, CD31 , CD50, E-selectin and P-selectin.
  • the anti- inflammatory agent is TGF-beta1.
  • the anti-inflammatory agent may also be an inhibitor of synthesis of oligosaccharides, such as Castanospermine.
  • It may alternatively be a glucosidase capable of removing oligosaccharides from glycoproteins.
  • anti-inflammatory agents have for effect to hinder the binding of pro-inflammatory cells to transplanted cells, or to inhibit the recruitment of pro-inflammatory cells on the transplantation site. Their effect results in the inhibition of pro-inflammatory cell action. The end effect is that pro-inflammatory cells either cannot activate themselves or at least cannot kill the transplanted cells.
  • the step of early death of transplanted cells being overcome, the next one to be addressed is the diffusion of the donor cells into the recipient tissue. To that effect, this inhibition of formation of glycoproteins is useful as well as the promotion of the formation and secretion of metalloproteases are useful.
  • the combinations of the effect of an anti-inflammatory and of a metalloprotease treatment constitute a second object of the present invention.
  • Metalloprotease treatment can be achieved by treating the donor cells with inducers like phorbol esters or concanavalin A, or directly co-injecting the donor cells with metalloproteases, or injecting the host tissue with metalloproteases, or else by genetically modifying the donor cells to produce the metalloproteases.
  • Combining treatment of donor cells with metalloproteases with the early anti- inflammatory treatment provides for a maximum of chances that donor cells colonize the recipient tissue.
  • Such combined treatments permit an optimal spreading of the transplanted cells, which can fuse with the recipient tissue cells and are ready to restore a functional protein and tissue function.
  • Metalloproteases promote the fusion of donor with recipient cells. Inhibiting the synthesis of oligosaccharides or detaching the oligosaccharide part from glycoproteins may also promote the diffusion of the transplanted cells by preventing their immediate fusion with recipient tissue cells. As well, avoiding the formation or degrading glycoproteins renders less possible the functional contacts of the donor cells with the recipient cells and connective tissue, and it further prevents adhesion to pro-inflammatory cells and immune system cells.
  • a third object of the present invention relates to the combination of one of the first two treatments, or both, with to a third step for increasing to any possible extend the number of cells available for transplantation: the ex vivo growth of donor cells.
  • 1 proposed to grow myoblasts in vitro in the presence of growth factors such as b-FGF.
  • Growing the donor cells in the presence of suitable growth factors increases the success of transplantation perhaps by increasing survival and diffusion of the cells.
  • the treatment of donor cells with b-FGF increased by 4-fold the number of functional cells resulting from their fusion with the recipient muscle cells.
  • This treatment was as efficient as another pre-treatment which consists of pre-conditioning the recipient tissue with a toxin or an irradiation.
  • the donor cells are stimulated to proliferate by an ex vivo treatment, and these cells are transplanted with no damaging pretreatment of the recipient tissue, , such as irradiation and notexin.
  • this does not exclude that both a trophic or growth factor and pretreatment with a myotoxic agent can be performed, which may improve the results.
  • Another way to promote cell proliferation has been tried with success, that is conditionally immortalized donor cells.
  • One way of doing this is to modify the donor cells to express a thermosensitive mutant of SV40 large T antigen. Proliferation occurs at 33°C. Proliferation is stopped and differentiation occurs after shifting at 37°C. These modified donor cells should not be tumorigenic upon transplantation. An additional precaution may be to remove the T antigen before transplanting the cells. This proliferation step may be done in the presence of growth factors.
  • the cell proliferation may also be increased by introducing the telomerase gene or the oncogene c-myc gene, as well as other oncogenes. These oncogenes should, however, be removed before the cell transplantation.
  • the best thing to do is to match the donor with the recipient cells for histocompatibility. It is almost impossible to achieve the perfect MHC (major histocompatibility complex) match and even with such a perfect MHC match immune problems due to minor antigens will occur.
  • Two solutions are readily apparent: on one hand, using a specific immunotherapy or the development of tolerance on the other hand, using autologous donor cells. Transplanting autologous cells having the same vocation or function appears for the moment the solution of choice, although the source of cells may be limited.
  • a first problem is the availability of myoblasts, because the muscles of these patients are atrophied and/or occupied by a larger than normal connective tissue and, moreover, the remaining myoblasts are senescent or near senescence.
  • the present invention relates to a new way by which other cell types such as mesenchymatous cells and fibroblasts can be converted into myoblasts, by inserting one or more members of a myogenic gene family.
  • the myoD family comprises at least the following six members: myoD, myogenin, herculin, myf-5, myf-6 and MRF-4.
  • Fibroblasts coming from a foreign donor or from the patient himself can be grown ex vivo and modified by the insertion of 1) a myogenic gene and of 2) a gene expressing a functional protein corresponding to the protein defective in the diseased subject. If the patient to be treated does not have any inherited defect in the protein of interest, the promoter of the gene encoding this protein may also be derepressed or replaced by an active one.
  • myoblasts transplantation may be used to treat muscular dystrophy, heart insufficiency, nanism, hemophilia and Parkinson disease, it will be readily appreciated that the transformation of mesenchymatous stem cells or of fibroblasts into myoblasts opens an interesting way by which a given source of tissue may be used in lieu of myoblasts for restoring a functional protein.
  • a fifth object of the invention is an alternative way by which a functional dystrophin-like protein may be restored.
  • the promoter of a dystrophin-related protein, utrophin may be mutated. The result of this mutation would be that utrophin is no longer confined to the synaptic junction, but is expressed through the whole muscle. Utrophin being closely related to dystrophin, a functional tissue is restored.
  • Heterologous and autologous transplantation may be therefore performed with genetically modified recipient cells. It is therefore possible that fibroblasts, for example, are converted into myoblasts by inserting myogenic genes, and by inserting a functional dystrophin gene or mutating the utrophin promoter.
  • the cells to be transplanted may be modified to express specific proteins. Some proteins are of course the proteins to restore for recovering a normal tissue function. Ischemia produced following occlusion of blood vessels triggers the death of many cells. In the heart, such infarctus leads to the death of many cardiomyocytes. The loss of these cells leads to atrophy of the cardiac tissue and ventricle dilatation. Transplantation of skeletal myoblasts in the heart could prevent this atrophy of the cardiac muscle as it does for the skeletal muscles. Although these skeletal myoblasts may not differentiate into cardiomyocytes, they may nevertheless form a more elastic and contractile tissue.
  • transplanted myoblasts are genetically modified to express connexin, this may permit electrical coupling between them and the cardiomyocytes and allow synchronous contraction with the rest of the heart compartment.
  • Some other proteins include trophic gene products, anti- inflammatory gene products, metalloproteases, glucosidases or inhibitor of synthesis of oligosaccharides, growth factors, hormones, coagulation factors or MHC proteins or any combination thereof.
  • the genetic modifications may include myogenic genes introduced in another cell type, which transforms the latter into myoblasts.
  • the genetic modifications can also include the expression of molecules involved in specific immunotherapy (i.e., development of specific tolerance).
  • Specific immunotherapy is the best way to avoid severe side effects and to encourage tolerance to the graft by the humoral and cellular immune system.
  • An increasing body of literature exists on the identity of cell surface determinants (stimulatory or co-stimulatory proteins and of receptors which are responsible for immune reactions). Once a specific set of antigens or receptors is identified as taking part to the graft rejection mechanisms, it is feasible to design suitable protein ligands which interfere with these cell surface determinants or their receptors.
  • Such ligands should prevent the recruitment of blood cells, inhibit their binding with the transplanted cells and the activation of blood cells which normally kills the transplanted cells.
  • the donor cells may therefore be modified to express such proteic ligands, which provides for the necessary specific immunotherapy.
  • a specific immunotherapy tolerance
  • An indication that a specific immunotherapy will work properly is already provided when considering the remarkable success achieved with an anti-LFA-1 antibody.
  • a donor cell which expresses a ligand for binding leukocytes does not necessarily have to express other foreign genes.
  • the cells expressing an anti-LFA-1 antibody may be co- transplanted with other cells expressing a metalloproteases, and with a third cell type expressing a functional tissue protein.
  • cells may also be genetically manipulated to express more than one foreign protein. A typical example of this would be fibroblasts transformed with both a myogenic gene and a functional tissue protein like dystrophin, to make a functional myoblast.
  • a mixture of different recombinant cells may be transplanted in a recipient tissue, each having a specific vocation, e.g., breaking down the connective tissue, keeping the pro-inflammatory cells away or preventing their activation, keeping the whole recipient subject tolerant to the graft, replacing a defective protein or expressing any protein of interest.
  • the present invention provides for improved methods to restore missing functional molecules in affected individuals, which restoration is greatly improved by solving each above pointed out problems.
  • transplantation of myoblasts e.g., restoration of dystrophin
  • non myogenic cells e.g., restoration of merosine
  • the invention is based on successful demonstrated restoration of these molecules (Vilquin et al., 1994 a,b, 1995a,b; Huard et al. 1991 a,b; 1992 a, b; 1993, 1994 a,b; Tremblay et al. 1991 b; 1993 a,b; Roy et al. 1993; Kinoshita et al.
  • mice affected of various hereditary diseases, in monkeys and in Duchenne Muscular Dystrophy patients see references above and Roy et al. 1991 ; Tremblay et al. 1991a; Labrecque et al. 1992; Satoh et al. 1992, 1993; Albert and Tremblay 1992; Meola et al. 1993; Sansone et al. 1993; Belles-Isles et al. 1993; Vilquin et al. 1995).
  • Dystrophic muscle fibers degenerate because of the lack of a normal gene product.
  • Cell transplantation can restore this missing gene product either in the case of myoblasts by forming new muscle fibers or forming hybrid muscle cells (i.e. by fusing with host myoblasts or muscle fibers) containing the missing gene product (e.g., dystrophin, sarcoglycans or dystroglycans).
  • the missing gene product e.g., merosin
  • Extracellular proteins may also be secreted by any other type of genetically modified cells either injected directly in the muscle or invading the muscles following their injection in the blood.
  • neutrophils and macrophages are neutrophils and macrophages.
  • extracellular proteins such as merosin
  • merosin could also be secreted around nerves or within any other tissues having damages due to the absence of this extracellular protein.
  • the same approach could also be used for any matrix protein.
  • the myogenic or non myogenic cells to be transplanted can be MHC compatible or incompatible with the host. In both of these cases and in cases where a new gene product is introduced by the transplanted cells, adequate immunosuppression will be required. This immunosuppression may be assured with pharmacological agents such as cyclosporine-A, rapamycin or FK506 (Kinoshita et al. 1994a, b; Vilquin et al. 1994, 1995a, b). The immunosuppression may also be induced by monoclonal antibodies against various lymphocytes or antigen presenting cell determinants or by genes encoding the antibodies or antibody fragments or recombinants proteins which are the receptors or part of a receptor for the binding proteins.
  • pharmacological agents such as cyclosporine-A, rapamycin or FK506 (Kinoshita et al. 1994a, b; Vilquin et al. 1994, 1995a, b).
  • the immunosuppression
  • a suitable regimen therapy using a combination of CTLA4-lg combined with an anti-CD4 mAb or combination of anti-CD4, anti-CD8 and anti-LFA-1 mAbs were both capable of increasing the number of positive cells temporarily in MHC- incompatible subjects. It is suggested that the regimen therapy should be carefully monitored in such a way that the dose is adjusted to still permit the formation of suppressor T-cells which may be responsible for the development of tolerance.
  • These immunosuppressive agents may maintain long-term graft survival and induce tolerance to the myoblasts and muscle fibers of donor origin. The immunosuppressive treatment may therefore be induced by non-specific suppressive agents or by more specific ones.
  • mAbs may eventually lead to permanent tolerance for the new antigens introduced by the cell transplantation procedure (see review by Waldman and Cobbold 1993).
  • the myogenic and the non myogenic cells may be grown and cloned using several different methods. Several examples of such methods have been described by the inventor (for human myoblast culture technique see Tremblay et al. 1991a, b; 1993a, b; Huard et al. 1992a, b). Experts in tissue culture may, however, modify these methods to reach the same purpose.
  • the cloning of cells may also be facilitated by introducing in the founder cell a gene to induce a conditional immortalization.
  • the use of cloned cells may also prevent undesired effects produced by other cell types present in primary muscle cell cultures.
  • a mouse cell line MB3 has been produced which contains the heat sensitive SV40 T antigen under control of the IFN-gamma inducible H-2 Kb promoter.
  • the MB3 cells were transfected using the retrovirus LNPOZ (kind gift of Dr. D. Miller, Fred Hutchinson, Cancer Research Center, Seattle, WA) which contains the LacZ reporter gene and a neomycin resistance gene driven by retroviral LTR (Adam et al. 1991).
  • the clone 3LN was obtained by further selection for resistance to geneticine (200 microg/ml) and beta-Gal expression, and expanded for use in transplantation experiments.
  • the survival of the beta-Gal labeled cells was assessed by dosing the beta-Gal activity (Sambrook et al. 1989) in homogenates of control muscles (obtained 3 hours after the transplantation) and in muscle homogenates obtained three days after the transplantation.
  • the method of increasing the proliferation of the transplanted cells by introducing a gamma-IFN inducible SV40 T antigen, c-myc gene or the telomerase gene may be subject to variation, as will be apparent to the skilled artisan, to reach the same goal. Animal models are applicable to other mammals including humans.
  • a PCR reaction may be made on a biopsy.
  • the messenger RNAs so amplified may have a differential restriction enzyme pattern on an electrophoretic gel. This has been shown to be the case for the dystrophin cDNA which is different in mdx mice and in C57BL/10SnJ donor mice, (Asselin et al.
  • transplanted myoblasts may be monitored byway of fluorescent latex microspheres labeled myoblasts (Satoh et al.,1993).
  • the amount of cells to be injected may depend of the type of disease to be treated, of the severity and stage of the disease and of the type of cells to be injected for the treatment. In the case of myoblasts it may be necessary to inject these cells throughout the skeletal or cardiac muscle because these cells do not diffuse very well - 1 f -
  • the distribution of the myoblasts throughout the skeletal or cardiac muscles may be facilitated by developing a device controlled by a robot.
  • This robotic device may receive from an imaging system (e.g., magnetic scanner, echography or low intensity X-rays) the information describing the exact muscle shape and size, and the position of bones, nerves and blood vessels. The robot will use this information to plan injection trajectories avoiding the major nerves and blood vessels and thus avoiding damaging them.
  • an imaging system e.g., magnetic scanner, echography or low intensity X-rays
  • the cells required to treat the muscle may also be inserted by some other routes.
  • the cells may also be distributed in the skeletal and cardiac muscles by injecting them in blood vessels of the patient and genetically modify them so that they not only attach to blood vessel cells but also cross the blood vessel and migrate in the tissue.
  • the expression of adhesion molecules or metalloproteinases may be increased by stimulating the cells with cytokines (e.g., proinflammatory cytokine, IL-1).
  • cytokines e.g., proinflammatory cytokine, IL-1
  • the secretion of these transplanted cells may also diffuse in the muscles and eventually in the blood. This could be the case for merosin or for factor VIII for example.
  • the solution to inject the cells may range from simple sterile saline isotonic solution, to sophisticated culture mediums containing growth factors such as bFGF) and/or metalloproteases to facilitate the cell survival and migration.
  • the injection solution may also contains pharmacological agents and monoclonal antibodies to prevent death of the injected cells. These solutions will have to be prepare using Good Laboratory Practice. Since one of the principal objects of this invention is to enhance the early survival rate of transplanted cells, monoclonal antibodies at least comprise an anti-LFA-l antibody (if administered directly to the patient) or an anti-CAM-l antibody fragment, alone or in combination with any anti-inflammatory agents.
  • the claimed invention can be clarified by examples of experimental treatments in animals and human patients.
  • the experiments as well as the appended figures are examples and should not be used to limit the scope of the invention.
  • FIG. 1 TGF- ⁇ 1 gene expression increased myoblast transplantation survival. Percentage of myoblast death 3 days after transplantation in Tibialis anterior muscle of CD1 mice. Transplantations were performed with CMV LacZ myoblasts (Group 1 : 8 mice), CMV LacZ myoblasts infected with TGF- ⁇ 1 retrovirus (Group 2: 10 mice) and CMV LacZ myoblasts co-transplanted with a TGF- ⁇ 1 G8 myoblast clone (Group 3: 10 mice). Each value is the mean ⁇ SEM. * : significantly different from group control (Group 1) at P ⁇ 0.05 (Student's t -test).
  • Figure 2 Products of RT-PCR amplification with TGF- ⁇ 1 primers of total RNA extracts from untransplanted TA muscle (lane b), TA muscle transplanted with CMV LacZ myoblasts (lane c), TA muscle transplanted with infected TGF- ⁇ 1 myoblasts (lane e) afetr 3 days.
  • the amplification products were digested with restriction enzyme Mae ⁇ .
  • Lane d TA muscle transplanted with CMV LacZ myoblasts.
  • Lane f TA muscle transplanted with infected TGF- ⁇ 1 myoblasts.
  • Lane a molecular weight standard.
  • FIG. 3 Decrease of neutrophil in the blood after transplantations with myoblasts expressing TGF- ⁇ 1.
  • Myeloperoxidase activity was determined in blood in control CD1 mice which were not transplanted with any myoblast (Control: 4 mice) or in CD1 mice 3 days after transplantation of myoblasts in Tibialis anterior muscle (Group 1 to 3).
  • Transplantations were performed with only normal CMV LacZ myoblasts (Group 1 : 4 mice), CMV LacZ myoblasts infected with a TGF- ⁇ 1 retrovirus (Group 2: 6 mice) and CMV LacZ myoblasts co-transplanted with TGF- ⁇ 1 G8 myoblast clone (Group 3: 6 mice).
  • Each value is the mean ⁇ SEM.
  • f significantly different from Control
  • * significantly different from Group 1 at P ⁇ 0.05 (Student's -test).
  • FIG. 4 Infection of myoblasts with TGF- ⁇ 1 retrovirus decreased the release of oxidant by neutrophils.
  • CMV LacZ myoblasts (Group 1), CMV LacZ myoblasts infected with TGF- ⁇ 1 retrovirus (Group 2) and CMV LacZ myoblasts mixed with a TGF- ⁇ 1 G8 myoblast clone (Group 3) were incubated with interleukin-1 ⁇ (IL-1 ⁇ ), loaded with C-DCF and mixed for one hour with neutrophils activated with zymosan activated serum (ZAS). The fluorescence intensity of the C-DCF was measured at different intervals after the addition of neutrophils. Each value is the mean ⁇ SEM of 6 distinct experiments. *: significantly different from Group 1 at P ⁇ 0.05 (Student's t -test).
  • Figure 5 Effect of castanospermine (1 ,5, and 10 mM) on myoblast oxidation by PMNs.
  • the positive control is myoblasts exposed to PMNs activated with Zymogen Activated Serum.
  • Figure 6 In vivo effect of castanospermine on myoblast ⁇ -gal marker activity. Myoblasts were treated with castanospermine during 24 hours prior to transplantation. Treated animals were sacrificed after 3 days. Control animals consist of animals sacrificed 1 hour after transplantation without pretreatment with castanospermine.
  • Figure 7 shows the number of muscle fibers positive for X-Gal counted after an injection of 500 000 donor's cells in one site of the tibialis anterior of recipient mice.
  • Imm 7 neo expresses neomycin.
  • Imm 7 Matrilysine expresses neomycin and matrilysine.
  • Tn I ⁇ gal untreated transgenic mouse myoblasts expressing ⁇ -Gal.
  • Tn I ⁇ Gal + TPA transgenic mouse myoblasts / ⁇ -Gal treated with phorbol ester.
  • Figure 8 Modification of dermal fibroblasts with MyoDI .
  • Expression of muscle markers, i.e. desmin (a) and MyoDI (b) is shown by immunostaining in cultures of dermal fibroblasts infected with a retroviral vector expressing MyoDI .
  • Dystrophin immunofluorescence in fused genetically modified fibroblasts d. Fusion and ⁇ -gal expression is not detected in uninfected fibroblasts (e).
  • ⁇ -gal expression in mdx mouse muscles 1 month post-transplantation of control (f) or MyoDI -expressing fibroblasts (g) and Tnl LacZ myoblasts (h).
  • No dystrophin was detected by immunofluorescence (i) on the ⁇ -gal + fibers (same fibers as in g) obtained by the fusion of the MyoDI -infected Tnl LacZ fibroblasts.
  • Hematoxylin-eosin coloration on transplanted muscle revealed the presence of abundant connective tissue (j). (a, b, h: 100x; c, d, e, f, g, 200x; i, 400x; j, 25x).
  • Myoblast transplantation is one of potential treatments of different muscular dystrophies. Myoblast transplantation in mice resulted in the expression of a reporter gene or of a lacking protein (i.e., dystrophin) in more than 90% of host fibers. These myoblast transplantations were performed either in muscle of histocompatible or immunosuppressed mice to avoid the specific immune response. Despite the control or the prevention of the specific immune response, there was an inflammatory reaction which occurred during the first 3 days after myoblast injection. Our group has recently presented evidences that this inflammatory reaction was responsible for the rapid myoblast death of up to 99% of the myoblasts observed by many laboratories.
  • a reporter gene or of a lacking protein i.e., dystrophin
  • Myoblast mortality has been previously reduced by treating the host with a monoclonal antibody against LFA-1 (CD11a/CD18).
  • LFA-1 monoclonal antibody against LFA-1
  • This protein is expressed on polymorphonuclear cells, on natural killer cells and lymphocytes and interacts with ICAM-1 present on myoblasts. Results of our group suggested that the fixation of the antibody on LFA-1 prevented the interaction of neutrophils with myoblasts and thus limited the cytotoxic activity against injected cells.
  • This very efficient method in mice will require high dose of mAb if it would be applied to DMD patients. It is therefore necessary to find a new medication to prevent inflammatory reaction before the next clinical trials.
  • a first solution is to genetically engineered the myoblasts to express and secrete anti-LFA-1 antibodies.
  • a second solution is to find other cytokines which alone or in combination will achieve an efficient anti-inflammatory effect.
  • TGF-beta1 Transforming Growth Factor-beta 1
  • TGF-beta1 is a 25 Kd cytokine produced by many cells including platelets, bone cells, macrophages and fibroblasts. This protein is a multifunctional cytokine that plays central roles in embryonic development, wound healing and particularly immunoregulation. Different studies have shown that TGF- betal has important anti-inflammatory properties. It decreases the expression of IL- 1beta and of its receptor and increases synthesis and secretion of IL-1 Ra, a natural receptor antagonist of the IL-1 receptor.
  • IL-1 Ra a natural receptor antagonist of the IL-1 receptor
  • TGF-beta1 has been shown to inhibit the transmigration of neutrophils through activated endothelium and the secretion of cytolytic product such as nitric oxide or granzyme B by polymorphonuclear and natural killer cells.
  • TGF-beta1 promotes opposite activities: applied locally, it promotes, in vivo, leukocyte adhesion, infiltration and activation. It has been shown to stimulate in vitro degranulation and oxidant release by adherent human neutrophils.
  • the aim of this study is the prevention of inflammatory reaction after myoblast transplantation.
  • the DFG retroviral vector containing human TGF-beta1 gene under control of the retroviral LTR was used to infect myoblasts before their transplantation.
  • the results show that expression of TGF-beta1 by injected cells reduced significantly their death by preventing the recruitment of PMNs and by decreasing the cytotoxic activity of neutrophils against myoblasts.
  • CMV LacZ transgenic mouse line (line 9) (generous gift of Dr K. E. Hasting, McGill University, Montreal, P.Q., Canada) was produced by pronuclear injection of a 5 Kb Xhol/Spel fragment (Cayla, Toulouse, France) including the human cytomegalovirus (CMV, Towne strain) immediate early enhancer/promoter region (-511 to +97 relative to IE1 transcription start site) driving expression of E. coli LacZ beta-gal.
  • CMV human cytomegalovirus
  • Towne strain immediate early enhancer/promoter region
  • the transgene was maintained on a CD1 mouse background.
  • Primary myoblast cultures were prepared from hemizygous transgenic 2 days old mice. After enzymatic dissociation with collagenase 0.2% and trypsin 0.25%, myoblasts were cultured in M199 medium at 37C for two days.
  • Retroviral DFG-TGF-beta1 vector encodes for recombinant human Transforming Growth Factor-betal under the control of the retroviral LTR.
  • the human TGF-beta1 gene was inserted into the MFG vector at the Ncol and BamHI cloning sites.
  • An IRES-Neo-cassette was inserted 3' to TGF-beta1 , allowing the expression of two genes from a polycistronic message.
  • Virus was prepared by transfection of BOSC23 cells.
  • This retroviral vector was directly added to myoblast culture (G8 or CMV LacZ) with polybrene (8 microg/mL) overnight.
  • CMV LacZ myoblasts were infected 3 times.
  • G8 myoblasts were infected only once but after 4 days in DMEM-FCS in presence of 400 microg/mL of G418, they were cloned by limited dilution in 96 well plates.
  • the resulting clones of G8 and primary CMV LacZ myoblasts cultures infected with this vector were grown and assayed for TGF-betal production.
  • TGF-beta1 was assayed by ELISA.
  • a 96-well flat-bottom plate was coated with human TGF-beta1 (Genzyme) for the standard curve, with culture supernatants or with mouse serum and incubated 2 hours at 37C. After 3 washes with PBS containing 0,5% Tween 20, non specific sites were blocked by PBS-milk 5% overnight at 4°C. Plates were again washed and incubated with 0.1 microg of a rabbit IgG against TGF-beta1 (R&D System inc.) for 2 hours at 37C.
  • This antibody reacts with human TGF-beta1 but also crossreacts with mouse TGF-beta1 , TGF-beta2 and TGF-beta3. Plates were washed and incubated with a goat anti-rabbit IgG coupled to biotin (1/1000) 1 hour at 37C. After 3 washes, plates were incubated with streptavidin-horse radish peroxidase (HRP) (1/500) for 1 hour at 37C. Plates were washed and 200 microL of OPD (o- phenylene-diamine-2HCI) (Kit OPD, Abbott Diagnostic) were added to each well. Reaction was developed for 15 min. at room temperature and stopped with H 2 SO 4 (2N). Plate ODs were measured at 492 nm. Results were expressed as means +/- SEM.
  • RNA isolation was performed using Trizol reagent (Gibco BRL, Burlington, Ontario, Canada). Muscles were homogenized in 1 mL of Trizol and 0.2 mL of chloroform was added to the homogenate. RNA fraction was isolated by centrifugation (12000 g, 5 min., 4°C). After precipitation with isopropyl alcohol (0.5 mL), RNA was washed in ethanol 75% and redissolved in RNAse free water. RNA was dosed at 280/260 nm, using a spectrophotometer.
  • RT-PCR was carry out using the antisense primer 5'-GCGCCCGGGTTATGCT GGTTGTA-3' and the sense primer 5'-CGGAGTTGTGCGGCAGTGGTTGA-3' producing a 449 bp sequence of mouse or human TGF-beta1.
  • 50 ng of antisense primer were added to 200 ng of total RNA sample and completed with RNAse free water. RNA was denatured at 60 °C for 5 min. and cooled for a few seconds on ice.
  • Complementary DNA was synthesized by adding 4 microL buffer 5X (Gibco BRL), 2.5 microL of dNTPs (10 mM) (Pharmacia, Baie d'Urfe, Quebec, Canada), 2 ⁇ L of 100 mM DTT (Gibco BRL), 0.5 microL of RNAse inhibitor (Boehringer Mannheim, Laval, P.Q., Canada) and 1 microL of reverse transcriptase (Gibco BRL) and incubating for 1 h at 42°C.
  • the cDNAs were amplified in a 100 microL reaction volume containing sense and antisense primers (400 ng), 10 microL of PCR buffer 10X and 0.5 microL of The polymerase (Boehringer Mannheim).
  • the PCR amplification was done at the following temperature : 94°C for denaturation, 65°C (30 cycles) for the annealing and 72°C for the extension.
  • PCR products were digested for 5 h at 55°C with the restriction enzyme Maelll (3 U/microg DNA) in a total volume of 20 microL containing 1 microL BSA (2 mg/mL), 2 microL buffer 10X completed to 20 microL with water. Only one Maelll restriction site was present in the 449 bp amplicon of mouse TGF-beta1. Thus the digestion with this enzyme produced two fragments (72 and 377 bp). Two Maelll restriction sites were present in the amplified segment of human TGF-beta1. Therefore after complete digestion of human 449 bp amplicon with Maelll, 3 fragments were obtained (72, 60 and 317 bp).
  • Myoblast transplantation Maelll (3 U/microg DNA) in a total volume of 20 microL containing 1 microL BSA (2 mg/mL), 2 microL buffer 10X completed to 20 microL with water. Only one Maelll restriction site was present in the 449 bp amplicon of mouse TGF-beta1. Thus the digestion
  • Myoblast transplantations were performed in Tibialis anterior (TA) muscle of CD1 mice (Jackson Laboratories, Bar Harbor, ME, USA). The skin of hind limb was cut to expose the TA muscle. Roughly 106 CMV LacZ or TGF-beta1 -CMV LacZ myoblasts were injected using a micropipette (Drummond Co., Broomal, PA, USA) at different sites in each muscle. For cotransplantation of CMV LacZ myoblasts and G8-TGF- betal , 10 6 cells of each type were injected. The skin was sutured with 6.0 silk (Johnson & Johnson, Peterborough, Ont., Can). Myeloperoxidase (MPO) assay
  • Myeloperoxidase enzyme activity in muscle was dosed by a modified method described by Bradley et al. Briefly, blood was diluted (1/2) in potassium phosphate buffer (50 mM, pH 6.0). After 30 sec of sonication, the solution was frozen in liquid nitrogen, thawed 3 times and centrifuged 5 min. at 12500 rpm. A solution of o- dianisidine dihydrochloride (0.0167%) and 0.0005% H 2 O 2 in KH 2 PO 4 (50 mM, pH 6.0) was added to the blood supernatant. Changes in absorbance at 460 nm were monitored with a spectrophotometer every 30 sec for 5 min. Beta-Galactosidase (beta- ⁇ al) assay
  • Muscles were homogenized in 800 microL of 0.25 M Tris-HCI pH 7.8. After centrifugation (5 min., 6500 rpm), supernatants were kept at -20°C until beta- galactosidase assay. Supernatant (100 microL) was mixed with 3 microL of a solution containing 0.1 M MgCI 2 , 4.5 M beta-mercaptoethanol, 66 microL of o-nitrophenyl- ⁇ -D- galactopyranoside (4 mg/mL) and 131 microL of sodium phosphate (0.1 M, pH 7.5). Samples were incubated 1.5 hour at 37°C. The reaction was stopped by adding 150 microL of 1 M Na 2 CO 3 .
  • optical density was read at 420 nm on spectrophotometer. Standard curve was made with a solution of beta-galactosidase extracted from Escherishia coli (Sigma, St-Louis, MO, USA). Neutrophil isolation and in vitro cvtotoxicity assay
  • Casein 8% (w/v) was injected intraperitoneally (IP) in CD1 mice. Six hours after injection, mice were injected IP with HBSS and massaged gently. Neutrophils were removed from the peritoneal cavity with a plastic pipet.
  • IL-1beta mouse interleukine-1 beta
  • C-DCF 6-carboxy-2',7' dichlorodihydrofluoroscein diacetate diacetoxymethyl-ester
  • myoblasts were incubated with neutrophils (50 neutrophils per myoblast) in presence or absence of 1% zymosan activated serum (ZAS). Oxidation of myoblasts by neutrophils was monitored with a fluorometer (excitation 485 nm, emission 530 nm) for 60 min. Results were expressed as DOD +/- S.E.M. Immunohistochemistry
  • TGF-beta1 production by myoblasts was assessed by ELISA. Culture supernatants were dosed for both types of cells (CMV LacZ and G8 myoblasts). TGF- betal production was similar for G8 and CMV LacZ myoblasts infected by DFG-TGF- betal retrovirus (respectively 171 ⁇ 30 and 191 ⁇ 3 ng/day/10 6 cells).
  • TA muscles of CD1 mice were transplanted either with 1 ) CMV LacZ myoblasts (10 6 cells) alone or 2) with CMV LacZ myoblasts co-transplanted with TGF- ⁇ 1 G8 cells (10 6 cells) or 3) with DFG-TGF ⁇ -1 infected CMV LacZ myoblasts (10 6 cells). Muscles were removed 1 hour or 3 days following transplantation, ⁇ -galactosidase activity was assayed for each muscle. The percentage of death of injected CMV LacZ myoblasts was defined as the difference between ⁇ -gal activity at 1 hour and 3 days divided by the ⁇ -gal activity at 1 hour multiplied by 100 ( Figure 1).
  • TGF ⁇ -1 expression was quantified in TA muscles by RT-PCR.
  • control transplantation of normal myoblasts in TA muscles induced only a slight activation of mouse TGF ⁇ -1 expression (lane c).
  • Digestion with the restriction enzyme Maelll produced a 377 bp fragment (lane d) characteristic of mouse TGF ⁇ -1.
  • transplantation of myoblast expressing human TGF ⁇ -1 increased markedly the expression of TGF ⁇ -1 (lane c).
  • digestion of the RT-PCR product with Maelll enzyme produced both the 317 and the 377 bp fragments (lane f). This clearly indicated the presence of two components: an endogenous mouse TGF ⁇ -1 and an exogenous recombinant human TGF ⁇ -1.
  • TGF ⁇ -1 The measurement of TGF ⁇ -1 by ELISA in the serum did not reveal any significant difference in the circulating TGF ⁇ -1 levels in mice transplanted with normal or infected CMV LacZ myoblasts (with respectively 653 ⁇ 112 ng/mL and 684 ⁇ 45 ng/mL).
  • MPO activity in blood characterized the number of circulating neutrophils. In untransplanted mice, the MPO activity was at basal level with 9.4 ⁇ 2.3 10 3 ⁇ OD/min. After transplantation of CMV LacZ myoblasts in TA muscles of CD1 mice, MPO activity in blood increased markedly up to 47.5 ⁇ 6.9 10 3 ⁇ OD/min at 3 days. This increase was related to recruitment of neutrophils following transplantation. For mice transplanted with CMV LacZ infected with the DFG-TGF ⁇ -1 retroviral vector, the increase in MPO activity in blood was lower than for mice transplanted with uninfected myoblasts (27.0 ⁇ 7.5 10 ⁇ 3 ⁇ OD/min.). Similar results were obtained for the cotransplantation of CMV LacZ myoblasts with TGF ⁇ -1 G8 myoblasts ( Figure 3).
  • Myoblast transplantation induced during the first week an inflammatory reaction in muscles.
  • the sites of cell injection were indeed infiltrated by many mononuclear cells such as neutrophils, NK cells, macrophages. This non specific response to transplantation was associated with an acute death of injected myoblasts.
  • Myoblast death occurred even following transplantation in immunosuppressed mice or following syngeneic transplantation. Neutrophils and NK cells were directly implicated in myoblast death (Merly et al.)
  • TGF ⁇ -1 the anti-inflammatory property of TGF ⁇ -1 was used to control myoblast death.
  • Myoblasts were infected with a retroviral vector containing the gene of TGF ⁇ -1. Survival of genetically modified myoblasts was increased at 3 days following transplantation and the same increase survival was obtained when normal myoblasts were co-transplanted with a G8 myoblast clone expressing TGF ⁇ -1.
  • the ⁇ -galactosidase assay measured only the survival of CMV LacZ myoblasts and not G8 myoblasts. Thus, TGF ⁇ -1 secreted by the G8 myoblasts protected the non infected CMV LacZ myoblasts.
  • TGF ⁇ -1 When applied locally, TGF ⁇ -1 have been shown to promote leukocyte recruitment and infiltration to sites of inflammation. However, when TGF ⁇ -1 was administrated systemically, it had the opposite effect, inhibiting infiltration of leukocytes.
  • human TGF ⁇ -1 was only expressed in transplanted muscles and that its level did not increase in serum. This local secretion of human TGF ⁇ -1 at the site of inflammation decreased the polymorphonuclear cell and macrophage infiltration in muscles.
  • the increased expression of human TGF ⁇ -1 in the muscle also resulted in a decreased number of neutrophils in blood (determined by MPO assay). This observation seems initially surprising since the total TGF ⁇ -1 (human ans mouse) was not increased in serum.
  • TGF ⁇ -1 reduces the release of proinflammatory cytokines in the muscle which diffuse in the blood and produce a systemic effect, i.e., reduction of release of neutrophils in the blood from bone marrow.
  • plasmid containing TGF ⁇ -1 gene Following the injection of plasmid containing TGF ⁇ -1 gene in a muscle, no infiltration with monocytes or neutrophils was observed.
  • TGF ⁇ -1 In animal model of gouty attacks, local injection of TGF ⁇ -1 in monosodium urate monohydrate crystal-induced inflammation was also associated with a decrease in leukocyte infiltration. Therefore, the effects of TGF ⁇ -1 are complex, being both pro and anti-inflammatory.
  • TGF ⁇ -1 In myoblast transplantation, the expression of TGF ⁇ -1 by myoblasts reduced the inflammatory response, in vitro, TGF ⁇ -1 inhibits the IL-8 dependent transmigration of neutrophils through activated endothelium and, in vivo, it inhibits endothelial cell adhesiveness for neutrophils. Following myoblast transplantation, decrease of leukocyte infiltration by the secretion of TGF ⁇ -1 could be due to an action directly on cell migration and/or indirectly by inhibition of chemoattractant expression in muscle.
  • TGF ⁇ -1 decreased in vitro cytotoxicity of neutrophils.
  • TGF ⁇ -1 stimulated in vitro degranulation and oxidant release by neutrophils
  • this cytokine inhibited superoxide release by neutrophils or macrophages.
  • Interaction of ICAM-1 and LFA-1 located on neutrophils induced liberation of free oxygen radicals by neutrophils and thus target cell oxidation, in our in vitro experiments, TGF ⁇ -1 decreased myoblast oxidation by neutrophils.
  • Expression of TGF ⁇ -1 could down regulate the expression of ICAM-1 on myoblasts and consecutively decrease the interaction between neutrophils and target cells, as it has been observed with E-selectin.
  • IL-1 plays a major role in the initiation and the amplification of the inflammatory response by activating a cascade of immune cells. It stimulates chemotaxis of leukocytes to the site of inflammation by inducing expression of chemotactic molecules and of adhesion molecules responsible for the migration of neutrophils and monocytes. IL-1 has been also shown to stimulate the production of nitric oxide by neutrophils. Systemic effects of IL-1 include leukocytosis (increased number of circulating mononuclear cells). Our results showed that secretion of TGF ⁇ -1 , by transplanted myoblasts in muscle, decreased neutrophil recruitment in muscles and their number in blood. This suggests that reduction by TGF ⁇ -1 of the inflammatory reaction and of the consecutive myoblast death after transplantation could be due to the inhibition of production or action of IL-1 at the transplantation site.
  • Use of genetically modified cells could be one of the possible way to release anti-inflammatory products such as TGF ⁇ -1. This procedure permits 1) a local release of anti-inflammatory molecules at the injection site, thus reducing systemic effects, 2) the use of transformed cells co-transplanted with normal myoblasts and 3) the use of cells co-transplanted with gene encoding TGF ⁇ -1 and another protein (structural, immunosuppressive and/or a complementary anti- inflammatory drug or cytokine). These genetically modified cells could eventually in future animal or human experiments be inactivated after the first week by using an inducible promoter, if indicated.
  • TGF ⁇ -1 secretion by myoblasts decreased inflammatory reaction, but did not abrogated it completely.
  • IL-1 blockade does not also completely prevent inflammation, emphasizing the importance of other inflammatory pathways.
  • TNF ⁇ an other pro-inflammatory cytokine secreted by activated macrophages, has been demonstrated to be an alternative pathway to initiate inflammatory reaction.
  • This mediator may comprise one or more of the following antagonistic ligands: IL-1 , IL-8, E-selectin, P-selectin, ICAM-1 , LFA-1 and TNF ⁇ .
  • Antagonist ligands are intended to include antisense nucleic acids, analogs or pharmacological antagonists, and antibodies.
  • the inhibition of intracellular oligosaccharide processing is a new approach to immunosuppression in allotransplantation.
  • the net effect of such inhibition is reduction in the membrane expression of some glycoproteins.
  • cell-cell interaction in allorejection may be impaired in the presence of glycoprotein processing inhibitors because the expression of key ligand-receptor pairs of glycoproteins including adhesion molecules is inhibited.
  • Grochowicz et al. (1996, 1997) found that Castanospermine, an alpha-glucosidase 1 inhibitor, reduced the membrane expression of adhesion molecules (LFA-1 alpha, LFA-1, ICAM-1), class I and class II MHC antigens and on other T cell associated molecules (CD4, CD8, CD39, CD45, W3/13).
  • Castanospermine could also inhibit inflammation through its ability to prevent the expression of adhesion molecules, which may be necessary for the capture and retention of leukocytes in the inflamed tissue (Willenborg et al. 1992). Indeed, Castanospermine inhibits the passage of leukocytes through vascular subendothelial basement membranes by inhibiting the expression of leukocyte cell surface-bound enzymes that are essential for such migration.
  • the activation of neutrophils which is responsible for myoblast death requires the interaction between adhesion molecules such as LFA-1 and ICAM-1.
  • the adhesions molecules are glycoproteins. It is possible that other unidentified glycoproteins are also involved in these cellular interactions.
  • One way of blocking these interactions involving the cell surface glycoproteins is to block the glycosylation of proteins. This can be done with several drugs, one of them being Castanospermine. Effects of Castanospermine on myoblast oxidation in vitro.
  • Myoblasts were obtained from newborn CMV-LacZ transgenic mice, expressing a beta-galactosidase gene under a CMV promoter. These myoblasts were grown in culture during two days in presence of various concentrations of Castanospermine (1 , 5 and 10 mM). The myoblasts were then stimulated with IL-1 during 6 hr. These cells were then loaded with a compound DCSF (Molecular Probes inc. ) which becomes fluorescent following oxidation. Polymorphonuclear (PMN) cells were obtained from the abdominal cavity of mice injected with casein. They were activated with ZAS (Zymogen Activated Serum). These PMNs were added to myoblasts at a ratio of 5 PMNs per myoblasts. Fluorescence was read at every 15 min. during 60 min. Results:
  • Figure 5 illustrates the results of the experiments.
  • the control is made of myoblasts not treated with Castanospermine.
  • the control fluorescence value is set at 100%.
  • the reduced glycosylation produced by Castanospermine resulted in a marked reduction of the myoblast oxidation by the PMNs. This should therefore reduce the death of the myoblast in vivo.
  • mice received with an osmotic mini pump 0.855 mg/day of Castanospermine during 4 days, starting the day before myoblast transplantation.
  • Myoblasts were grown from a muscle biopsy of CMV-LacZ mice. They were treated or not with 1 mM Castanospermine during 24 hours. Roughly 800,000 cells treated with Castanospermine were transplanted in the right Tibialis anterior muscle of 6 mice.
  • 12 control mice not treated with Castanospermine were injected with 800,000 myoblasts not treated with Castanospermine. Six of these control mice were sacrificed after 1 hour. All the other mice were sacrificed after three days. Their right side was taken and frozen immediately in liquid nitrogen. The muscles were later homogenized and the beta-galactosidase activity was determined. Results:
  • Dog myoblasts were grown in a fusion medium (MCDB with 15% FCS) with or without Castanospermine. The number of nuclei incorporated in myotubes was counted in each group. Results:
  • Metalloproteases are enzymes involved in tumor invasion and migration of cells (Stetler-Stevenson et al FASEB J. 7: 1434-1441 ; 1993). There are also involved in extracellular matrix restructuration during tissue remodelling (Gaire et al, JBC, 269, no 3 pp.1032-2040 1994). Matrilysin and Gelatinase A are metalloproteases involved in tumor invasion (Sato et al, FEBS Letters, 385 (1996) 238-240). Gelatinase A have been associated with muscular degenerescence and regeneration (Kherif et al, Colloque sur les maladies neuromus vide lymph).
  • Gelatinase A induces cell migration by cleaving laminin-5, exposing a pro-migration kryptic site (Giannelli et al, Science 97 (277) pp.225-228).
  • concanavalin A a lectin that stimulates the expression of metalloproteases
  • TPA a phorbol ester
  • MMP-7 matrilysin
  • myoblasts which express matrilysin (MMP-7) as one example of a useful metalloproteases, which myoblasts are also conditionally immortalized by the expression of a thermosensitive mutant of SV40 large T antigen.
  • MMPs are involved in the normal tissue turnover and remodulation during growth and development.
  • ECM extracellular matrix
  • proteases such as MMPs or protease activators such as urokinase at their leading edge, where complex proteolysis can direct migration, preserve ECM attachment, and avoid unwanted tissue damage. It is still unclear whether proteases remove physical barriers or if they modify the ECM in such a way that it is suitable for migration.
  • metalloproteinases are found in abundance in their inactive precursor form in normal tissues and fluids, suggesting that their activities are limited by physiological activation rather than by expression alone (37). These enzymes are made of common domains and fall into four main groups called the collagenases, gelatinases, stromelysins and membrane-type metalloproteinases. Their catalytic mechanism depends on a zinc ion bound at the active center; each enzyme is secreted with a propeptide which is removed proteolytically before the enzyme becomes active.
  • MMP-7 Matrilysin
  • PUMP-1 putative metalloproteinase
  • MMP-7 degrades type IV collagen, laminin-1 , fibronectin, proteoglycan, type I, III, IV, V gelatin and insoluble elastin. It is also known to activate collagenase (MMP-1) to its fully activated form. MMP-1 degrades type I, II, III, VII, X collagen and gelatins.
  • MMP-7 can be purified in its inactive zymogen form of 28 kDa and as an active species of 19 kDa. It lacks the C-terminal domain common to all other metalloproteinases and is therefore the smallest.
  • the extracellular matrix has a profound influence on muscle morphogenesis. Although damaged muscle fibers regenerate from a resident population of satellite cells that are wedged between the basal lamina (BL) and the muscle fiber, these satellite cells apparently penetrate poorly the BL unless it is mechanically damaged or proteolytically digested. Laminin, fibronectin, proteoglycan, collagen I, III, IV, V, VI are all associated with the muscle fiber BL.
  • the BL limits the growth and migration of activated satellite cells, orients the regeneration of new muscle fibers and could be involved in repressing satellite cell mitosis and differentiation in the absence of damage.
  • the aim of the present study was to investigate the effects of constitutive MMP-7 expression in mouse myoblasts on the migratory and fusogenic potential of these cells in the muscle tissue.
  • the parental Immortomyoblast cell line clone (clone #7) was derived from muscle biopsies of a newborn transgenic TnlLacZ immortomouse.
  • the TnlLacZ immortomouse was obtained by interbreeding H-2k -tsA58 transgenic mice with TnlLacZ transgenic CD1 mice.
  • H-2k b -tsA58 transgenic mice harbor a thermolabile immortalizing gene (the tsA58 mutant of SV40 large T antigen) under the control of an inducible promoter (H-2k b ).
  • the expression of SV40 large T antigen is induced through application of interferon gamma (INF-g).
  • Immortomyoblast clones are grown in DMEM Low Glucose medium containing 15% Donor Calf Serum (Gibco), 2% Chick Embryo (Gibco/BRL, Burlington, Ontario, Canada), 20 U/mL interferon gamma (INF-g), 1% (10 000 U/mL) penicillin, 1% (10000 U/mL) streptomycin in 8% CO 2 at 32°C in permissive conditions for the expression and function of the tsA58 gene product.
  • DMEM Low Glucose medium containing 15% Donor Calf Serum (Gibco), 2% Chick Embryo (Gibco/BRL, Burlington, Ontario, Canada), 20 U/mL interferon gamma (INF-g), 1% (10 000 U/mL) penicillin, 1% (10000 U/mL) streptomycin in 8% CO 2 at 32°C in permissive conditions for the expression and function of the tsA
  • Immortomyoblast clones were grown in DMEM High Glucose medium containing 15% Donor Calf Serum (Gibco), 1% (10 000 U/mL) penicillin, 1% (10 000 U/mL) streptomycin in 5% CO 2 at 37°C for non-permissive conditions.
  • Human myoblasts expressing normal dystrophin were obtained from fresh biopsies of patients as previously described and grown in MCDB120. Human primary culture myoblasts from a 12-year-old female and cultures from a cloned human myoblasts isolated from a 13-month-old patient were used for co- transplantation studies. Plasmids and cell transfection.
  • the matrilysin expression vector pbAprMUTM and the control vector pRSVneo were transfected using Lipofectamine (GIBCO/BRL).
  • pbAprMUTM contains the neomycin selectable marker gene and a mutated form of the human matrilysin cDNA driven under the human beta-actin promoter.
  • pbAprMUTM was provided by J.D. Knox and is derived from the eucaryotic expression vector pH-b-APr-neo-1.
  • pRSVneo contains the neomycin selectable marker gene under the RSV promoter.
  • Immortomouse myoblasts derived from the pbAprMUTM transfected cells were designated 7mat and pRSVneo transfected myoblasts 7neo, while the parental cell line was called clone #7. In vitro behavior of the Immortomyoblast clones.
  • mice Six SCID and 20 Balb/C mice (all females between 5 and 8 weeks old) were used as hosts for these experiments. Both Tibialis anterior (TA) were used in Balb/C mice for the implantation of Immortomouse myoblasts pellets (clone #7, 7mat or 7neo), each containing 5x10 5 myoblasts. SCID mice were used for the transplantation of mix pellets containing 1x10 6 human primary culture myoblasts and 5x10 5 Immortomyoblasts (7mat, 7neo or none). The day of transplantation, the myoblasts were detached using 0,125% trypsin and washed three times in HBSS. These cells were kept on ice until transplantation.
  • TA Tibialis anterior
  • Pellets were obtained by centrifugation at 6500 rpm for 5 minutes. The skin was opened to expose the TA muscle and the myoblast pellets, resuspended in 10 mL of Hank's balanced salt solution (HBSS), were slowly injected into the belly of the TA along the rostrocaudal axis. A 1/2 cc U-100 Insulin Syringe (Becton Dickinson, NJ) was used for these injections, applying low pressure while the muscle was examined for leakage. The skin was then closed with fine sutures. FK 506 (Fujisawa Pharmaceuticals Co. Ltd, Osaka, Japan) was administered daily at 2.5 mg/kg to immunosuppress Balb/C mice. Muscle biopsy and preparation.
  • HBSS Hank's balanced salt solution
  • mice 21 days after myoblast transplantation, mice were deeply anesthetized with 0.15 mL of a solution containing ketamine (10 mg/mL) and xylazine (10 mg/mL). They were then killed by intracardiac perfusion with 0.9% saline containing heparin (2 lU/mL). The TA muscles were dissected out and immersed in 30% sucrose solution at 4°C for 12 to 18 h. The specimens were imbedded in OCT and frozen in liquid nitrogen. Serial cryostat cross sections (12 mm for immunochemistry and 25 mm for X-Gal staining) of the muscles were thawed on gelatin-coated slides. Reverse transcriptase polymerase chain reaction (RT-PCR).
  • RT-PCR Reverse transcriptase polymerase chain reaction
  • a DNAse treatment was performed to avoid contamination by the cDNA from the vector in the PCR reaction.
  • the reverse transcription was performed at 42°C for 1-hr using MMLV reverse transcriptase (BRL) on 500 ng of sample RNA as described previously.
  • the cDNA was amplified in a Perkin Elmer Cetus Thermal cycler using Tth DNA polymerase (Boehringer Mannheim, Montreal, Canada). Multiplex amplification of the cDNA was performed, using the beta-Actin cDNA as an internal control for the amplification of the human matrilysin cDNA.
  • Primer sequences for mouse beta-Actin cDNA were as follows : sense, 5'-GTGGCGCGCTCTAGGCACCAA-3'; antisense, 5'- CTCTTTGATGTCACGCACGATTTC-3' as previously described.
  • Primer sequences for the human matrilysin were designed to amplify a 397 bp fragment from the cDNA according to the published sequence (GenBank/EMBL data bank accession number: L22519) and were as follow: sense, 5'-CAGATGTGGAGTGCCAGATG-3'; antisense, 5'-CCAAGTTCATGAGTTGCAGC-3'.
  • the samples were amplified for 30 cycles with the following parameters: 94°C, 1 min.; 60°C, 1.5 min.; 72°C, 2 min.
  • the primers amplified a 540 bp (b-Actin) and 397 bp (matrilysin) fragments. Immunohistochemistry and ⁇ -Galactosidase staining.
  • NCLDys3 Human dystrophin was detected using the NCLDys3 monoclonal antibody which does not react with mouse dystrophin.
  • the sections were incubated after TBS/BSA rinses respectively: 1.5 h with NCLDys3 (dilution 1 :10; Novocastra, New Castle Upon Tyne, UK), 1 h with a rabbit antimouse antibody biotin conjugate (dilution 1 :250; Dako) and 1.5 h with streptavidin Cy3 (dilution 1 :500; Sigma, St. Louis, Mo).
  • ⁇ -Gal expression in muscle fibers was detected on slides containing 25 mm thick cross sections of TA muscles and myotubes (in cultured cells) following a 5 min. fixation in 0.25% glutaraldehyde and staining in 0.4 mM X-gal (Sigma) at room temperature in a dark box overnight (12-18h). Image and statistical analysis.
  • RT-PCR products densitometry was performed with NIH Image analysis software, using molecular weight marker VI (Boehringer Mannheim) as internal standard for curve fitting. Significant differences were evaluated using an analysis of variance (ANOVA) followed by a Fischer's PLSD test on a Stat View 512 software (Brain Power, Calabasas, CA) with a level of p ⁇ 0.05 being considered significant.
  • ANOVA analysis of variance
  • Fischer's PLSD test on a Stat View 512 software
  • the transfection on the parental cell line, the Immortomyoblast clone #7, with pbAprMUTM vector generated 13 Geneticin resistant colonies (7mat).
  • the transfection with pRSVneo yielded 8 Geneticin-resistant control clones (7neo).
  • the resistant transfectants of each type were pooled together to avoid drifting from the parental cell line.
  • Myoblast cultures obtained by the transfection of the matrilysin expression vector (7mat) and the control vector (7neo) were observed and compared with the parental cell line (clone #7). None of the stably transfected cell lines exhibited any significant modifications in their doubling time compared to clone #7 once the geneticin selection was stopped.
  • FIG. 7 also shows that transgenic mouse myoblasts transfected with Tnl ⁇ -gal had a significant increase in the ⁇ -gal positive muscle fibers after activation with phorbol ester. The same results are observed with concanavalin A (not shown). These agents stimulate the production of metalloproteases. The diffusion of transplanted cells is better (3 to 4 fold increase) with concanavalin A that with MMP-7 probably because other metalloproteases are produced in response to concanavalin A. Co-transplantation of matrilysin expressing Immortomyoblasts with primary human myoblast cultures in SCID mice.
  • matrilysin expressing myoblasts were co-injected with human myoblasts. This has also given us the opportunity to evaluate the potential of matrilysin to increase graft success for cells of a human origin.
  • matrilysin is able to promote graft success in this system.
  • the increase in graft success by matrilysin expressing cells could be explained by numerous potential mechanisms divided in three groups.
  • the localized degradation of the ECM surrounding the injected cells could stimulate myogenesis.
  • Myoblasts are normally anchored to the ECM through integrins. Therefore the degradation of the ECM could increase cell-cell or cell-fiber interactions. Myoblasts could penetrate more easily through the basal lamina of the host muscle fibers and generate more hybrid myotubes following the partial degradation of the ECM.
  • meltrin-alpha a metalloprotease-desintegrin found on myoblast surface, was shown to confer increased fusogenic properties and to be essential for myotube formation. Thus, it is likely that muscle cell fusion involves metalloproteinase-sensitive mechanisms, as previously suggested.
  • an increased amount of transduced fibers could result from the increased migration of transplanted myoblasts following the degradation of the ECM.
  • the matrilysin activity might diminish the physical impairment of the ECM, therefore augmenting the diffusion area covered by the grafted cells.
  • the modification of the ECM in a structure promoting the migration of the myoblasts over their quiescent embedding into the matrix could also explain increased cell diffusion.
  • matrilysin activity is not restricted to the extracellular matrix.
  • MMPs are involved in the processing of TNF-alpha, heparin-binding epidermal growth factor, and Fas ligand. Therefore it has been speculated that matrilysin may activate lumenal or membrane-bound cytokines or growth factors, such as HGF or FGFs, to perturb locally the growth of the responding cells. Because of the complex interactions between the grafted myoblasts and the living tissue, we cannot clearly evaluate the relative importance of the potential mechanisms listed above.
  • the substrates may be used directly in the injectable compositions, or cells entering the compositions may be genetically engineered to produce the substrates (and metalloproteases, if necessary), during their in vitro culturing and/or their in vivo transplantation.
  • matrilysin in the surroundings of grafted cells could prove to be of significant help, not mainly in helping the migration of the myoblasts but in increasing the regenerating myogenic capacity of myoblasts after multiple passages.
  • the use of metalloproteinase transgenes or ECM degrading enzymes and/or their substrates could possibly reduce the efficiency gap observed between freshly isolated and multiple passage myoblasts.
  • compositions of this invention therefore include one or more of gelatinase A (MMP-2), gelatinase B (MMP-9), coliagenase-1 (MMP-1), collagenase-2 (MMP-8), collagenase-3 (MMP-13), collagenase-4 (MMP-18), stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), stromelysin-3 (MMP-11), metalloelastase (MMP-12), matrilysin (MMP-7), MT1-MMP (MMP-14), MT2-MMP (MMP-15), MT2-MMP (MMP-16), MT3-MMP (MMP-16) and MT4-MMP (MMP-17). They may also include inducers like concanavalin A, phorbol esters and urokinas
  • the donor myoblasts may be grown from a muscle biopsy of an histocompatible or an histo-incompatible mouse.
  • the host may be immuno-suppressed with cyclosporin, but more effective immuno-suppression may be obtained with rapamycin, FK506 (Kinoshita et al. 1994; Vilquin et al. 1994, 1995) or monoclonal antibodies against molecules located on the membrane of lymphocytes or of antigen presenting cell, as described above.
  • Myogenic cells have been shown to be able to form new or hybrid (containing donor and host nuclei) muscle fibers in monkeys.
  • the injected myoblasts can be obtained either from the animal itself or from another animal. In both cases, the host has to be adequately immunosuppressed. Immunosuppression is required at least for a short term because foreign antigens are present in the culture medium.
  • This experiment demonstrated that new muscle fibers can be formed in a primate.
  • the injected cells were labelled with a retrovirus vector containing the ⁇ -galactosidase gene. The muscle fibers formed partially or totally by the injected myoblasts expressed that gene demonstrating that new genes can be effectively introduced in muscle cells of primates by myoblast transplantation.
  • Myoblasts originate from primary myoblast cultures obtained from muscle biopsies taken from two monkeys approximately two weeks before the transplantation. Biopsies were performed in the quadriceps femori by opening the skin and aponeurosis and exposing the belly of the muscle under general anesthesia. A biopsy of approximately 0.5 cm 3 was taken and thereafter the muscle, the aponeurosis and the skin were closed separately with absorbable sutures.
  • the biopsies were dissociated as in the routine, cells were cultured in MCDB supplemented with 15% of bovine calf serum and infected three times with the retrovirus LNPOZ, containing a ⁇ -galactosidase reporter gene. The final percentage of cells expressing the ⁇ -galactosidase was approximately 30%.
  • All the transplantations were allogeneic.
  • the number of cells transplanted by site were 4x10 6 (one site) 8x10 6 (two sites) and 24x10 (One site).
  • the final pellets of cells were resuspended in 100 ⁇ l of HBSS (4x10 6 , 8x10 6 and 24x10 6 cells) or in 100 ⁇ l of notexin diluted 5 ⁇ g/mL in HBSS (8x10 6 cells).
  • the FK506 was increased to 0.3 mg/kg 10 days after the transplantation, taking into account that cell infiltration was seen in the other two monkeys when the first biopsies were analyzed.
  • the sites grafted were biopsied under general anesthesia in the same way as it was described for the culture of cells.
  • the biopsies of muscles were placed in sucrose 30% overnight at 4°C and thereafter frozen in liquid nitrogen. Sections of 30 ⁇ m and 10 ⁇ m were performed in a cryostat at -25°C.
  • Hematoxylin-eosin stain was performed to show the general histology of the grafted muscle (eventual presence of cell infiltration and central nucleus as evidence of regeneration). ⁇ -Gal in transplanted cells was revealed. Hematoxylin-eosin slides were mounted in Permount and X-Gal slides mounted in a glycerin-gelatin medium. Results:
  • EXAMPLE 5 Transplantation SV40 of large T antigen - immortalized myogenic cells
  • the C57BL/6J dy/dy mice (Jackson Lab.) were used as recipients for myoblast transplantation.
  • the C57BL/6J +/+ normal newborn mice (Jackson) were used as compatible donors for some myoblast transplantations.
  • These strains are used to study the effect of the marker LacZ gene.
  • the marker gene is an indicator of transformation success.
  • the protein may be, factor VIII, merosin, dystrophin, connexin, etc. depending on the disease to cure.
  • the transgenic Tnl-LacZV29 mice (Tn-LacZ, gift from K. Hasting, McGill University, Montreal, Canada) contain the LacZ gene under the control of the quail fast troponin I promoter, thus, differentiated muscle cells express a cytoplasmic ⁇ - galactosidase ⁇ (-gal) protein (Hallauer et al., 1993; Kinoshita et al., 1994a,b). ⁇ -gal expression is not restricted to the nucleus in this model. Both male and female parents are heterozygous animals, and newborn mice were used as donors for some immunologically noncompatible myoblast transplantations.
  • thermolabile tsA58 mutant of SV40 large T antigen under the control of the H-2K b promoter (Jat et al., 1991).
  • Interferon y IFN- ⁇
  • the thermolabile protein is functionally active at 33°C but not at 39°C.
  • Offsprings were all heterozygous for the H-2Kb-tsA58 transgene and some were heterozygous for the Tn- LacZ transgene. Thus, newborns were tested for ⁇ -gal expression, and only ⁇ -gal positive animals were used for the establishment of myogenic cell lines (see below). Primary cell cultures
  • Mouse primary myoblast cultures were obtained from biopsies from newborn skeletal muscle as previously described (Vilquin et al., 1995c). The cell suspension was cultured in 199 medium (Gibco, Grand Island, NY) supplemented with 15% FBS (Gibco) and antibiotics. Cells were harvested at 70% confluence, that is, 2 days after plating, either for immediate grafting or for freezing until grafting. These cultures were not pure and contained several cell types, with 30% to 40% being committed myoblasts as assessed by desmin immunostaining (personal results).
  • mice transplanted with these batches have been gathered under the letters A, B ( ⁇ -gal cells), and H (histocompatible cells).
  • A, B ⁇ -gal cells
  • H histocompatible cells.
  • the myogenicity of colonies and clones was assessed by desmin immunostaining and by the ability to fuse and form myotubes in vitro. Fusion was obtained by reducing the FCS concentration to 5% and growing the cells at 37°C in 5% CO 2 in the absence of IFN- ⁇ .
  • the muscle cells isolated as colonies or single clones from newborn muscles of (Tn-LacZ) X (H-2K b -tsA58) mice were able to grow and proliferate at 33°C in a 10% CO 2 atmosphere when stimulated by murine IFN- ⁇ .
  • the muscle cells When shifted to differentiation medium the muscle cells fused together and formed giant myotubes.
  • These cells either myotubes or myoblasts alone, expressed the intracellular filament desmin, which is an early marker of myoblasts (Lin et al., 1994).
  • These cells were able to fuse and to form new or hybrid muscle fibers expressing ⁇ -gal in vivo in immunodeficient SCID mice without forming solid tumors.
  • the colony # 24 (two cells in the original cloning well) and the clones MB3, MB7 and MB27 were selected based on their in vitro myogenic characteristics and were injected into d
  • HBSS Gibco
  • TA Tibialis anterior
  • the left TA of the mouse received only myogenic clones, while the right TA received both myogenic clones and a histocompatible primary muscle cell culture (i.e. obtained from normal C57BL6J+/+ newborn mice).
  • the TA muscles were collected and immersed in a sucrose solution (Tremblay et al., 1993). Muscles were embedded, frozen in liquid nitrogen, and serially sectioned at 8 ⁇ m. Adjacent serial sections were thus spaced by 8 ⁇ m, while the series of sections were separated by 180 ⁇ m. -gal histochemistry
  • Newborns originating from Tn-LacZ mice were tested for ⁇ -gal expression using the X-gal reagent, except that no fixation was necessary and incubation of a small muscle piece was performed at 37° C for 1 h.
  • Muscle sections were fixed in acetone at -20°C for 10 min. then nonspecific Ig binding was blocked with 10% FBS in PBS for 30 min.
  • the rabbit polyclonal anti- mouse LAMA2 (Xu et al., 1994a) was used 1/300 in PBS containing 1% FBS for 2 h at 37°C.
  • the second antibody was a biotinylated goat anti-rabbit Ig (1/100 in PBS containing 1% FBS for 1 h; Dako, Copenhagen, Denmark).
  • the next step was incubation with streptavidin-HRP or streptavidin- FITC in some cases (1/200 in PBS containing 1% FBS; Dako).
  • Conditionally immortalized myogenic cells were grown on 2% gelatin-coated plates and allowed to fuse in 5% FBS at 37°C. The cells were fixed and permeabilized with methanol at -20°C . Nonspecific binding was blocked using 10% FBS in PBS for 30 min. The cells were incubated with a mouse anti-desmin antibody (1/50 in PBS containing 1% FBS for 1 h; Dako). The second antibody was FITC-conjugated rabbit anti-mouse IgG (1/100 in PBS containing 1% FBS for 1 h; Dako).
  • LAMA2 was absent from muscles of dyldy mice (Arahata et al., 1993; Sunada et al., 1994; Xu et al., 1994a, b) and was never expressed in muscles only injected with HBSS. LAMA2 surrounded normal mouse muscles. LAMA2 was expressed around some muscle fibers following Tn-LacZ primary muscle cell culture transplantation. The total and relative numbers of LAMA2-positive fibers, however, depended on the age of the mouse at transplantation and on the pretreatment of the muscle . The percentage of LAMA2-positive fibers was low (mean ⁇ SD, 6.4 ⁇ 4.4) when older animals (i.e., more than 3 months), were used as recipients.
  • the percentage of LAMA2-positive fibers was higher (15.9 ⁇ 9.0) when younger animals (i.e., 6 week old) were used as recipients. Notexin alone, or Y- irradiation alone, did not increase the number of LAMA2-positive fibers. The combination use of notexin and ⁇ -irradiation increased the percentage of LAMA2- positive fibers in transplanted muscles (27.8 ⁇ 14.9). There was an overall but not significant increase in the percentage of LAMA2-positive fibers with the duration of the experiment (the mice in group A all received the same myoblast preparation). Colocalization of ⁇ -gal and LAMA2
  • the Tn-LacZ primary muscle cultures originate from mice with normal LAMA2 expression.
  • both the normal gene for LAMA2 and the reporter gene coding for ⁇ -gal are present in the donor cells.
  • LAMA2 and ⁇ -gal were characterized on serial sections only spaced by 8 ⁇ m. The percentages of LAMA2-positive and ⁇ -gal positive fibers were comparable when old animals (more than 3 months) were used for transplantation. LAMA2 and ⁇ -gal labeling were localised in the same clusters of fibers. Some LAMA2-positive fibers, however, were negative for ⁇ -gal expression, and some ⁇ -gal positive fibers were negative for LAMA2 expression on serial sections. Most of the smallest fibers were ⁇ -gal positive. These results indicated that ⁇ -gal and LAMA2 expression were not regulated similarly, or that the same type of cells were not responsible for their expression.
  • LAMA2 seemed to have spread centrifugally from the center of the injection site to its periphery. While LAMA2 completely surrounded normal mouse muscle fibers, its localization was frequently incomplete and disrupted in transplanted dyldy muscles, especially in case of the largest fibers. Thus, LAMA2 deposition seemed to be localized. LAMA2 deposition also presented important variations in transplanted mice as compared to normal mice. Most of the smallest fibers were totally surrounded by LAMA2. Overall transplantation success
  • LAMA2 which is the only known difference between dyldy host mice and +/? donor mice, is not sufficient to trigger efficient acute rejection of LAMA2- expressing cells. Chronic rejection was not assessed in this study, but the overall highest numbers and percentages of LAMA2-surrounded fibers were observed 11 weeks after grafting. LAMA2 is undetectable in immunohistochemistry but very low amounts of LAMA2 mRNA are detected in untreated dyldy mice by RT-PCR (Arahata et al., 1993; Xu et al., 1994a). These small amounts of LAMA2 would be sufficient to make the animals tolerant to their self antigens and to avoid immunological rejection of the transplanted LAMA2.
  • dystrophin-positive fibers are frequently obtained in only 2 months following histocompatible primary muscle culture transplantation in mdxlmdx mice (Vilquin et al., 1995c), whereas it is difficult to obtain more than 300 to 400 laminin-2- surrounded muscle fibers in the same time in dyldy mice.
  • the dyldy muscle fibers are smaller than normal muscle fibers and they show important size variation within a single muscle. This may reflect trophic problems in dyldy muscles related to the absence of LAMA2 in basal lamina that could explain the variation in efficiency between the mdxlmdx and the dyldy models.
  • the initial lack of LAMA2 could hamper myoblast migration or alignment either with other myoblasts or with host muscle fibers, because basal lamina is known to play important roles in the control of migration, proliferation and differentiation of various cell types (Engvall et al., 1992).
  • an impaired muscle innervation due to the absence of LAMA2 around peripheral nerve fibers or neuromuscular junctions could induce a muscle atrophy.
  • the myoblast proliferation and/or migration could be impaired by the abundant connective tissues, present especially in the oldest dyldy mice. Taken together, these problems may explain why muscle regeneration was relatively poor following transplantation, whatever the pretreatment used.
  • LAMA2 and ⁇ -ga/ colocalization after transplantation of transgenic primary cells were generally more widely distributed than ⁇ -gal. This could indicate differences in the regulation of expression of these two proteins, that LAMA2 but not ⁇ -gal is diffusible, or that the cell type responsible for LAMA2 expression is not the cell type responsible for ⁇ -gal expression, ⁇ -gal expression is restricted to skeletal muscle cells as an intracellular protein, and its nuclear domain is about 1000 ⁇ m.
  • laminins and likely LAMA2 are secreted outside of the cells, and may have a relatively long half-life in vivo (Engvall, 1993) as components of muscle cells basal lamina. Thus, the fate of ⁇ -gal and LAMA2 in vivo is probably different. Whereas ⁇ -gal expression requires myoblast fusion and formation of hybrid or new muscle fibers, the developmental mechanisms of LAMA2 expression are still unclear. LAMA2 expression pattern
  • LAMA2 generally deposited continuously around small caliber muscle fibers, whereas this deposition was frequently discontinuous around normal diameter muscle fibers, even two months after transplantation.
  • dystrophin was expressed along the complete inner circumference of muscle fibers by some weeks after transplantation, and dystrophin disruption was rarely observed one or several months after transplantation in the mdxlmdx or SCID mouse models (Partridge et al., 1989; Morgan et al., 1990, 1993, 1994; Huard et al., 1994; Kinoshita et al., 1994b; Vilquin et al., 1995c).
  • the expression of LAMA2 seemed to be centrifuge relative to injection sites.
  • transplantation models and results presented above should prove useful to study in vivo interactions between muscle cells and extracellular matrix, and interactions or coordinate regulations between nerve and muscle cells. These models should allow to study the regulations of extracellular matrix protein interactions in vivo.
  • the critical steps for normal and pathologic muscle regeneration, that is, myoblast proliferation, alignment or fusion, or the stability of nerve-muscle regulations, should be discriminated.
  • LAMA2 extracellular matrix protein
  • fibroblasts fibroblasts, macrophages or neutrophils genetically modified to secrete an extracellular matrix protein.
  • Long-term experiments should also be designed to further explore the feasibility of myelination restoration in the peripheral nervous system of the dyldy mouse by these genetically modified cells (Bradley and Jenkison, 1973; Madrid et al., 1975; Montgomery and Swenarchuk, 1977).
  • another protein like merosin is secreted by non-myogenic cells, which stabilizes the muscle fibers.
  • Dystrophin Proteins of interest to be expressed as restored structural proteins: Dystrophin:
  • Myogenic cells may be transplanted to restore the expression of dystrophic mdx dystrophic mice
  • the mdx mice are an animal model of Duchenne muscular dystrophy (Bulfield et al. 1984). As the human patients, this animal model lacks dystrophin due to a mutation of that gene located in the X-chromosome (Sicinski et al. 1989). The absence of this protein under the sarcolemma lead to muscle fiber degeneration and to eventual muscle weakness (Pastoret and Sebille, 1995). The development of this disease may be prevented by transplanting normal myoblasts which restore the expression of dystrophin in the muscle fibers. Merosin:
  • the dyldy dystrophic mice are an excellent model of Congenital Muscular Dystrophy (Arahata et al. 1993; Sunada et al. 1994, 1995; Xu et al. 1994 a,b).
  • the expression of merosin can be eventually restored by the transplantation in the muscle of dyldy non-myogenic cells. Since this protein is present in the extracellular matrix it has to be secreted by the cells injected either directly in the muscles or injected in the blood and migrating in the muscles or in any other affected tissues, e.g., the peripheral nerves in the case of the dyldy mice to restore the expression of merosin in the extracellular matrix surrounding the muscle fibers.
  • the presence of merosin in the extracellular matrix stabilizes the muscle fibers and helps recovering a normal muscle function while the presence of merosin near the nerve could restore a normal propagation of action potentials.
  • proteins of interest that can be expressed in transplanted myoblasts or other cell types exist.
  • the cytoplasmic restoration of glucose-6-phosphate dehydrogenase has been described in stable hybrid myotubes (Sansone et al.,1993; Meola et al.,1993). Therefore, the present invention applies at least to different types of myopathies of a genetic origin or other origins. Since other types of cells (neurons, islets, hepatocytes, macrophages and neutrophils) may be transplanted to cure diseases like Huntington, Parkinson diseases, diabetes and liver diseases, it will be readily appreciated that the present invention applies to all types of diseases that may be cured by transplanting healthy donor cells.
  • Duchenne muscular dystrophy is an X-linked recessive disease characterized by the absence of dystrophin, a protein located beneath the sarcolemma of muscle fibers. Transplantation of normal myoblasts into dystrophic muscles is a potential therapy to compensate the lack of dystrophin expression in muscles of DMD patients.
  • DMD Duchenne muscular dystrophy
  • Skeletal muscles are derived from the dorsal portion of somites, the dermomyotome, which is the source of muscle precursor cells and dermal fibroblasts of the dorsal region of the body. Proliferating myoblasts migrate from the dorsomedial region of the dermomyotome to form the myotome which will become the first differentiated skeletal muscle formed in the embryo. Mononuclear myoblasts, the precursors of skeletal muscle fibers, differentiate into myocytes under the control of a group of myogenic regulatory genes. Those myocytes fuse together to form multinucleated myotubes. MyoD1 is a master regulatory gene for skeletal myogenesis.
  • the product of this gene a nuclear phosphoprotein, contains a basic-helix-loop-helix motif and a c-myc homology domain in a 68-amino acids region. This latter region is both sufficient and necessary to engage a variety of non-muscle cells into the myogenic lineage.
  • MyoD is a DNA-binding protein that binds to the enhancer sequences of a number of muscle specific genes. Furthermore, it has been shown to activate its own transcription. This autoactivation could provide a positive feedback loop to keep cells engaged in the myogenic pathway following MyoD activation or a control mechanism to increase MyoD levels once the gene has been activated.
  • MyoDI transcription factor 1
  • myf-5 transcription factor 2
  • herculin transcription factor 3
  • All of these factors show a large degree of homology with the MyoDI gene, but in at least some cases MyoDI alone has been shown to activate the muscie-program in a variety of differentiated cell lines.
  • the introduction in dermal fibroblasts of the retrovirally encoded MyoDI gene has been shown to convert these cells to myoblasts under specific conditions.
  • MyoD expression is repressed by trans-acting factors.
  • the forced expression of this gene with a retroviral vector could down-regulate the endogenous program of these non-myogenic cells and permit the conversion to myogenic program.
  • the use of dermal fibroblasts to eventually complement the dystrophin deficiency into dystrophic muscle is interesting because these cells are not functionally compromised by DMD and thus retain a high proliferative potential.
  • the formation of normal muscle fibers in DMD patients could result from the graft of their own fibroblasts transduced with a myogenesis regulatory gene, an hypothesis first proposed by Tapscott et al.
  • mouse dermal fibroblasts can be induced to express muscle markers, are able to form myotubes in vitro and fuse with host muscle fibers in vivo once infected with the retroviral vector containing MyoDI cDNA.
  • Transgenic Tnl LacZ1/29 mice contain the ⁇ -galactosidase cDNA under the control of the quail fast troponin I promoter.
  • Primary fibroblast cultures were established from the dermis of newborn transgenic mice identified by the X-gal histochemical staining (Boehringer Mannheim Canada, Laval, Can.) of a small muscle biopsy. Dermal fibroblasts were isolated with a modified technique described elsewhere (32). Precisely, the skin of hind limbs was minced into 0,5 cm2 pieces and then digested in 0,25% trypsin (GIBCO BRL, Canada) overnight at 4°C.
  • the retroviral vector pLMDSN containing the MyoD coding region under the MoMLV LTR promoter and a neomycin resistance gene under the SV 40 early promoter was used to create the amphotrophic retrovirus PA317 LMDSN producing cell lines (generously donated by S. J. Tapscott from the Hutchinson Cancer Research Center). These PA317 LMDSN cells were grown in Dulbecco's modified Eagle's medium with high glucose and with 10% fetal calf serum. The culture supernatant was harvested after an overnight exposure to a confluent layer of PA317 LMDSN and stored at -70 °C.
  • Infected fibroblasts were grown to confluence on Labtek slides (Nunc) in DMEM high glucose (GIBCO BRL, Canada) containing 10'c horse serum. These conditions were maintained for 10 days to allow sufficient cell fusion. Non-infected dermal fibroblasts were used as control.
  • X-Gal staining Cells were fixed on Labtek slides with glutaraldehyde 0,25" for 3 min. After 3 washes with PBS, cells were incubated overnight in the dark, at room temperature, with a X-Gal solution (3 mM X-Gal of potassium ferrocyanide, 3 mM of potassium ferricyanide, 0,1 mM of MgCI 2 and 0,4 mM of X-Gal in dimethyl formamide; Boehringer Mannheim Canada, Laval, Can.). Cell preparations were mounted in PBS-glycerol.
  • Desmin immunocytolluorescence Fibroblast cultures were first fixed with 95'7c ethanol for 15 minutes and permeabilized with 5'7c Tnton X-100 for 25 minutes. Desmin was then detected with the monoclonal DE-R-11 antibody (1 :40; Dako, Copenhagen, Denmark) followed by a biotinylated anti-mouse antibody (1 :250; Dako) and Streptavidin-Cy3 (1 :700; Sigma).
  • Dystrophin and MyoDI immunocytochemistries were detected in the infected fibroblast cultures with a polyclonal rabbit antibody that recognizes the dystrophin COOH-terminal portion of many species (generous gift from Dr. J. S. Chamberlain). The cells were first fixed in 95% ethanol for 15 minutes and permeabilized with 0,5'7s Triton X-100 for 15 minutes. Fixed cultures were then incubated with the anti-dystrophin antibody (1 :1000) followed by a biotinylated anti-rabbit antibody (1 :250) and Streptavidin-Cy3 (1 :700). MyoDI staining was done as previously described. in vivo Assay
  • the skin of hind limbs was cut to expose the TA muscle and roughly 40 sites were injected with a total of 4 x 106 transfected fibroblasts using a micropipette (Drummond Co., Broomal, PA, USA).
  • Non-infected dermal fibroblasts were grafted as negative and Tnl LacZ myoblasts were transplanted as positive controls.
  • the skin was sutured using 6.0 silk (Johnson & Johnson, Peterborough, Ont., Can.).
  • mice were immunosuppressed with a daily dose of FK506 (2,5 mg/kg i.m.; Fujisawa Pharmaceutical Co. Ltd. Osaka, Japan).
  • mice were sacrified 30 days after transplantation under deep anesthesia (0,1 ml of a solution containing ketamine (10 mg/ml) and xylazine (10 mg/ml)). After perfusion with a NaCI 0,9% solution containing heparin (2 lU/ml) (Organon Teknika, Toronto, Ont., Can.), muscles were removed and placed in PBS with 30% (W/V) sucrose overnight at 4°C. Muscles were then embedded in cryomatrix (Shandon, Pittsburgh, PA, USA) and frozen in liquid nitrogen. Muscles were cut in 10 Hm thick sections using a cryostat microtome and picked up on gelatin coated slides.
  • Dystrophin immunohistochemistry The endogenous peroxidase activity in muscle sections was first blocked using 1% hydrogen peroxide. Nonspecific binding sites were blocked with 10% calf serum. Muscle sections were then incubated with the anti-dystrophin antibody (1 :1000) followed by a biotinylated anti-rabbit antibody (1 :250) and Streptavidin-Cy3 (1 :700).
  • Dermal fibroblasts were grown in culture for six passages to avoid myogenic cell contamination prior to in vitro and assays. The purity of the fibroblast cultures was confirmed by immunostaining using antibody to the intermediate filament protein desmin. All cultures were found negative for desmin. Moreover, cells failed to fuse and form myotubes when cultured for 10 days in a differentiation medium containing 10% horse serum. These cells were mononuclear with the usual fibroblast-like morphology. Cells were then infected with the LMDSN amphotrophic retrovirus and selected in G418 for 10 days. We observed that 30% of the infected fibroblasts survived G418 selection. The expression of muscle-specific proteins and morphological characteristics in MyoD-infected and control cells were studied in vitro.
  • Irradiated TA muscles of mdx mice were transplanted either with 4X10 6 uninfected dermal fibroblasts or with 4X10 6 LMDSN-infected fibroblasts.
  • the infected fibroblasts were not selected with G418 prior to their transplantation to preserve their proliferative and myogenic capacities.
  • Some mice were grafted with 4X10 6 myoblasts as positive controls. Thirty days following transplantation, mice were perfused and the engrafted muscles were frozen and cut in 10 Hm thick sections for subsequent analysis.
  • ⁇ -galactosidase positive fibers had a centrally located nucleus indicating a recent regenerative process: a phenomenon not observed in revertant fibers. Except for the overall reduced efficiency, muscle fibers originating from MyoD converted fibroblasts were indistinguishable from those derived from the Tnl LacZ myoblasts. No fibers expressing ⁇ -galactosidase were found in muscles injected with dermal fibroblasts (Fig. 8f) although some of them expressed dystrophin. As described before, we consider those fibers as revertants.
  • MyoDI is a master regulatory gene for skeletal myogenesis. Forced expression of this transcription factor in a variety of differentiated cell types allow their conversion to myoblasts by the activation of muscle differentiation markers.
  • MyoDI myogenic potential of MyoDI was used to convert dermal fibroblasts to myoblasts, as previously reported by Weintraub.
  • Fibroblasts were infected with a retroviral vector containing the coding section of the MyoDI gene. Differentiation of some cells was observed and muscle-specific proteins were detected, even if the transformation rate was low. Cell fusion also occurred.
  • ⁇ - galactosidase in these fibers indicate that they originated from the implanted cells which encode the ⁇ -galactosidase bacterial enzyme under a muscle specific promoter. These results are in accordance with those recently obtained by Lattanzi et al. following the injection of adeno-MyoD-converted fibroblasts in the TA muscle of SCID/bg mice. In that study, despite an infection of the fibroblasts with a high MOI (2000), only a few muscle fibers were formed by the infected fibroblasts.
  • the difference in muscle fiber formation efficiency between myoblasts and MyoD-infected fibroblasts could be explained in part by 1) the low percentage (30%) of fibroblasts infected with the retrovirus encoding MyoD and/or by 2) a premature growth arrest in MyoD-expressing fibroblasts early after infection which could decrease the amount of myogenic cells.
  • a third possible explanation could be that only a small portion of MyoD-expressing fibroblasts were efficiently transformed into the myogenic lineage as previously described. However, no muscle fibers expressing ⁇ -gal were seen in muscles transplanted with non-infected dermal fibroblasts.
  • connective tissue in sections obtained from muscles engrafted with normal and infected dermal fibroblasts. In comparison, the connective tissue was less abundant in muscles transplanted with myoblasts. It is well known that fibroblasts are the major cellular constituent of the dermis, a layer of connective tissue. The role of dermal fibroblasts is to synthesize collagen. In our experiments, one problem encountered is that roughly 30% of fibroblasts were converted to myoblasts following infections with the LMDSN retrovirus. It seems that following their transplantation, MyoD-converted fibroblasts lost their proliferative capacity, exited the cell cycle and entered the myogenic differentiation pathway early. Therefore, the non-converted fibroblasts still present in the culture divided more rapidly than transformed cells and produced connective tissue in the grafted muscle.
  • Another possibility to obtain a higher myogenic conversion rate is the injection of a replication-defective adenovirus containing MyoD following the graft of dermal fibroblasts.
  • Murry et al. have shown that the in vivo injection of a high dose adenovirus encoding MyoD in cardiac granulation of an healing heart induced the differentiation of these cells into skeletal muscle.
  • the MyoD-retroviral vector may also be used to infect mesodermal progenitors, which are less differentiated than fibroblasts, providing an alternative strategy to the myogenic conversion of dermal fibroblasts.
  • the in vitro myogenic conversion rate and the in vivo fusion of dermal fibroblasts transformed with the coding section of the MyoDI gene must be greatly improved to enhance the formation of normal muscle fibers in mdx mouse muscles.
  • the use of genetically modified fibroblasts could then be considered as potential therapy to increase the formation of skeletal muscle at the onset of the muscle weakness characteristics of Duchenne muscular dystrophy and other myopathies.
  • myoblasts are transplanted as xenografts, it is possible to insert a gene of different species, while results in a transformed donor myoblasts to be transplanted in another species.
  • a suitable immuno suppressive therapy (specific or not) may be undertaken to support graft survival.
  • Duchenne and Becker muscular dystrophies are due to mutation of the dystrophin gene (Hoffman et al 1987). The mutation leads to the absence of a functional dystrophin molecule in the case of the Duchenne dystrophy and to a defective dystrophin in the case of Becker dystrophy (Arahata et al. 1988; Sugita et al. 1988; Worton et al. 1988; Zubrzycka-Gaam et al 1988).
  • Utrophin also called dystrophin related protein, DRP
  • DRP dystrophin related protein
  • mice expressing the utrophin gene have normal muscles and no increase of the creatine kinase (CK) activity in their serum.
  • CK creatine kinase
  • Utrophin gene is expressed only at the neuromuscular junction in the adult muscle fibers (Gramolini et al. 1997). Utrophin can bind with the dystrophin associated glycoprotein complex (Mizuno et al. 1994). If this protein would be expressed throughout the muscle fibers, the results of Davies group indicates that this would reduce the vulnerability of muscle fibers of dystrophic animals or humans.
  • N box which is responsible for the specific synaptic expression (Dennis et al. 1996). Similar N boxes conferring specific synaptic expression have also been described for the d and the e subunit of the Acetylchloline Receptor genes (Koike et al. 1995; Dueled et al. 1996). A single base mutation of the second base of that N box suppress the specific synaptic expression and the e subunit is expressed everywhere on the muscle fiber (Dueled et al. 1996).
  • Myoblasts are the cells which form muscle fibers by their fusion (Trupin et al. 1982). Following transplantation in a muscle, myoblasts fuse with the host muscle fibers. The muscle fibers formed by the fusion of myoblasts express the genes present in the donor myoblasts (Bartlett et al. 1990, 1995; Huard et al. 1991 , 1993, 1995; Kinoshita et al. 1994 a, b; Partridge et al. 1989; Vilquin et al. 1995; Yao and Kurachi1992; Zatloukal et al. 1994).
  • Duchenne and Becker muscular dystrophies by mutating one or several nucieotides in the N box of the utrophin promoter.
  • Such a mutation would permit a non synaptic expression of utrophin which would compensate for the absence of dystrophin.
  • This mutation can be done in vitro by mutating myoblasts before their transplantation or in vivo by injection of the mutating oligonucleotide in the muscle or in the blood circulation. To effect such a mutation, 1 to 6 nucieotides of the N box of the utrophin promoter are mutated.
  • the second nucleotide of the N box of the utrophin promoter is changed from a C to a T when oligonucleotides are used to effect such a mutation they have the following two sequences: 5'GGC TGA TCT TCC AGA ACA AAG TTG CTT TTg caa cuu ugu TCT GGa aga uca gcc GCG CGT TTT CGC GC3'
  • An in vivo mutation is achieved by injecting an oligonucleotide in the muscle or in the blood circulation.
  • the mutation of the utrophin promoter can also be done in myoblasts in vitro.
  • the myoblasts were obtained from a donor, and may be the patient's own myoblasts.
  • the same oligonucleotides as above may be used.
  • the mutation of the utrophin promoter is also done in fibroblasts in vitro.
  • the fibroblasts were obtained from a donor. These may be the patient's own fibroblasts. In that case also, the same oligonucleotide are used.
  • the fibroblasts are converted in myoblasts before their transplantation in the patient muscles, by inserting a myogenic gene.
  • the myogenic gene is that of MyoD, myogenin or Myf5.
  • the mutation of the utrophin promoter is also done in stem cells in vitro.
  • the stem cells were obtained from a donor. They may be patient's own stem cells. The same oligonucleotides as above are used.
  • the stem cells are converted in myoblasts before their transplantation in the patient muscles by inserting a myogenic gene.
  • the myogenic gene is that of MyoD, myogenin or Myf5.
  • Merosin a protein specific for basement membranes of Schwann cells, striated muscle, and trophoblast, is expressed late in nerve and muscle development. Proc. Natl. Acad. Sci. USA 85: 1544-1548.
  • Dystrophic mice show age related muscle fibre and myelinated axon losses. Nature 267: 167-169.
  • Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle.Neuron 7: 499-508.
  • CM.; Sodek, J. Concanavalin A produces a matrix-degradativephenotype in human fibroblasts. J Biol Chem 1990; 265: 21141-21151.
  • Tremblay J.P., Bouchard, J.P., Theau, D., Cottrell, F., Collin, H., Tremblay, M., Richards, C.L., Malouin, F., Tome, F. and Fardeau, M. Myoblast transplantation between monozygotic twin girls which are carriers of Duchenne muscular dystrophy. Neuromus. Disorders 3, 583-593, 1993a.
  • TGF-beta transforming growth factor beta

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Abstract

L'invention concerne des améliorations importantes relatives à chaque élément contribuant à la réussite de la transplantation ou de la survie d'un greffon. Ces améliorations, prises séparément ou combinées entre elles, améliorent sensiblement la récupération d'un tissu pour qu'il atteigne un fonctionnement normal. Ces améliorations consistent a) à réduire la mort prématurée de cellules transplantées par des agents anti-inflammatoires tels que TGFbeta1, un inhibiteur de la synthèse d'oligosaccharides, une glucosidase, IL-10, vIL-10, Il-4, INFgamma, IL-2R, IL-1Ra, Fas-L, sCR1, une super oxyde dismutase, un facteur d'inhibition de neutrophiles (NIF), une liaison antagoniste des ligands aux LFA-1, MAC-1, ICAM-1, CD-18, CD-31, CD-50, E-sélectine, P-sélectine, TNFalpha, IL-1 et IL-8, les agents anti-inflammatoires pouvant comprendre un anti-LFA-1 ou -ICAM-1, b) à améliorer la diffusion et la fusion de cellules transplantées avec le tissu hôte par métalloprotéases, c) à proliférer ex vivo les cellules transplantées avec les facteurs de croissance ou oncogènes, d) à utiliser des fibroblastes ou des cellules souches au lieu de myoblastes, par la transformation des premiers en ces derniers avec des gènes myogènes, e) à exprimer l'utrophine au lieu de la dystrophie lors de dystrophies musculaires et e) à maintenir la survie du greffon à long terme par des agents immunosuppresseurs.
PCT/CA1998/001176 1997-12-15 1998-12-15 Procedes et compositions permettant d'ameliorer la reussite de la transplantation cellulaire chez un receveur WO1999030730A1 (fr)

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WO2001024828A2 (fr) * 1999-10-06 2001-04-12 Basf Aktiengesellschaft MODULATEURS DE VOIES DE SIGNALISATION ASSOCIEES A LA CYTOKINE ET ANTAGONISTES DU RECEPTEUR αVβ3 D'INTEGRINE POUR THERAPIE COMBINATOIRE
US6403555B1 (en) 1999-12-08 2002-06-11 Xcyte Therapies, Inc. Depsipeptide and congeners thereof for use as immunosuppressants
US6436909B1 (en) * 1999-09-17 2002-08-20 Isis Pharmaceuticals, Inc. Antisense inhibition of transforming growth factor-β expression
WO2002066069A1 (fr) * 2001-02-20 2002-08-29 Kansai Technology Licensing Organization Co., Ltd. Remedes contre l'inflammation/les maladies tumorales
EP1379139A2 (fr) * 2000-12-19 2004-01-14 Insight Strategy & Marketing Ltd. Utilisation d'enzymes de degradation de l'ecm en vue de l'amelioration de greffes de cellules
WO2004090120A2 (fr) * 2003-04-08 2004-10-21 Yeda Research And Development Co. Ltd Cellules souches presentant une sensibilite accrue a un chimioattracteur, et leurs procedes d'obtention et d'utilisation
US6828302B1 (en) 1999-12-08 2004-12-07 Xcyte Therapies, Inc. Therapeutic uses of depsipeptides and congeners thereof
WO2013165304A1 (fr) * 2012-04-30 2013-11-07 Yo Proteins Ab Procédés pour une modification de tissus
WO2015038058A1 (fr) * 2013-09-16 2015-03-19 Elastomics Ab Modification des propriétés mécaniques d'un tissu vivant

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EP1296643A4 (fr) * 1999-09-17 2005-07-27 Isis Pharmaceuticals Inc Modulation antisens de l'expression du tgf-beta
US6436909B1 (en) * 1999-09-17 2002-08-20 Isis Pharmaceuticals, Inc. Antisense inhibition of transforming growth factor-β expression
EP1296643A1 (fr) * 1999-09-17 2003-04-02 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression du tgf-beta
WO2001024828A3 (fr) * 1999-10-06 2002-05-02 Basf Ag MODULATEURS DE VOIES DE SIGNALISATION ASSOCIEES A LA CYTOKINE ET ANTAGONISTES DU RECEPTEUR αVβ3 D'INTEGRINE POUR THERAPIE COMBINATOIRE
WO2001024828A2 (fr) * 1999-10-06 2001-04-12 Basf Aktiengesellschaft MODULATEURS DE VOIES DE SIGNALISATION ASSOCIEES A LA CYTOKINE ET ANTAGONISTES DU RECEPTEUR αVβ3 D'INTEGRINE POUR THERAPIE COMBINATOIRE
US6403555B1 (en) 1999-12-08 2002-06-11 Xcyte Therapies, Inc. Depsipeptide and congeners thereof for use as immunosuppressants
US6548479B1 (en) 1999-12-08 2003-04-15 Xcyte Therapies, Inc. Therapeutic uses of depsipeptides and congeners thereof
US7041639B2 (en) 1999-12-08 2006-05-09 Xcyte Therapies, Inc. Depsipeptide and congeners thereof for use as immunosuppressants
US6828302B1 (en) 1999-12-08 2004-12-07 Xcyte Therapies, Inc. Therapeutic uses of depsipeptides and congeners thereof
EP1379139A2 (fr) * 2000-12-19 2004-01-14 Insight Strategy & Marketing Ltd. Utilisation d'enzymes de degradation de l'ecm en vue de l'amelioration de greffes de cellules
EP1379139A4 (fr) * 2000-12-19 2004-12-01 Insight Strategy & Marketing Utilisation d'enzymes de degradation de l'ecm en vue de l'amelioration de greffes de cellules
WO2002066069A1 (fr) * 2001-02-20 2002-08-29 Kansai Technology Licensing Organization Co., Ltd. Remedes contre l'inflammation/les maladies tumorales
WO2004090120A3 (fr) * 2003-04-08 2005-05-12 Yeda Res & Dev Cellules souches presentant une sensibilite accrue a un chimioattracteur, et leurs procedes d'obtention et d'utilisation
WO2004090120A2 (fr) * 2003-04-08 2004-10-21 Yeda Research And Development Co. Ltd Cellules souches presentant une sensibilite accrue a un chimioattracteur, et leurs procedes d'obtention et d'utilisation
US8252588B2 (en) 2003-04-08 2012-08-28 Yeda Research And Development Co. Ltd. Stem cells having increased sensitivity to SDF-1 and methods of generating and using same
WO2013165304A1 (fr) * 2012-04-30 2013-11-07 Yo Proteins Ab Procédés pour une modification de tissus
WO2015038058A1 (fr) * 2013-09-16 2015-03-19 Elastomics Ab Modification des propriétés mécaniques d'un tissu vivant

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