METHOD OF RESTORING A FUNCTIONAL PROTEIN IN A TISSUE BY CELL TRANSPLANTATION

Abstract: The present invention relates to a method for improving the success of a transplantation which at least addresses to one of the most serious problems: transplant survival. The method comprises the steps of transplanting donor cells in a patient in need for such a transplantation, the transplantation being made in the presence of an agent blocking the interaction between a ICAM-1 molecule located at the membrane surface of the donor cell and a LFA-1 molecule located at the membrane surface of the patient's leucocytes, which blocking increases the viability of said transplanted donor cells by at least about four fold. The agent blocking this interaction is an anti-LFA-1 antibody which is injected to the host at the time of transplantation or an anti-ICAM-1 antibody fragment which is not capable of fixing complement, such as a F(ab)2 fragment.

Description: THEE OF THE INVENTION Method of restoring a functional protein in a tissue by cell transplantation. FIELD OF THE INVENTION This invention relates to a method of restoring a functional protein in a tissue by transplanting healthy donor cells or genetically modified donor cells. More particularly, the present method reduces the mortality rate of the transplanted cells by at least about 4-fold. BACKGROUND OF THE INVENTION Cell transplantation is a potential treatment for several diseases. Myoblast transplantation may indeed be use to treat Duchenne Muscular Dystrophy (Partridge 1991) , 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 use for diabetic (Hering et al. 1993), hepatocytes for liver diseases (Raper and Wilson, 1993) . The success of these transplantations has been so far rather limited. This limited success has been attributed in part to the specific immune responses (Huard, 1992, Tremblay, 1993) . However, some research groups have also reported high levels of cellular death during the first three days following cellular transplantations (Beauchamp et al . 1994; Huard et al. 1994) . This time course is too rapid to be attributed to a specific immune response in naive animals. All the treatments used so far to improve the success of a transplantation never overcome the problem of the early mortality of transplanted cells. The principal problems to overcome during cell transplantation are the following: a) a host immune reaction towards donor cells; b) the number of cells expressing the non- defective protein; c) the amount of non-defective protein expressed in the cells participating to tissue function recovery; and d) the high donor cell death rate. a) A host immune response towards donor cells: It is now known that an adequate immunosuppressive therapy should be undertaken in the case wherein host and donor are histoincompatible, to avoid rejection of the transplanted cells. Shortly after transplantation, infiltrating cells may be observed at the site of transplantation. Even in the case wherein histocompatible host and donor are selected (matched for MHC class I and class II antigens) , other non-MHC antigens might be responsible for an observed immune response. Although the cellular immunoresponse may decrease with time, the humoral response starts building after one week and may be largely responsible for the lack of long-term success of transplantation, especially in immunohistoincompatible subjects. Nonspecific immunosuppression has been found successful for improving and maintaining the success of transplantation. Immunosuppressors like FK506, rapamycin, cyclosporin A and cyclophosphamide did improve the residence time of the transplant . Among these, 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. Histocompatibility is not the only factor to consider for increasing the success of transplantation. When identical twins are matched, there is still a poor regeneration of muscle function. It appears to be due to the low spontaneous muscle regeneration or to inflammation. b) The number of cells expressing the non defective protein: The number of cells expressing a non-defective protein, e.g. fused donor cells or hybrid donor- recipient cells, are usually quite low in non- immunosuppressed individuals. Beside the beneficial effect of an adequate immunosuppressive therapy or of an adequate match of donors and recipients on the number of functional cells, 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. Up to date, the best results have been obtained by combining immunosuppressive and tissue treatment with notexin and irradiation. However, notexin and irradiation remain unacceptable from a clinical point of view because they have severe undesirable side effects. In my co-pending application which serial number is US 08/404,888, a pre-treatment step consisting of culturing yoblasts in the presence of a trophic factor is described. Particularly, the obtention of primary cultured myoblasts in the presence of bFGF (basic fibroblast growth factor) is shown to increase by four¬ fold the number of dystrophin-positive cells. This represents a clear progress to enhance the success of transplantation in absence of notexin and irradiation pre-conditioning step in dystrophic individuals. Even with this progress, the level of cell mortality still remains very high (more than 90%) . The high level of early cell mortality has not yet been solved. c) The amount of non-defective protein expression in the cells participating £Ω tissue function recovery; 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. d) The hiσh donor death cell rate: This problem remains one of the most serious to overcome. Immunosuppressive treatment has been successful in inhibiting the humoral immune response which appears more lately than the cellular immune response. However, there is still a very high early mortality of the transplanted cells (more than 90%) . Inflammation may be one of the major factors involved in the important early mortality e.g. before the building of a cell 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. Despite all the attempts made to improve the success of transplantation, there still a need for increasing the number of cells which survive after the first five days of transplantation, beyond which immunosuppressive treatments as well as an enhanced proliferation of the cells which have survived may exert their beneficial effects to improve long-term recovery of tissue function. U.S. Patent 5,130,141 to Peter Law describes and claims a method for treating muscle degeneration and weakness similar to the one that we are presenting. All the scientific experiences which are the basis for this patent have been made on a dystrophic mouse model (as indicated column 2, line 35; column 3, line 25) . The dystrophic mouse which was used was (C57BL/6J-dy 2 /dy 2J ) as indicated in column 6, line 2 and in column 7, line 40. The author of patent 5,130,141 indicated that the dystrophy in that mouse model was treated by the injection of myogenic cells (column 1, lines 17 and 18; column 2, lines 34 and 35; column 3, line 26; column 4, line 39; column 5, line 67; column 6, line 54; column 9, line 5; column 10, line 64) . In fact, it is stated quite clearly that myogenic cells are the "active ingredient" (column 6, line 54) . It is also clearly stated that other cell types such as fibroblasts interfere with the practice of the claimed invention and cause detrimental effects (column 4, line 66) . The main problem with this patent is that its scientific basis is not sound. Since the experiments made by Dr. Law's group, it has been established that in the dystrophic mouse dy is missing a protein called merosin (Arahata et al . 1993; Sunada et al. 1994, 1995; Xu et al. 1994a, b) . The dy mice have an intact dystrophin. We clearly demonstrate in this application that merosin is not restored by myoblast transplantation but that merosin is, however, restored by another type of cell present in primary muscle culture. Therefore the interpretation of the experiments of Dr. Law's group is erroneous and thus the scientific basis of the patent is not sound, Dr. Law's patent does not further describe any method to solve the problem of early mortality of transplanted cells. STATEMENT OF THE INVENTION It is now provided a method for improving the success of a transplantation which at least addresses to one of the most serious problems: transplant survival. It is therefore an object of the invention to provide a method of restoring a functional protein in a tissue which comprises the steps of transplanting donor cells in a patient in need for such a transplantation, said transplantation being made in the presence of an agent blocking the interaction between a ICAM-1 molecule located at the membrane surface of the donor cell and a LFA-l molecule located at the membrane surface of the patient's leucocytes, which blocking increases the viability of said transplanted donor cells. In a preferred embodiment, the agent blocking this interaction is an anti-LFA-l antibody which is injected to the host at the time of transplantation. In an alternatively preferred embodiment, the blocking is effected by using an anti-ICAM-1 antibody fragment which is not capable of fixing complement, such - li ¬ as a F(ab) 2 fragment. The advantage of using the latter resides in the capability of treating with F(ab) 2 the cells before their transplantation which would impede the recognition of leucocytes towards the transplanted cells. In the above method, it is still preferable to match the donors and the patients for their histocompatibility (MHC class I and class II antigens) . Concurrently, immunosuppressive therapy should be undertaken particularly in the case wherein a MHC- incompatible donor is used. The preferred immunosuppressive agents are rapamycin or FK506, although a combination or other suppressive agents may achieve similar good results. When a MHC- histocompatible donor is used, the immunosuppressors are mostly selected amongst those inhibiting the building of an immune humoral response towards antigens other than the MHC antigens . The donor cells might be selected from the group consisting of primary cultured donor cells, cloned donor cells and genetically modified donor cells. To increase the number of cells that may be transplanted from a donor biopsy, these cells may be grown in vi tro in the presence a trophic factor. When primary cultures of myoblasts are contemplated, the use of bFGF is preferred, for a short period to avoid growing of fibroblasts. bFGF has been shown to increase by four-fold the number of dystrophin-positive cells in a mdx mouse model. When cloned cells are used, longer time of growing in the presence of bFGF is contemplated. Before transplantation, the proliferation of donor cells may be alternatively encouraged by the use of cloned cells which have been transfected with a recombinant vector containing a gene encoding a molecule encouraging cell proliferation, which gene is under the control of a promoter compatible with the donor cells. The used promoter might be an inducible promoter. In a preferred embodiment, the molecule encouraging cell proliferation is SV40-T antigen. The promoter is a promoter of a major histocompatibility gene. Preferably, this promoter is a MHC Class II gene promoter. A MHC Class II promoter is inducible by γ- interferon (γ-IFN) . To ascertain that the introduction of SV4T0 antigen does not render the transplant tumorigenic, thermosensitive mutants thereof may be used to encourage the proliferation of cells in vi tro at 33°C, which cells are returned to 37 or 39°C, and tested for cell differentiation prior to transplantation. At 37°C no functional SV40T antigen would be produced. The above invention is particularly applicable to treat myopathies, dystrophies, natural muscle weakness due to aging, and motoneuron diseases which are due to a lack of trophic substances. Some of the muscle weaknesses may be eventually treated by gene therapy when the gene mutation responsible for each of these diseases is known. These diseases can, however, be treated by the transplantation of cells present in normal muscle. For some diseases, myogenic cells may be required while for other diseases other non-myogenic cell may be necessary. In the case of muscle weakness due to natural aging or following mechanical or other type of physical injuries to the muscles, gene therapy will not be effective since new muscle fibers have to be formed by injecting new myogenic cells. In these cases, it would be envisageable that cells of an individual may be banked at a younger age, proliferated in vi tro and transplanted when needed as an autologous transplant. DETAILED DESCRIPTION OF THE INVENTION; The present invention provides for the first time, methods to restore missing functional molecules in affected individuals, which restoration is greatly improved by solving the problem of the early mortality of transplanted donor cells. In dystrophic patients, transplantation of myoblasts (eg. restoration of dystrophin) or the transplantation of non myogenic cells (eg. restoration of merosin) will achieve such restoration. The invention is based on successful demonstrated restoration of these molecules (Vilquin et al., 1994a, 1995a, ; Huard et al. 1991a,b; 1992a, b; 1993, 1994a,b; Tremblay et al. 1991; 1993a,b; Roy et al . 1993; Kinoshita et al. 1994a, b,c; Asselin et al. 1994; 1995) . The claimed methods have been demonstrated in 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; Kinoshita 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 (eg. dystrophin) . In the case of transplantation of non-myogenic cells, the missing gene product (eg. merosin) may be secreted in the extracellular matrix. The myogenic or non myogenic cells to be transplanted can be histocompatible or histoincompatible with the host. In cases of histoincompatibility 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 cyclosporin-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. A suitable regimen therapy using a combination of CTLA4-Ig 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. The used of 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 SV40T antigen under control of the IFN-γ inducible H-2Kb 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 μg/ml) and β-Gal expression, and expanded for use in transplantation experiments. The survival of the β-Gal labelled cells was assessed by dosing the β-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 γ-IFN inducible SV40 T antigen 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. To evaluate how a non-defective protein is produced in a recipient individual, a PCR reaction may be effected on a biopsy. The messenger cDNAs 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. (1994 and 1995)) . Furthermore, the fate of transplanted myoblasts may be monitored by way 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 muscle because these cells do not diffuse very well in the muscle. The distribution of the myoblasts throughout the muscle may be facilitated by developing a device controlled by a robot. This robotic device may receive from an imaging system (eg. magnetic scanner) 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. The cells required to treat the muscle may also be inserted by some other routes. Some type of cells may even be injected in the blood vessels of the patients and migrate inside the muscles. The secretion of these cells may also diffuse in the muscles. The secretion of these cells may also diffuse in the muscle. 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 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-I antibody (if administered directly to the patient) or an anti-CAM-I antibody fragment . The claimed invention can be clarified by examples of experimental treatments in animals and human patients. The experiments described are examples and should not be use to limit the scope of the invention. EXAMPLES 1) Myogenic cells transplanted in mdx dγ.q rophic mice to restore the expression of dystrophin 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. The donor myoblasts may be grown from a muscle biopsy of an histocompatible or an histo-incompatible mouse. In the cases where myoblasts are obtained from an histoincompatible donors, the host may be im uno- 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. 2) Myogenic cells transplanted in monkeys 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. In this experiment, the injected cells were labelled with a retrovirus vector containing the β-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. 3) Myogenic c_e_lls injected in Puchenne muscular dystrophy patients Duchenne muscular dystrophy is due to the absence of dystrophin in the muscle fibers. The protein can be restored in the muscle fibers of these patients by the transplantation of myoblasts obtained preferentially but not exclusively from an MHC-histocompatible donor. This has been demonstrated by the inventor's research team (Huard et al . 1992a,b; Tremblay et al . 1993a,b) . 4) Injection of non-myoαenic cells in dv/dγ dystrophic mice The muscular dystrophy in dy/dy mice is due to the absence of an extracellular matrix protein called merosin. This animal is an excellent model of Congenital Muscular Dystrophy (Arahata et al . 1993; Sunada et al . 1994, 1995; Xu et al . 1994a,b) . The expression of merosin can be restored by the transplantation in the muscle fibers of non-myogenic cells. Since this protein is present in the extracellular matrix it has to be secreted by the injected cells 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. Animals 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. The transgenic TnI-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 β-galactosidase (β-gal) protein (Hallauer et al . , 1993; Kinoshita et al . , 1994a,b) . β-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. The H-2K b -tsA58 transgenic mice carry the thermolabile tsA58 mutant of SV40 large T antigen under the control of the H-2K D promoter (Jat et al . , 1991) . Interferon Y (IFN-γ) increases the transcription of this promoter. The thermolabile protein is functionally active at 33°C but not at 39°C. These characteristics facilitate the derivation of conditionally immortalized cell lines (Morgan et al . , 1994) . An homozygous H-2K -tsA58 male mouse (Charles River Lab., Wilmington, MA) was crossed with a heterozygous transgenic Tn-LacZ female mouse. Offsprings were all heterozygous for the H-2K b -tsA58 transgene and some were heterozygous for the Tn-LacZ transgene. Thus, newborns were tested for β-gal expression, and only β-gal positive animals were used for the establishment of myogenic cell lines (see below) . Primary cell cul tures 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 commited myoblasts as assessed by desmin immunostaining (personal results) . Cultures were also obtained from newborn transgenic Tn-LacZ. Because parents were both heterozygous, newborns were individually tested for the expression of β-gal using X-gal (see below) . Only β-gal-positive newborns were used for primary myoblast cultures . Different batches of primary cell cultures have been used for this work. The mice transplanted with these batches have been gathered under the letters A, B (β-gal cells) , and H (histocompatible cells) . Establishment of permanent myogenic cell lines from (TnI-LacZl/29J X (H-2tf > - tsA68) offsprings . Primary muscle cell cultures were started using the β-gal-positive offsprings. Following preplating, the cells were plated at 50 cells/cm 2 in gelatin-coated wells and grown in DMEM (Gibco) supplemented with 20% FCS and 2% chick embryo extract (Gibco) at 33°C in 10% C0 2 . Mouse recombinant IFN-γ (Genzy e Co., Cambridge, MA) was added at the final concentration of 20 U/ml (Morgan et al . , 1994) . Colonies of typical myogenic morphology were subsequently cloned in 96-well plates at the limit dilution of 1 cell per well. The myogenicity of colonies and clones was assessed by desmin immunostaining and by the ability to fuse and form myotubes in vi tro . Fusion was obtained by reducing the FCS concentration to 5% and growing the cells at 37°C in 5% C0 2 in the absence of IFN-γ. Cell transplantation On the day of transplantation, the cells were harvested by trypsinization or thawing, washed three times in HBSS (Gibco) , and concentrated as pellets. Cell viability was assessed using trypan blue staining. The Tibialis anterior (TA) muscles were exposed and injected with approximately 4 x IO 6 (primary muscle cell cultures) , or IO 6 (myogenic cell lines # 24, MB7, MB27) or 3 x 10 s (myogenic cell line MB3) viable cells suspended in 10 μl of HBSS. When the role of myoblasts in LAMA2 restoration was explored by clonal cell transplantation, 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) . Some protocols included γ~irradiation and/or notexin treatment of the muscles before cell transplantation: three days before transplantation, one or both hind legs of the dy/dy mice were Cobalt- irradiated (20 Gy) . This level of irradiation has been shown to block host myoblast proliferation and to favour donor myoblast implantation (Wirtz et al . , 1982; Morgan et al . , 1990; Wakeford et al . , 1991) ; one day before transplantation, one or both TA were exposed and injected with 10 μl of notexin venom (5 μg/ml) , which has been shown to trigger muscle fiber degeneration without damaging myoblasts (Harris et al . , 1975) . FK506 immunosuppression was started on the day of transplantation in noncompatible grafting models (2.5 mg/kg/d i.m., Fujisawa Co, Osaka, Japan; Kinoshita et al . , 1994b) . The transplantation schedules are presented in Fig. 1. Muscle collection At the times indicated, 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 μm. Adjacent serial sections were thus spaced by 8 μm, while the series of sections were separated by 180 μm. -gal histochemis try Differentiated conditionally immortalized myogenic cells and muscle cryostat sections were fixed with 0.25% glutaraldehyde for 3 min and incubated overnight at room temperature with 0.4 mM X-gal (5-bromo-4-chloro- 3-indolyl-β-D-galactopyranoside, Boehringer Mannheim, Laval, Canada) (Kinoshita et al . , 1994a) . Preliminary experiments indicated that the X-gal enzymatic histochemistry was as sensitive as three-step immunohistochemistry using monoclonal anti-β-galactosidase antibodies to localize muscle fibers formed by the fusion of donor myoblasts (not shown) . Newborns originating from Tn-LacZ mice were tested for β-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. LAMA2 immunohistochemistry 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) . Binding was revealed with DAB (0.5 mg/ml, Sigma) and 0.015% hydrogen peroxide. Slides were mounted in PBS-glycerol. Immunoperoxidase-positive and -negative fibers were counted by microscopic examination of each muscle on the section with the most positive fibers. Desmin im unocytochemis try 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) . Primary muscle cel l cul ture transplanta tion under FK506 i mmunosuppre ssion β-gal expression Primary muscle cell cultures from transgenic mice expressing β-gal under the control of a muscle-specific promoter were able to develop inside the TA muscles of dy/dy mice immunosuppressed with FK506. Some myoblasts fused together or with host muscle fibers to form new or hybrid muscle fibers expressing β-gal. These β-gal expressing fibers were not numerous and they were not dispersed throughout muscles. They were presumably located only near the injection sites. As seen in Table I, the percentage of β-gal-positive fibers was rarely higher than two. These low percentages were obtained whatever the age of the recipient mice at the time of transplantation, a slight but not significant increase was observed when animals were kept longer after transplantation. LAMA2 expression As previously reported, LAMA2 was absent from muscles of dy/dy 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 (Table I) . 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 γ~ irradiation alone, did not increase the number of LAMA2-positive fibers. The combination use of notexin and Y-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 (Table I, the mice in group A all received the same myoblast preparation) . Colncali7.at.ion of β-σal and LAMA2 The Tn-LacZ primary muscle cultures originate from mice with normal LAMA2 expression. Thus, both the normal gene for LAMA2 and the reporter gene coding for β-gal are present in the donor cells. LAMA2 and β-gal were characterized on serial sections only spaced by 8 μm. As shown in Table I, the percentages of LAMA2-positive and β-gal positive fibers were comparable when old animals (more than 3 months) were used for transplantation. LAMA2 and β-gal labeling were localised in the same clusters of fibers. Some LAMA2-positive fibers, however, were negative for β-gal expression, and some β-gal positive fibers were negative for LAMA2 expression on serial sections. Most of the smallest fibers were β-gal positive. These results indicated that β-gal and LAMA2 expression were not regulated similarly, or that the same type of cells were not responsible for their expression. When experiments were designed in younger animals (i.e. 6 weeks old) , the LAMA2-positive fibers were much more abundant than β-gal positive fibers (Table I) . Surprisingly, the pattern and intensity of β-gal and LAMA2 expression greatly differed. Some LAMA2-positive clusters of fibers were totally devoid of β-gal. The β-gal positive fibers were restricted to smaller areas than LAMA2-surrounded fibers. LAMA2-positive fibers were observed over long distances throughout the muscle, i.e. more than lOOOμm, whereas no β-gal was observed in most of these fibers over all length of LAMA2 expression. Long-term histocompatible transplantations Transplantation of histocompatible, isogenic primary mouse muscle cells in 6 week-old male dy/dy mice led to long-term LAMA2 expression (i.e. 11 weeks) in the transplanted muscles of all the animals. The percentage of LAMA2-positive muscles was greater when muscles were treated using notexin and γ~irradiation before transplantation (14.75 ± 6.4 without pretreatment versus 41.2 ± 14. 3 with pretreatment) . Notexin alone (12.75 ± 1.2 positive fibers) , or irradiation alone (19.25 ± 6.9 positive fibers) , followed by transplantation, was not sufficient to increase dramatically the percentage of LAMA2-positive fibers (Table II) . Given the small number of dy/dy mice available for these experiments, however, no statistical analysis was performed. 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 dy/dy 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. Condi tional ly immortal ized myoblast cul ture transplan ta tion under FK506 immunosuppression Characteri ation of immortalized myogenic cell lines. The problem of tumorigenicity was frequently reported upon the use of the classical C2 mouse myoblast cell line (Wernig et al . , 1991; Morgan et al . , 1992) . Thus, Morgan et al . (1994) developed a technique to obtain pure, immortalized myoblast clones with the ability to form new muscle fibers after transplantation into immunodeficient and dystrophin-deficient nu/mdx mice, without inducing tumors. The transgenic myoblast cultures developed in the present study have the additional advantage that differentiated cells express β-gal under the control of a specific muscle promoter. This allows the rapid identification of new or hybrid fibers formed by the fusion of these myoblasts in vi tro or in vivo in any mouse model. 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% C0 2 atmosphere when stimulated by murine IFN-γ. 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 myotubes and some myoblasts expressed also β-gal. These cells were able to fuse and to form new or hybrid muscle fibers expressing β-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 vi tro myogenic characteristics and were injected into dy/dy muscles. Lack of LAMA2 expression despite muscle fiber formation after myoblast clone transplantations Two, 3 and 4 weeks after injection of conditionally immortalized myoblast clones into left TA of dy/dy mouse, some β-gal positive fibers were formed using clones MB27 and MB3, and Table III) . The number of β-gal positive fibers was low possibly because the muscles were not pretreated with notexin and irradiation. On adjacent sections, however, no LAMA2 could be detected in the same TA muscle, neither surrounding β-gal positive fibers or β-gal negative fibers (using clone MB27) . LAMA2 was also absent from the series of sections preceding and succeeding β-gal-positive sections, thus indicating that a putative difference in the restriction domain of LAMA2 and β-gal was not responsible for the lack of LAMA2 expression in this model. A very faint labeling could be observed around some, but not all muscle fibers (using clone MB3) and some of these faintly-positive fibers were β-gal-negative. Pure myoblasts were thus able to fuse together or with host muscle fibers and to trigger β-gal expression, but in most of the cases they did not trigger LAMA2 formation or extracellular deposition at least at levels detectable by immunohistochemistry or immunohistofluorescence (Table III) . The right dy/dy TA of the same mouse, which was injected with a classical histocompatible primary cell culture together with the same myogenic clone MB27, however, contained many LAMA2- surrounded muscle fibers and/or β-gal-positive fibers. The β-gal and LAMA2 proteins were not always located in and around the same fibers. Overall transplanta tion success Three transplantation models were used in this study. In the first one, nonhistocompatible primary muscle cells expressing the β-gal under control of a muscular promoter were transplanted into dy/dy mice. This allogenic transplantation required an efficient immunosuppression, which was obtained using FK506 (Kinoshita et al . , 1994b; Vilquin et al . , 1995b) . The use of β-gal expressing cells showed the exogenous origin of the labelled muscle fibers and allowed the comparison between LAMA2 and β-gal localization after transplantation. In the second model, histocompatible syngeneic primary cultures from normal littermates were transplanted into dy/dy mice. This model did not require immunosuppression and allowed the study of muscle regeneration over longer time. In the third model, pure but nonhistocompatible myoblasts were transplanted into one leg of FK506-immuno-suppressed dy/dy mice, whereas the other leg received histocompatible primary culture as a control of LAMA2 expression. This model allowed the direct comparison of the outcome of transplantation between pure myoblasts and primary muscle cells and allowed the investigation of the potential role of myoblasts in LAMA2 deposition. Cell transplantation allowed the restoration of a structural, extracellular protein. In these immunologically controlled models the transplantation of primary muscle cells lead to LAMA2 expression in variable amounts in all animals. The number of LAMA2-surrounded fibers was shown to depend on the age of the animal at the time of transplantation and on the muscle pretreatment . The highest numbers of LAMA2-surrounded fibers were obtained when young animals (6 week old) received histocompatible cells after Y-irradiation and notexin pretreatment of the muscle. Irradiation was shown to hamper myoblast proliferation and thus reduce normal muscle regeneration, and to favour extensive fibrosis in dy/dy (Wirtz et al . , 1982) and mdx/mdx (Wakeford et al . , 1991) mice. This treatment greatly increased donor myoblast permeation into host degenerating fibers and the formation of new or hybrid dystrophin-positive fibers in mdx/mdx mice (Morgan et al . , 1990) . In our models, it is thus likely that irradiation and notexin necrosis favour donor cell development in vivo over the dy/dy recipient cells. In the histocompatible model LAMA2 , which is the only known difference between dy/dy 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 dy/dy 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. The intensity of β-gal staining and the number of β-gal positive fibers after transgenic Tn-LacZ culture transplantation was low as compared to results obtained in other, non dy/dy mice strains under the same FK506 immunosuppressive treatment. In a previous report, up to 90% of mdx/mdx TA muscle fibers could express β-gal only one month after transplantation (Kinoshita et al . , 1994b) . The present results suggest that muscle cell transplantation is less efficient in dy/dy mouse than in mdx. Actually, more than 1000 dystrophin-positive fibers are frequently obtained in only 2 months following histocompatible primary muscle culture transplantation in mdx/mdx 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 dy/dy mice. The dy/dy muscle fibers are smaller than normal muscle fibers and they show important size variation within a single muscle. This may reflect trophic problems in dy/dy muscles related to the absence of LAMA2 in basal lamina that could explain the variation in efficiency between the mdx/mdx and the dy/dy models. First, 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) . Second, an impaired muscle innervation due to the absence of LAMA2 around peripheral nerve fibers or neuromuscular junctions (Leivo and Engvall, 1988; Sunada et al . , 1994; Xu et al . , 1994a) could induce a muscle atrophy. Third, the myoblast proliferation and/or migration could be impaired by the abundant connective tissues, present especially in the oldest dy/dy mice. Taken together, these problems may explain why muscle regeneration was relatively poor following transplantation, whatever the pretreatment used. LAMA2 and β-gral colocalization after transplantation of transgenic primary cells In transplanted muscle, LAMA2 was generally more widely distributed than β-gal. This could indicate differences in the regulation of expression of these two proteins, that LAMA2 but not β-gal is diffusible, or that the cell type responsible for LAMA2 expression is not the cell type responsible for β-gal expression, β-gal expression is restricted to skeletal muscle cells as an intracellular protein, and its nuclear domain is about 1000 μm. In contrast, 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 β-gal and LAMA2 in vivo is probably different. Whereas β-gal expression requires myoblast fusion and formation of hybrid or new muscle fibers, the developmental mechanisms of LAMA2 expression are still unclear. β-gal and LAMA2 did not always colocalize in and around the same segments of muscle fibers. On serial sections β-gal positive segments of fibers could be LAMA2-negative and vice-versa. Thus, either myoblasts and muscle fibers do express both β-gal and LAMA2, or these proteins are expressed by several or only partially overlapping different cell types, or by cells at different stages of differentiation. If muscle cells express both LAMA2 and β-gal, then β-gal positive fibers should be surrounded by LAMA2, even on short muscle fiber segments. This was not always observed. The other hypothesis implies that myoblasts and muscle fibers should express β-gal, whereas other cells should secrete LAMA2. Depending on the presence and development of myoblasts, or fibroblast-like, or both cell types in the vicinity of host muscle fibers after transplantation, β-gal positive, or LAMA2-positive, or both β-gal and LAMA2-positive fibers should be observed. LAMA2 expression pa t tern 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 (Fig. 5e, f) . In contrast, 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 mdx/mdx 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. This observation suggests that competent cells could proliferate and diffuse, or that the secretion product may diffuse, thus producing a gradient of LAMA2 expression from center to periphery, that would progressively accumulate around the most proximal muscle fibers expressing the appropriate receptors and extracellular matrix components, allowing LAMA2 sequestration around these muscle fibers. This hypothesis could explain the polarization of LAMA2 deposition around the biggest fibers in the particular orientation from periphery to center. Small-diameter fibers would be more completely surrounded by LAMA2 because they were located at the injection sites in close contact with LAMA2-secreting cells. The surface to be covered by LAMA2 is also less in small-diameter than in normal-diameter fibers. The role of myoblasts in LAMA2 restoration in dy/dy mice Cloned myogenic cells formed new or hybrid β-gal-positive muscle fibers following transplantation into dy/dy mice. Small and normal caliber p-gal positive fibers were obtained already 2 weeks after transplantation. Small-caliber fibers were likely entirely of donor origin. Normal-caliber fibers probably originated from the fusion of donor myoblasts with host muscle fibers undergoing a segmental degeneration-regeneration process at the site of injection. The β-gal positive fibers, however, were very rarely surrounded by LAMA2, thus indicating that myoblasts and muscle fibers formed by them were not able to secrete LAMA2, at least to levels detectable by immunohistochemistry and immunohistofluorescence. As a control for LAMA2 expression, primary cells from mixed cultures injected in the contralateral leg were able to trigger LAMA2 deposition around new and hybrid muscle fibers within 2 weeks. Thus, LAMA2 may be secreted by some competent cells present in primary culture, but not in cloned myoblasts. Another hypothesis which cannot be ruled out yet, is that none of the myogenic clones used in this study was able to produce LAMA2 in vivo, despite their ability to express desmin, to express β-gal under control of a muscular promoter, to form myotubes in vi tro and to form muscle fibers in vivo . Some distinct populations of satellite cells have been described in regenerating rat skeletal muscle (Rantanen et al . , 1995) . Last, the secretion of LAMA2 could be dependent on cell dif erentiation status. Indeed, LAMA2 deposition was observed in vitro in cultures of myogenic clones and colonies (personal results) . Some, but not all myoblasts produced LAMA2 and small myotubes did not, as observed on immunocytochemistry preparations (data not shown) . This suggests that LAMA2 secretion could be regulated in myogenic cells, as are other muscle proteins and myogenic factors (Rantanen et al . , 1995) . This hypothesis would be supported by the very faint labeling observed around 2 or 3 fibers following clone MB3 and clone MB7 transplantation, and is currently under investigation. The 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. The above results suggest that the dy/dy mouse develops a pathology of the extracellular matrix. Some clinical signs of this matrix pathology are similar to those observed in some neuromuscular diseases primarily originating from muscle or nerve cell deficiencies. These observations should prove useful in the investigation of human CMD aetiology. The identification of the cell type responsible for LAMA2 secretion and its relation with other muscle constituents are important future issues, and could help to categorize clinical cases of CMD and develop new strategies for the correction of muscular diseases by gene complementation. The technologies will differ from those used for myoblast culture and transplantation in other types of muscular dystrophies if the LAMA2-secreting cell is a connective cell, with no fusion ability but adhesion and migration properties. Long-term experiments should also be designed to further explore the feasibility of myelination restoration in the peripheral nervous system of the dy/dy mouse (Bradley and Jenkison, 1973; Madrid et al . , 1975; Montgomery and Swenarchuk, 1977) . Alternatively, the transducibility of the LAMA2-secreting cell type by different gene vectors (adenoviruses, retroviruses, herpesviruses) should indicate if it could constitute a good target candidate for in vivo or ex vivo gene therapy of CMD. 5) Increasing the earlv survival of cells by blocking the interaction of LFA-l and ICAM-1 molecules: Cell transplantation is a potential treatment for several diseases . Myoblast transplantation may indeed be use to treat Duchenne Muscular Dystrophy (Partridge 1991) , 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 (E erich, 1995) and Parkinson disease (Borlongan and Sandberg, 1995) ; islet may be use for diabetic (Hering et al . 1993), hepatocytes for liver diseases (Raper and Wilson, 1993) . The success of these transplantations has been so far rather limited. This limited success has been attributed in part to the specific immune responses (Huard, 1992, Tremblay, 1993) . However, some research groups have also reported high levels of cellular death during the first three days following cellular transplantations (Beauchamps et al . 1994; Huard et al . 1994) . This time course is too rapid to be attributed to a specific immune response in naive animals. Our group now demonstrates that abundant neutrophils and macrophages infiltrate within hours the sites of the cellular transplantation. Respiratory burst of these infiltrating cells could be responsible for the early damage to the transplanted cells. We demonstrate that the rapid death of transplanted cells is indeed due to an inflammatory reaction which can be largely reduced by treatments with a monoclonal antibody against LFA-l. β-Galactosidase labelled myoblasts were initially transplanted in the muscles of non-immunosuppressed C57BL10J mice. The reporter gene expression was reduced by 74 ± 12.4% by the third day following the transplantation. This was attributed to necrosis of the transplanted cells containing this reporter gene. The reduction of the β-Gal labelled cells in the injected muscle was also confirmed by histochemistry. When myoblasts were transplanted in a host previously irradiated over their whole body, their death was significantly reduced suggesting that some host cells were contributing to their mortality. Two experiments were therefore made to verify whether this cell death could be due to a specific cellular immune reaction. β-Gal labelled myoblasts were injected in mice immunosuppressed with FK506 starting one day before the transplantation. FK506 has already been shown to control very effectively both the cellular and humoral reaction following myoblast transplantation in mice and thus to permit very successful myoblast transplantation as indicated by a high percentage (95%) β-Gal positive muscle fibers one month after the myoblast transplantation (Kinoshita et al . 1994) . In the present experiment, the short term mortality of the injected myoblasts was however not reduced by the FK506 immunosuppression. The mortality of the injected cells was also not reduced when they were injected in immunodeficient SCID mice. These two results rule out a rapid cellular immune reaction by lymphocytes as the cause of the rapid cell death. During the first three days following myoblast transplantation, the injected muscles were, however, infiltrated by Mac-l positive cells (cells/section) (i.e. macrophages and neutrophils) . The presence of neutrophils was also confirmed by immunohistochemistry using a specific mAb (RB6-8C5, Pharmingen, San Diego, Ca) and by dosage of myeloperoxidase activity. This observation suggests that the death of the transplanted cell is due to an inflammatory reaction rather than to a specific immune reaction. Attempts to reduce cell death by injecting the host with a monoclonal antibody (mAb) against Mac-l (CDllb) were not successful. The death of the transplanted cells was, however, significantly reduced to only 18.2 ± 7.2% by injecting the host with a mAb against LFA-l (CDlla) before the transplantation. This is in sharp contrast with the mortality rate already observed (more than 90%) . This mAb blocks the interaction of LFA-l located on leukocytes with ICAM-1 located on target cells and thus blocks the attachment of macrophages and neutrophils to their target cells (ref ) . This binding is required for the infiltrating cells to produce oxidative damage to these target cells (ref ) . Thus the anti-LFA-l treatment reduced the inflammatory damages and improved the myoblast survival . When cells are transplanted into a tissue there is always some damage to the transplanted cells and to the host tissue by the mechanical process, i.e. the injection. This initial cell death would trigger an inflammatory process which leads to an infiltration of the host tissue by macrophages and neutrophils. These infiltrating cells would then release free radicals and thus trigger much more damage to the transplanted cells and the host tissue during the following three days. This situation is similar to reperfusion damages previously reported following short ischemia of the heart (Entman et al. , 1992) . The presence of such inflammatory reactions could seriously limit the effectiveness of different cellular transplantations. They could be especially detrimental when the transplanted cells are either not abundant, non or poorly proliferating. This inflammatory damage is largely reduced by blocking neutrophils and macrophages attachment to the transplanted cell with an anti-LFA-l mAb. Although the importance of the inflammatory process has been demonstrated in these experiments for myoblast transplantation, inflammation may also limit the transplantation success of other cell types. An anti- LFA-I Mab reduced by at least about four fold the mortality rate of transplanted myoblasts, and there is no apparent reason why an anti-CAM-I antibody or fragment thereof could not achieve similar results, and this, in other types of cells to be transplanted. 6) Other defective proteins may b£ replaced by transplanted corresponding non-defective proteins The above examples described the recovery of merosin and dystrophin in deficient animals or humans by transplanting healthy donor cells expressing the corresponding non-deficient proteins. This invention should not be restricted in any way to these two proteins. For example, 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) 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. Laminin α2 chain restoration in dy/dy mouse (Vilquin e t al . ) Table I. Outcome of transplantation of transgenic primary muscle cell culture in FK506-immunosuppressed animals Cultured cells were injected in one or both TA at the ages indicated, with or without muscle pretreatment. At the times indicated, the mice were sacrificed. The β-gal- and LAMA2-positive fibers were counted on the best section " Tκ, transplantation b Notexin indicates notexin injection and Y, /ray irradiation Mouseft Age at Tx a Days post Tx Leg Muscle Total nb β-gal- LAMA2- % β-gal- % LAMA2- (days) treatment 6 of fibers positive positive positive positive Al 92 21 Left None 1655 53 85 3 2 5.1 A2 92 21 Left None 1187 88 97 7 4 8 2 A3 92 21 Left None 1165 28 45 2 4 3 9 Right Notexin 875 5 55 0 6 6 3 A4 92 21 Left None 774 15 24 1.9 3.1 A5 92 39 Left Notexin 920 58 128 6 3 13 7 1 A6 92 39 Left None 1125 103 165 9 1 14 7 o r A7 92 39 Left None 1277 25 47 1 9 3 7 Bl 45 27 Left Y + Notexin 932 3 162 0 3 17 4 Right None 1454 70 204 4.8 14 B2 45 27 Left Y + Notexin 1161 5] 511 4 4 44 Right None 1375 58 355 4 2 25 8 B3 45 27 Left Y + Notexin 1505 85 285 5.6 18 9 Right None 1468 48 118 3 3 8 B4 45 27 Left Y 502 24 97 4 8 19 3 Right Notexin 895 8 115 0.9 12 8 B5 45 27 Left Y 651 2 101 0 3 15 5 Right Notexin 953 21 193 2 2 20 3 Laminin 2 chain restoration in dy/dy mouse (Vilquin et al . ) Table II. Outcome of long-term histocompatible primary muscle cell transplantation without jm unosupression. Cell cultures were injected in both legs after muscle pretreatment. The mice were killed 11 weeks after transplantation, and LAMA2-positive fibers were counted on the best section Λ : Tx, transplantation. b : Notexin indicates notexin injection, and Y, yray irradiation. Mouse)) Age at Days at Leg Muscle Total inb LAMA2- %LAMA2- Tχ a post Tx treatment 13 of fibers positive posi ive (days) HI 45 77 Left Y 1238 178 14.4 Right Notexin 1655 225 13.6 H2 45 77 Left Y 1396 336 24.1 Right Notexin 1475 175 11.9 H3 45 77 Left Y + Notexin 1007 517 51.3 Right None 942 182 19.3 H4 45 77 Left Y + Notexin 711 221 31.3 Right None 1280 130 10.2 Laminin 2 chain restoration in dy/dy mouse (Vilquin et al . ) Table III. Characteristics of the conditionally immortalized myogenic cells. Myogenic cells were selected in vi tro based on desmin expression and the ability to form β-gal positive myotubes i differentiation medium. The cells were injected in FK506-immunosuppressed, 4 to 8 week=old dy mice with or without addition of primary muscle cell culture. 2, 3 or 4 weeks later the mice were sacrificed, β-gal and LAMA2 expression were investigated on adjacent sections. Typical β-gal and LAMA2 expression are presented in Fig. 6. a : only the myogenic clone or colony injected. b : myogenic clone or colony injected together with primary muscle cell culture. c : number of mice expressing LAMA2; (n) total number of mice in one experiment. d : number of mice expressing β-gal; (n) total number of mice in one experiment. e : 2 or 3 fibers were faintly surrounded. ND: not done. In vi tro characterisation Injection in left Injection in right leg leg to Cell Passage# Desmin β-gal LAMA2- β-gal LAMA2- β-gal culture expression expression positive positive positive positive __!_ _i_ _____ (n)d MB3 9 pos (>95%) myotubes 0(1) e 1(1} ND ND MB7 7 pos (>95%) myotubes 0(3) e 3(3) 2(2) 2(2) Colony #24 4 pos (>90%) myotubes 0(1) 1(1) 1(1) l(l) MB27 8 pos (>90%) myotubes 0(3) 3(3) 3(3) 3(3) REFERENCES Adam, M., Ramesh, N. , Miller, D.A. and Osborne, W.R.A. Internal initiation of translation in retroviral vectors carrying picornavirus 5' nontranslated regions. J. Virol. 65:4985-4990, 1991. ALBERT, N. and TREMBLAY, J.P. Evaluation of various transfection methods of gene into human myoblast clones. Transpl. Proc. 24, 2784-2786, 1992 Andre, F., P. Filippi, and H. Feracci. 1994. Merosin is synthesized by thyroid cells in primary culture irrespective of cellular organization. J " . Cell Sci . 107: 183-193. Anton, E.S., A.W. Sandroc , and W.D. Matthew. 1994. Merosin promotes neurite growth and Schwann cell migration in vitro and nerve regeneration in vivo: evidence using an antibody to merosin, ARM-l. Dev. Biol . 164: 133-146. ARAHATA, K., HAYASHI, Y.K., KOGA, R., GOTO, K., LEE, J.H., MIYAGOE, Y., ISHII, H., TSUKAHARA, T., TAKEDA, S., WOO, M., NONAKA, I., MATSUZAKI, T. and SUGITA, H. Laminin in animal models for muscular dystrophy. Defect of laminin M in skeletal and cardiac muscles and peripheral nerve of the homozygous dystrophic dy/dy mice. Proc. Japan Acad. Ser B: 259-264, 1993. ASSELIN, I., TREMBLAY, M. , VILQUIN, J.T., GUERETTE, B., ROY, R. and TREMBLAY, J.P. Quantification of normal dystrophin mRNA following myoblast transplantation. Muscle and Nerve 18:980-986, 1995. ASSELIN, L, TREMBLAY, M. , VILQUIN, J.T., GUERETTE, B. and TREMBLAY, J.P. Polymerase chain reaction-based assay to assess the success of myoblast transplantation in mdx mice. Trans . Proc. 26:3389, 1994. Beauchamp, J.R., Morgan, J.E., Pagel, CM. and Partridge, T.A. 1994. Quantitative studies of efficacy of myoblast transplantation. Muscle and Nerve Suppl. 4:S261 (Abstract) . BELLES-ISLES, M., ROY, R., DANSEREAU, G., GOULET, M., ROY, B., BOUCHARD, J.P. and TREMBLAY, J.P. Rapid selection of donor myoblast clones for muscular dystrophy therapy using cell surface expression of NCAM. Eur. J. Histochem. 37, 375-380, 1993. Borlongan, C.V. and Sandberg, P. 1993. Microtransplantation of nigral dopamine neurons in a rat model of Parkinson's disease. Cell Transplantation 4:347. Bradley, W.G., and M. Jenkison. 1973. Abnormalities of peripheral nerves in murine muscular dystrophy. J. Neuroi . Sci . 18: 227-247. Buckle, V.J., J.L. Guenet, D. Simon-Chazottes, D.R. Love, and K.E. Davies. 1990. Localisation of a dystrophin-related autosomal gene to 6q24 in man, and to mouse chromosome 10 in the region of the dystrophia muscularis (dy) locus. Hum . Genet . 85: 324-326. BULFIELD G. SILLER WG, WIGHT PAL and MOORE KJ. X chromosome- linked muscular dystrophy (mdx) in the mouse. Proc. Natl Acad. Sci. 81:1180-1192, 1984. Burgeson, R.E., M. Chiquet, R. Deutzmann, P. Ekblom, J. Engel, H. Kleinman, G.R. Martin, G. Meneguzzi, M. Paulsson, J. Sanes. R. Timpl, K. Tryggvason, Y. Yamada, and P.D. Yurchenko. 1994. A new nomenclature for the laminins. Ma trix Biol . 14: 209-211. Campbell, K.P. 1995. Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage. Cell 80: 675-679. Dai, Y., Roman, M. , Navlaux, R.K. and Ver a, I.M. 1992. Gene therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo. Proc. Natl. Acad. Sci. USA 89:10892-10895. Dhawan, J., Pan, L.C, Pavlath, O.K., Travis, M.A. , Lanctot, A.M. and Blau, H.M. 1991. Systemic delivery of human growth hormone by injection of genetically engineered myoblasts. Science 254:1509-1512. Dubowitz, V., and M. Fardeau. 1995. Workshop report: Proceedings of the 27th ENMC sponsored workshop on congenital muscular dystrophy. Neuromusc . Disord. 5: 253-258. Ehrig, K. , I. Leivo, W.S. Argraves, E. Ruoslahti, and E. Engvall. 1990. Merosin, a tissue-specific basement membrane protein, is a laminin-like protein. Proc . Na tl . Acad. Sci . USA 87: 3264-3268. Emerich, D.F. 1993. Cell transplantation of Huntington's disease. Cell Transplantation 4:348. Engvall, E. 1993. Laminin variants: why, where and when? Kidney Int . 43: 2-6. Engvall, E., D. Earwicker, A. Day, D. Muir, M. Manthorpe, and M. Paulsson. 1992. Merosin promotes cell attachment and neurite outgrowth and is a component of the neurite-promoting factor of RN22 schwannoma cells. Exp . Cell Res . 198: 115-123. Entman, M.L. Youker, K. , Shaji, T. , Kukielka, G., Shappell, S.B., Taylor A.A. , Smith, CW. , Neutrophil induces oxidative injury of cardiac myocytes - J. Clin. Invest. 1992, 1335-1345. Grounds, M., T.A. Partridge, and J.C Sloper. 1980. The contribution of exogenous cells to regenerating skeletal muscle: an isoenzyme study of muscle allografts in mice. J. Pa thol . 132: 325-341. Guerette, B., I. Asselin, J.T. Vilquin, I. Kinoshita, R. Roy, and J.P. Tremblay. 1995. Lymphocyte infiltration following allo and xeno myoblast transplantation in mdx mice. Muscle Nerve . 18: 39-51. GUERETTE, B., GINGRAS, M., WOOD, K., ROY, R. and TREMBLAY J.P. Immunosuppression with monoclonal antibodies following myoblast transplantation in mice. Transplantation (submitted) . GUERETTE, B., ASSELIN, I., ROY, R., TREMBLAY, J.P. Lymphocyte infiltration following allo and xeno myoblast transplantation in mdx mice. Muscle and Nerve 18:39-51, 1995. GUERETTE, B., ROY, R., TREMBLAY, M., ASSELIN, I., KINOSHITA, I. and TREMBLAY, J.P. Increased granzyme B mRNA following alloincompatible myoblast transplantation. Transplantation (in press) . Hallauer, J.B., H.L. Bradshaw, and K.E.M. Hasting. 1993. Complex fiber-type-specific expression of fast skeletal muscle troponin I gene constructs in transgenic mice. Development 119: 691-701 Harris, J.B., M.A. Johnson, and E. Karlsson. 1975. Pathological responses of rat skeletal muscle to a single subcutaneous injection of a toxin from the venom of the Australian tiger snake, Notechis scutatus scutatus. Clin. Exp . Pharm . Phys . 2: 383-404. Hayashi, Y.K., E. Engvall, E. Arikawa-Hirasawa. K. Goto, R. Koga, I. Nonaka, H. Sugita, and K. Arahata. 1993. Abnormal localization of laminin subunits in muscular dystrophies. J. Neuroi . Sci . 119: 53-64. Hering, B.J., Browatzki, CC, Schultz, A., Bretzel, R.G. and Federlin, K.F. Clinical islet transplantation - registry report, accomplishments in the past and future research needs. Cell Transplantation 4:269. Hillaire, D., A. Leclerc, S. Faure, H. Topaloglu, N. Chiannilkulchai, P. Guicheney, L. Grinas, P. Legos, J. Philpot, T. Evangelista, M.-C Routon, M. Mayer, J.-F. Pellissier, B. Estournet, A. Barois, F. Hentati, N. Feingold, J. S. Beckmann, V. Dubowitz, F.M.S. Tome, and M. Fardeau. 1994. Localization of merosin-negative congenital muscular dystrophy to chromosome 6q2 by homozygosity mapping. Hum. Mol . Genet . 3: 1657-1661. Hironaka, T. , and Y. Miyata. 1973. Muscle transplantation in the aetiological elucidation of murine muscular dystrophy. Nature 244: 221-223. Huard, J., Bouchard, J.P., Roy, R., Malouin, F., Dansereau, G., Labrecque, C, Albert, N., Richards, CL., Lemieux, B. and Tremblay, J.P. 1992. Human myoblast transplantation: Preliminary results of 4 cases. Muscle and Nerve 15:550-560. Huard, J., Acsadi, G., Jani, A., Massie, B. and Karpati, G. 1994. Gene transfer into skeletal muscles by isogenic myoblasts. Human Gene Therapy 5:949-958. HUARD, J., ROY, R. # BOUCHARD, J.P., LABRECQUE, C , MALOUIN, F., RICHARDS, C and TREMBLAY, J.P. Human myoblast transplantation between immunohistocompatible donors and recipients produce immune reactions. Transpl . Proc. 24, 3049-3051, 1992b. HUARD, J., ROY, R., GUERETTE, B., VERREAULT, S., TREMBLAY, G., TREMBLAY, J.P. Human myoblast transplantation in immunodeficient and immunosuppressed mice: evidence of rejection. Muscle and Nerve 17, 224-234, 1994a. HUARD, J., TREMBLAY, G. , VERREAULT, S., LABRECQUE, C, TREMBLAY, J.P. Utilization of an antibody specific for human dystrophin to follow myoblast transplantation in mice. Cell Transpl. 2, 113-118, 1993. HUARD, J., VERREAULT, S., ROY, R., TREMBLAY, J.P. High efficacy of muscle regeneration following human myoblast transplantation in SCID mice. J. of Clin. Invest. 93, 586-599, 1994b. HUARD, J., LABRECQUE, C, DANSEREAU, G., ROBITAILLE, L., TREMBLAY, J.P. Dystrophin expression in myotubes formed by the fusion of normal and dystrophic myoblasts. Muscle and Nerve 14, 178-182, 1991b. HUARD, J., BOUCHARD, J.P., ROY, R., LABRECQUE, C, DANSEREAU, C, LEMIEUX, B. and TREMBLAY, J.P. Myoblast transplantation produces dystrophin positive muscle fibers in a 16 year old Duchenne patient. Clinical Science 81, 287-288, 1991a. Jat, P.S., M.D. Noble, P. Ataliotis, Y. Tanaka, N. Yannoutsos, L. Larsen and D. Kioussis. 1991. Direct derivation of conditionally immortal cell lines from an H-2K b -tsA58 transgenic mouse. Proc . Na tl . Acad. Sci . USA 88: 5096-5100. Kinoshita, I., J.T. Vilquin, B. Guerette, I. Asselin, R. Roy, and J.P. Tremblay. 1994b. Very efficient myoblast allotransplantation in mice under FK506 immunosuppression. Muscle Nerve . 17: 1407-1415. KINOSHITA, I., VILQUIN, J.T. and TREMBLAY, J.P. Pretreatment of myoblast cultures with basic fibroblast growth factor increases the efficacy of their transplantation in mdx mice. Muscle and Nerve 18:834-841, 1995. KINOSHITA, I., VILQUIN, J.T., GRAVEL, C, ROY, R. and TREMBLAY, J.P. Myoblast allotransplantation in primates. Muscle and Nerve 18:1217-1218, 1995 KINOSHITA, I., ROY, R., DUGRE, F.J., GRAVEL, C, VILQUIN, J.T., ROY, B., GOULET, M., ASSELIN, I. and TREMBLAY, J.P. Myoblast transplantation in monkeys: control of immune response by FK506. J. Neuropath, and Exp. Neuroi. submitted. KINOSHITA, I., HUARD, J. and TREMBLAY, J.P. Utilization of myoblasts from a transgenic mouse to evaluate the efficacy of myoblast transplantation. In Muscle and Nerve, 17:975-980, 1994a. KINOSHITA, I., VILQUIN, J.T. and TREMBLAY, J.P. Mechanism of increasing dystrophin-positive myofibers by myoblast t ansplantation: study using mdx/beta-galactosidase transgenic mice. Acta Neuropathologica (in press) . KINOSHITA, I., VILQUIN, J.T., GUERETTE, B., ASSELIN, I., ROY, R., LILLE, S., TREMBLAY, J.P. Immunosuppression with FK506 insures good success of myoblast transplantation in mdx mice. Trans. Proc. 26:3518, 1994c. KINOSHITA, I., VILQUIN, J.T., GUERETTE, B., ASSELIN, I., ROY, R. and TREMBLAY, J.P. Very efficient myoblast allotransplantation in mice under FK506 immunosuppression. Muscle and Nerve 17, 1407-1415, 1994b. LABRECQUE, C, ROY, R. and TREMBLAY, J.P. Immune reactions following myoblast transplantation in mouse muscles. Transplantation Proc. 24, 2889-2892, 1992. Law, P.K. 1982. Beneficial effects of transplanting normal limb-bud mesenchyme into dystrophic mouse muscles. Muscle Nerve 5: 619-627. Law, P.K. , T.G. Goodwin, and H.-J. Li. 1988a. Histoincompatible myoblast injection improves muscle structure and function of dystrophic mice. Transpl . Proc . 20 (Suppl 3) : 1114-1119. Law, P.K., T.G. Goodwin, and M.G. Wang. 1988b. Normal myoblast injections provide genetic treatment for murine dystrophy. Muscle Nerve . 11: 525-533. Leivo, I., and E. Engvall. 1988. 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. Lin, Z., M.-H. Lu, T. Schultheiss, J. Choi, S. Holtzer, C. DiLullo, D.A. Fischman, and H. Holtzer. 1994. Sequential appearance of muscle-specific proteins in myoblasts as a function of time after cell division: evidence for a conserved myoblast differentiation program in skeletal muscle. Cell Motil . Cytoskel . 29: 1-19. Madrid, R.E., E. Jaros, M.J. Cullen, and W.G. Bradley. 1975. Genetically determined defect of Schwann cell basement membrane in dystrophic mouse. Na ture 257: 319-321. MEOLA, G., TREMBLAY, J.P., ROTONDO, G., BRESOLIN, N., HOARD, J. and SCARLATO, G. Muscle glucose-6-phosphate dehydrogenase deficiency: restoration of the enzymatic activity in hybrid myotubes. Muscle and Nerve 16:594-600, 1993. Michelson, A.M., E.S. Russell, and P.J. Harman. 1955. Dystrophia muscularis: a hereditary primary myopathy in the house mouse. Proc . Natl . Acad. Sc . USA 41:1079-1084. Montgomery, A., and L. Swenarchuk. 1977. Dystrophic mice show age related muscle fibre and myelinated axon losses. Nature 267: 167-169. Morgan, J.E., S.E. Moore, F.S. Walsh, and T.A. Partridge. 1992. Formation of skeletal muscle in vivo from the mouse C2 cell line. J. Cell Sci . 102: 779-787. Morgan, J.E., CN. Pagel, T. Sherratt, and T.A. Partridge. 1993. Long-term persistence and migration of myogenic cells injected into pre-irradiated muscles of mdx mice. J. Neuroi . Sci . 115: 191-200. Morgan, J.E., E.P. Hoffman, and T.A. Partridge. 1990. Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse. J. Cell Biol . Ill: 2437-2449. Morgan, J.E., J.R. Beaucha p, CN. Pagel, M. Peckham, P. Ataliotis, P.S. Jat, M.D. Noble, K. FarMerosinj and T.A. Partridge. 1994. Myogenic cell lines derived from transgenic mice carrying a thermolabile T antigen: a model system for the derivation of tissue-specific and mutation-specific cell lines. Develop . Biol . 162: 486- 498. Partridge, T.A. 1991. Myoblast transfer: A possible therapy for inherited myopathies? Muscle and Nerve 14 :197-212. Partridge T.A., M. Grounds, and J.C Sloper. 1978. Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Nature. 273: 306-308. Partridge, T.A., J.E. Morgan, G.R. Coulton, E.P. Hoffman, and L.M. Kunkel. 1989. Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature . 337: 176-179. PASTORET C and SEBILLE A. Mdx mice show progressive weakness and muscle deterioration with age. J. Neuroi. Sci. 129:97-105, 1995. Philpot, J., C Sewry, J. Pennock, and V. Dubowitz. 1995. Clinical phenotype in congenital muscular dystrophy: correlation with expression of merosin in skeletal muscle. Neuromusc . Disord. 5: 301-305. Rantanen, J. , T. Hurme, R. Lukka, J. Heino, and H. Kalimo. 1995. Satellite cell proliferation and the expression of myogenin and desmin in regenerating skeletal muscle: evidence for two different populations of satellite cells. Lab . Inves t . 72: 341-347 Raper, S.E. and Wilson, J.M. 1993. Cell transplantation in liver-directed gene therapy. Cell Transplantation 2:381. Rosenstein, J.M. 1993. New horizons in neural transplantation. Cell Transplantation 3:183. ROY, R., DANSEREAU, C, TREMBLAY, J.P., BELLES-ISLES, M., LABRECQUE, C, BOUCHARD, J.P. Expression of major histocompatibility complex (MHC) antigens on human myoblasts. Transplantation Proceeding 23, 799-801, 1991. ROY, R., TREMBLAY, J.P., HUMD, J., RICHARDS, C.L., MALOUIN, F., BOUCHARD, J.P. Antibody formation after myoblast transplantation in Duchenne dystrophic patients, donor HLA compatible. Transpl. Proc. 25, 995997, 1993. Salatsky, B. 1971. Functional studies of regenerated muscles from normal and dystrophic mice. Nature 229: 270-272. Sambrook, J., Fritsch, E.F. and Maniates, T. 1989. Molecular cloning 2nd ed. Cold Spring Harbor Laboratory Press, pp 16.66-16.67. SANSONE, V., ROTONDO, C, BOTTIROLI, G., TREMBLAY, J.P. AND MEOLA, G. Cytoplasmic restoration and persistence of glucose-6-phosphate dehydrogenase activity in stable hybrid myotubes. Eur. J. Histochem. 37: 241-248, 1993. SATOH, A., LABRECQUE, C and TREMBLAY, J.P. Myotubes can be formed within implanted adipose tissue. Transplantation Proc, 24, 3017-3019, 1992. SATOH, A., LABRECQUE, C and TREMBLAY, J.P. Use of fluorescent latex microspheres (FLMs) to follow the fate of transplanted myoblasts. J. Histochem. Cytochem. 41:1579-1582, 1993. Sewry, A. , J. Philpot, D. Mahony, L.A. Wilson, F. Muntoni, and V. Dubowitz. 1995. Expression of laminin subunits in congenital muscular dystrophy. Neuromusc. Disord. 5: 307-316. SICINSKI P. RIDER-COOK AS, GENG YA, BARNARD KG, DARLISON M and BARNARD J. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 244:1578-1579, 1989. Skuk, D., Asselin, I., Celestin, F., Entman, M. and Tremblay, J.P. 1996. Neutrophil infiltration after myoblast transplantation in mice. Muscle and Nerve, submitted. SUNADA, Y., BERNIER, S.M., KOZAK, C.A., YAMADA, Y. and CAMPBELL, K.P. Deficiency of Merosin in dystrophic dy mice and genetic linkage of laminin M chain gene to dy locus. J. Biol. Chem. 269:13729-13732, 1994. SUNADA, Y., BERNIER, S.M., UTANI, A., YAMADA, Y. and CAMPBELL, K.P. Identification of a novel mutant transcript of laminin α2 chain gene responsible for muscular dystrophy and dysmyelination in dy 2d mice. Hum. Mol. Genet. 6:1055-1061,1995. Toda, T., M. Segawa, Y. Nomura, I. Nonaka, K. Masuda, T. Ishihara, M. Suzuki, I. Tomita, Y. Uriguchi, K. Ohno, M. Misugi, Y. Sasaki, K. Takada, M. Kawai, K. Otani, T. Murakami, K. Saito, Y. Fukuyama, T. Shimizu, I. Kanazawa, and Y. Nakamura. 1993. Localization of a gene for Fukuyama type congenital muscular dystrophy to chromosome 9q31-33. Nature Genet. 5: 283-286. Tome, F.M.S., T. Evangelista, A. Leclerc, Y. Sunada, E. Manole, B. Estournet, A. Barois, K.P. Campbell, and M. Fardeau. 1994. Congenital muscular dystrophy with merosin deficiency. C. R. Acad. Sci . Paris 317: 351-357. Tremblay, J.P., Malouin F., Roy, R. , Huard, J., Bouchard, J.P., Satoh, A. and Richards, CL. 1993. Results of a triple blind clinical study of myoblast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy. Cell Transplantation 2:99-112. TREMBLAY, J.P., ROY, B. and GOULET, M. Human myoblast transplantation: a simple assay for tumorigenicity. Neuromuscular Disorders 1, 341-343, 1991a. 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. TREMBLAY, J.P., ROY, R., BOUCHARD, J.P., HUARD, J., LABRECQUE, C, DANSEREAU, C, LEMIEUX, B. Human myoblast transplantation. Proceeding of Satellite Symposium on Muscular Dystrophy Research 1990: From Molecular Diagnosis toward Therapy. Elsevier Science Publisher 1991b, C Angelini et al. eds, pp 123-130. Vilquin, J.T. , Kinoshita, I., Roy, B., Goulet, M., Engvall, E., Tome, F., Fardeau, M. and Tremblay, J.P. Partial laminin α2 chain-deficient dy/dy mouse by primary muscle cell culture transplantation. J. Cell Biol. (in press) . Vilquin, J.-T., I. Asselin, B. Guerette, I. Kinoshita, S. Lille, R. Roy, and J.P. Tremblay. 1994. Myoblast allotransplantation in mice: degree of success varies depending on the efficacy of various immunosuppressive treatments. Transpl . Proc . 26: 3372-3373. Vilquin, J.-T., B. Guerette, I. Kinoshita, B. Roy, M. Goulet, C Gravel, R. Roy and J.P. Tremblay. 1995b. FK506 immunosuppression to control the immune reactions triggered by first generation adenovirus-mediated gene transfer. Hum. Gene Therapy 6: 1391-1401. Vilquin, J.-T., E. Wagner, I. Kinoshita, R. Roy, and J.P. Tremblay. 1995c. Successful histocompatible myoblast transplantation in dystrophin-deficient mdx mouse despite the production of antibodies against dystrophin. J. Cell Biol . 131: 1-14. VILQUIN, J.T., KINOSHITA, I., ROY, R. and TREMBLAY, J.P. Cyclosphosphamide versus FK506 for mice immunosuppression after myoblast allotransplantation. Neuromuscular Disorders 5, 511-517, 1995b. VILQUIN, J.T., ASSELIN, I., GUERETTE, B., KINOSHITA, I., LILLE, S., ROY, R., TREMBLAY, J.P. Myoblast allotransplantation in mice: the degree of success varies depending on the efficacy of various immunosuppressive treatments. Trans. Proc. 26: 3372-3, 1994 VILQUIN, J.T., ASSELIN, I., GUERETTE, B., KINOSHITA, I., LILLE, S., ROY, R. and TREMBLAY, J.P. Successful myoblast allotransplantation in mdx mice using rapamycin. Transplantation 59:422-449, 1995a. Vuolteenaho, R. , M. Nissinen, K. Sainio, M. Byers, R. Eddy, H. Hirvonen, T.B. Shows, H. Sariola, E. Engvall, and K Tryggvason. 1994. Human laminin M chain (merosin) : complete primary structure, chromosomal assignment, and expression of the M and A chain in human fetal tissues. J. Cell Biol . 124: 381-394. Wakeford, S., D.J. Watt, and T.A. Partridge. 1991. X-irradiation improves mdx mouse muscle as a model of myofiber loss in DMD. Muscle Nerve . 14: 42-50. WALDMANN H K and COBBOLD S. The use of monoclonal antibodies to achieve immunological tolerance . Trends Pharmacol. Sci. 14:143-148, 1993. Watt, D.J., J.E. Morgan, and T.A. Partridge. 1984. Long term survival of allografted muscle precursor cells following a limited period of treatment with cyclosporin A. Clin . Exp . Immunol . 55: 419-426. Wernig, A., A. Irintchev, A. Hartling, G. Stephan, K. Zimmerman, and A. Stariznski-Powitz . 1991. Formation of new muscle fibres and tumours after injection of cultured myogenic cells. J. Neurocy tol . 20: 982-997. Wirtz P., H. Loermans, and E. Rutten. 1982. Effects of irradiation on regeneration in dystrophic mouse leg muscles. Br . J. Exp . Pa th . 63: 671-679. XU, H., CHRISTMAS, P., WU, X.-R., WEWER, U.M. and ENGVALL. Defective muscle basement membrane and lack of M-laminin in the dystrophic dy/dy mouse. Proc. Natl. Acad. Sci. USA 91:5572-5576, 1994a. XU, H., WU, X.-R., WEWER, U.M. and ENGVALL, E. Murine muscular dystrophy caused by a mutation in the laminin 2 (Lama2) gene. Nature Genetics 8:297-302, 1994b.

Claims: WHAT IS CLAIMED IS: 1. A method of restoring a functional protein in a tissue producing a corresponding defective protein, by transplanting donor cells which express said functional protein in a patient in need of such a transplantation who expresses said defective protein, said method comprises the step of blocking the interaction between a ICAM-I molecule located at the membrane surface of the donor cells and a LFA-I molecule located at the membrane surface of the patient's leucocytes, which blocking increases the viability of the transplanted donor cells. 2. A method according to claim 1, wherein said blocking is effected by treating said patient with of an anti-LFA-I antibody prior to transplantation. 3. A method according to claim 1, wherein said blocking is effected by treating said patient with an anti-ICAM-I antibody. 4. A method according to claim 3, wherein said antibody is an antibody fragment which is non-capable of fixing complement. 5. A method according to claim 4, wherein said fragment is a F(ab) 2 fragment. 6. A method according to claim 1, which further comprises matching the donor and the patient for their MHC-histocompatibility. 7. A method according to claim 1, which further comprises the step of concurrently immunosuppressing the patient . 8. A method according to claim 7, wherein the immunosuppressing is achieved by administrating rapamycin or FK506. 9. A method according to claim 1, wherein said donor cells are cultured as primary cultured cells in the presence of a trophic factor prior to transplantation. 10. A method according to claim 1, wherein said donor cells are cloned cells grown in the presence of a trophic factor prior to transplantation. 11. A method according to claim 10, wherein said cloned cells are transfected with a recombinant vector comprising a gene encoding a molecule encouraging cell proliferation under the control of a promoter compatible with the donor's cells, said promoter being inducible by an activating factor, prior to or following transplantation. 12. A method according to claim 11, wherein said molecule encouraging cell proliferation is SV40 T antigen. 13. A method according to claim 12, wherein said promoter is a promoter of a major histocompatibility gene. 14. A method according to claim 13 , wherein said promoter is a MHC class II gene promoter. 15. A method according to claim 14, wherein said activating factor is γ-IFN. 16. A method according to claim 1, wherein said patient suffers of muscle cell degeneration or weakness. 17. A method according to claim 16, wherein said patient suffers of a myopathy. 18. A method according to claim 17, wherein said patient suffers of muscular dystrophy. 19. A method according to claim 18, wherein said patient suffers of Duchenne muscular dystrophy. 20. A method according to claim 16, wherein said donor cells are myogenic cells or non-myogenic cells. 21. A method according to claim 17, wherein said donor cells are myogenic cells or non-myogenic cells. 22. A method according to claim 19, wherein said donor cells are myogenic cells.

Inventors: GUERETTE BENOIT, ROY RAYNALD, TREMBLAY JACQUES P, VILQUIN JEAN-THOMAS, GUERETTE, BENOIT, ROY, RAYNALD, TREMBLAY, JACQUES, P., VILQUIN, JEAN-THOMAS
Applicants: GUERETTE BENOIT, ROY RAYNALD, TREMBLAY JACQUES P, UNIV LAVAL, VILQUIN JEAN THOMAS, GUERETTE, BENOIT, ROY, RAYNALD, TREMBLAY, JACQUES, P., UNIVERSITE LAVAL, VILQUIN, JEAN-THOMAS
Application year: 1996
Main classification: A61K35
IPC codes: A61K35, A61K38, C07K16