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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0656—Adult fibroblasts
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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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
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• This invention relates to the treatment of muscular disorders, in particular those resulting from the wastage of muscle such as muscular dystrophy or from muscle degeneration, e.g. as a result of traumatic injury.
• Skeletal muscle cells are composed of multinucleated cylindrical structures 10-100 ⁇ m in diameter and of lengths of the order of millimetres or even centimetres, surrounded by a plasma membrane.
• the cells are capable of contraction and arise by fusion of myoblasts, which are immature muscle cells.
• DMD Duchenne muscular dystrophy
• X-chromosome-linked genetic defect which results in a lack of production of the protein dystrophin. Since it is unlikely that the defect will occur in linkages to both X chromosomes of a female, and since it is recessive in character, it is transmitted only to males through the defective X-linkage.
• Becker muscular dystrophy (BMD) also results from an X-linked genetic defect.
• myoblast transfer therapy a form of therapy, termed myoblast transfer therapy, to alleviate the myopathic condition in DMD muscles.
• myoblast transfer therapy a form of therapy, termed myoblast transfer therapy.
• myoblasts irradiated muscular dystrophic mice.
• Mdx mice unlike humans, spontaneously regenerate the affected muscle. Irradiation of an area of the skeletal muscle makes it unable to regenerate the muscle in this area.
• non- diseased mice These authors inhibited the natural regeneration of muscle in mdx mice by applying a high dose of X-rays locally to a leg of the mice 3 days before injecting them with the culture of the neonatal cells.
• the proposed therapy is to take myoblasts from normal donors and insert them into the muscles of DMD boys.
• the source of donor myoblasts would be from a histocompatibility-matched donor (often the father) where problems of immune rejection would be reduced and where the presence of the normal counterpart of the dystrophin gene and its protein product would replace that missing in the DMD muscle.
• the object is to effect fusion of these cells with the muscle fibres of the DMD boys which are themselves dys rophin-negative.
• the myogenic cells of DMD boys are further down the senescence pathway than "normal" non-DMD myoblasts, for they have already passed through several bouts of degeneration/regeneration and hence mitoses. Given the Hayflick phenomenon, that cells divide a quantum number of times (50) before death, the strategy of using these "older" cells is not ideal.
• the present invention is based on the radically different idea of using fibroblasts of dermal origin as the donor cells. Surprisingly, it has been found that when dermal fibroblasts are implanted in muscles of an animal which has a muscular disorder, these donor fibroblasts appear to fuse together to make a multinucleate cell which behaves like a muscle cell. Since these newly-formed cells express the same products (e.g. dystrophin, desmin, carbonic anyhdrase III) as are specific to native muscle cells in the normal animal (and, therefore, not found inter alia in the donor fibroblasts), they are considered to have been converted to the myogenic lineage.
• the same products e.g. dystrophin, desmin, carbonic anyhdrase III
• An important aspect of the invention is therefore defined as a method of treatment of muscular disorder in a patient, which comprises administering to or adjacent to the muscle cells of the patient immunologically compatible dermal fibroblasts under conditions effective to convert the dermal fibroblasts to myogenic cells capable of producing products expressed by muscle- specific genes.
• a method of therapeutic treatment is not protectable, the use of the donor cells for the stated purpose or in the preparation of a formulation of the cells for the stated purpose should be substituted for the above method definition, as legally appropriate or conventional.
• the treatment is based on the notion that at least some of the donor fibroblasts fuse with each other, express muscle-specific genes, and thereby become cells of the myogenic lineage, notably muscle cells or satellite cells thereof.
• the effect is most easily seen in the irradiated mdx mouse.
• the same human fibroblasts as may be administered to a human patient, but possibly carrying a marker, are administered to irradiated mdx mice, the effect can be observed and such a test in mice may be considered as a test of the competence of the dermal cells (if any be needed) to be effective in the treatment of humans.
• the treatment aims to increase the number of dystrophin-positive cells found in the muscle of the treated patient, e.g. by muscle biopsy, the production of the muscle-specific protein dystrophin being regarded as a measure of success in conversion to normal muscle.
• Example 8 This invention has been particularly well demonstrated by Example 8, in which dermal fibroblast cells, from a human dystrophin gene-positive transgenic mouse, were implanted into an mdx mouse lacking dystrophin-positive muscle fibres.
• the mdx mouse muscle fibres became dystrophin positive and this gene could only have been provided by the donor cells.
• the gene was chromosomally incorporated into the dermal fibroblasts and expressed in a different cell type, viz. muscle cells.
• the invention relates primarily to skeletal muscle but could be applied to cardiac or smooth muscle. It is of interest mainly in relation to muscular dystrophy, particularly DMD or BMD, but could be applied to any wasting or degenerative disorder of the muscle, including injured or damaged muscle. This includes traumatic injury, but not, of course an injury so severe that the muscle "architecture" is wholly destroyed. Where the patient lacks or substantially lacks dystrophin-producing or another muscle-specific gene or possesses such a gene in a sub-normal concentration, the dermal fibroblasts administered contain the relevantly required DNA. The dermal cells may be transfected with this DNA in any of the usual ways, preferably so that it is incorporated within the cell nucleus.
• a retroviral vector could alternatively be used.
• Dermal fibroblasts have been infected with retroviral vectors for gene therapy, see e.g. J. H. Axelrod et al., Proc. Natl. Acad. Sci. USA, 87, 5173-5177 (1990), D. St. Louis and I. M. Verma ibid., 85, 3150-3154 (1988), T.D. Palmer et al, Blood 73, 438-445 (1989) and R. I. Garver Jr.
• dystrophin or other muscle-specific DNA for the factor IX and alpha- 1 anti-trypsin genes of these references.
• Another way is direct injection of the muscle-specific DNA into the fibroblasts.
• the manner of incorporation of the relevant DNA into the fibroblasts is not critical and other methods will be apparent to those skilled in the art.
• the relevant DNA may be a full length gene or only part thereof.
• a partial gene such as the so-called dystrophin minigene, see M. Dunckley et al, FEBS 296, 128-134 (1992) is often useful, for both BMD and DMD, especially where a retroviral vector is being used.
• the full length dystrophin gene which has been described by N. Wells et al, Human Mol. Gen. 1, 35-40 (1992), is useful in methods where no vector can present or package DNA of such a long length.
• Appropriate regulatory sequence is included as may be required by the technique used, as is well known in the art.
• the patient is not totally lacking in muscle cells when the treatment of the present invention is applied.
• a chemotactic factor present in the muscle cells may aid in the conversion of fibroblasts to muscle cells.
• the invention includes specifically dermal fibroblasts containing, especially within the cell nucleus, a muscle-specific DNA, especially DNA encoding part or all of the dystrophin-protein.
• the DNA may be a full length or a partial gene, as noted above.
• Cell suspensions and biologically pure, especially serum-free, culture media containing such cells are also included within the invention. None in this specification is to be construed as laying claim to rights in a part of the human body except when isolated therefrom.
• the donor fibroblasts need not contain any foreign DNA.
• the donor cells must be immunologically compatible with the patient to be treated in order to prevent their being rejected as foreign. Thus, some attempt will normally be made to match HLA type, as in other kinds of treatment involving the donation of body tissue.
• the donor cells are from the patient being treated. These cells are very easily harvested, e.g. by biopsy, to remove tissue from the dermis.
• the route of administration of the donor cells is not critical so long as they are brought into intimate or near contact with the patient's muscle cells. Intramuscular injection (into the muscle) is the preferred route; intermuscular injection (between muscles) is possible.
• the cells can be prepared for administration as follows. Skin biopsies are taken from the DMD patient to include the dermal layer. Dermal fibroblasts are grown in culture, but in serum-free medium, in case when the dermal cells are re-introduced to the patient, elements of animal serum proteins cause any immune response or contamination problems. Thus, a medium using serum-free supplements is normally required. Following growth of the dermal cells in culture, the normal counterpart of the defective gene (when required) is delivered to the cells. The cells are formulated as a suspension in a serum-free medium or as a cell pellet. Virological and bacteriological testing is compulsory before re- implantation.
• the donor cells will normally be administered at a single time and at more than one site in the affected muscle. Generally, they will be administered, normally by injection, at 30 or more sites, especially 30-100. The total amount of dermal cells administered will usually be in the range 10 6 to 10 10 for humans.
• the invention is mainly of interest for treating human patients, it includes treatment of non-human animals such as pets, livestock and animals used in the production of food.
• the dosage of cells will need adjustment with size of animal, but this is well within the competence of those skilled in the art.
• the skin from neonatal normal mice of strain C57Bl/10ScSn was harvested by ventral incision along the length of the anterior ataominal wall and removing the skin from the underlying tissues.
• the skin was placed for 2 minutes in a petri-dish containing 70% alcohol to remove contaminants from the skin which would cause infection of cells in tissue culture.
• the skin was then removed to a petri-dish containing phosphate buffered saline (PBS) containing the antibiotics 80 IU/ml penicillin and 80 mg/ml streptomycin and 1% "Fungizone" (250 ⁇ g ml amphotericin). Any adherent fat was scraped from the hypodermic surface of the skin.
• PBS phosphate buffered saline
• the skin was placed in a petri dish containing Basal Salt Solution (BSS) containing penicillin and streptomycin (as above) and cut using crossed scalpel blades into 1 mm-' pieces.
• BSS Basal Salt Solution
• the small pieces were removed singly into a tissue culture flask providing a 25 cm** growth area containing the minimum amount of growth medium.
• Minimum amount of medium (1 ml for the 25 cm? growth area TC flask) allows attachment of skin pieces to substratum.
• the growth medium was "DMEM” (Dulbecco's Minimal Eagle's Medium) supplemented with 2 inM L-glutamine, 88 IU/ml penicillin/88 mg/ml streptomycin (all supplied by Life Technologies Ltd) and 10% Fetal Calf Serum (Labtech International).
• DMEM Dulbecco's Minimal Eagle's Medium
• the culture vessel was placed in a 37 ⁇ C incubator in 5% CO2- Three hours after transplanting the skin pieces an additional 1-2 ml of growth medium was added to ensure that the skin did not dry out.
• the skin was left in culture until fibroblasts grew out from explanted skin. The medium was changed every 3 days during this time. When sufficient outgrowth had occurred, the skin pieces were removed to allow the culture to become sub-confluent. On reaching sub-confluency, cells were washed in lx phosphate buffered saline (PBS) and detached from the substratum using 0.25% Trypsin (Life Technologies). The action of trypsin was stopped after detachment of cells by the addition of growth medium containing 10% fetal calf serum. Cells were pelleted by centrifiigation at 350 g for 10 minutes and resuspended in growth medium (as above) to a concentration of 2 x 10-> cells/ml. This suspension was then plated into culture vessels and grown in the same growth medium as above until sub-confluency. This cycle of operations constituted a single passage.
• the cells were passaged thus at least 6 times before implantation into mouse muscle. Some cells were ring-cloned, after passage 19, as follows.
• Muscle cells were prepared from muscle tissue of neonatal mice by disaggregation with trypsin and pangestin, exactly as detailed in D. J. Watt et al, J. Neurological Science 57, 319-331 (1982). The cells were plated out and left for 40 minutes in an incubator, during which time the fibroblastic cells adhered to the plate, but myogenic cells remained in suspension. Growth medium containing myogenic cells was removed and fresh growth medium added to the original flask with attached fibroblasts. Contaminating cells other than fibroblasts were still present in these cultures, but after passage at least 6 times the fibroblast became the predominant cell in the culture and such fibroblasts were used for inj ection .
• nude mdx mice were used as recipients.
• the athymic nude mouse is immunologically compromised, possesses no thymus and hence will accept tissue from any other strain of mouse and indeed xenografted material without the fear of rejection of the implanted foreign tissue.
• each host x-linked muscular dystrophic (mdx) mouse was subject to 1,800 rads X-ir ⁇ adiation. This treatment knocks out the endogenous muscle precursor cells and renders the mdx muscle incapable of regeneration and hence more akin, histopathologically to DMD muscle.
• the method for such irradiation is as indicated in Wakeford et al, Muscle & Nerve, 14, 42-50 (1991).
• Sections for histological examination were washed in distilled water for 2 minutes, stained in haematoxylin for 2 minutes and washed in ⁇ * unning water for 1 minute. After being differentiated in 70% alcohol containing 1% v/v hydrochloric acid for 5 seconds (to differentiate nuclei from cytoplasm) they were "blued” in running tap water for 5 minutes ("blueing" converts acid haematoxylin to neutral haematoxylin which gives the stain its characteristic blue colour). Sections were then counterstained in eosin for 1-2 minutes before being rinsed briefly in water and briefly dipped in 70% alcohol.
• Immunofluorescence images were acquired using a Photonics Science Labstar intensified CCD camera attached to an Olympus BH2 fluorescence microscope. Following acquisition, images were stored on an optical disk. Quantitation of the area of the section that was immunofluorescently labelled was carried out and the territory occupied by fluorescent muscle fibres expressed as a volume fraction (percentage of the frame labelled). Results are shown in Table 1. Mdx mice are dystrophin-negative and in irradiated muscle remain so. Dermal fibroblasts implanted into irradiated mdx muscle resulted in strikingly higher proportions of dystrophin-positive fibres as compared with non-irradiated muscle, when examined after 21 days.
• the lower percentage of dystrophin positivity at 42 days in irradiated muscle reflects the organisation and compactness of the positive fibres seen histologically.
• Lower proportions of dystrophin-positive fibres observed after implanting cloned dermal fibroblasts could be the result of using passage 19 cloned cells which may have lost some pluripotency.
• Dermal Fibroblasts 21 27 8 cloned 21 20 2 21 17 0.2 21 14 2 Muscle Fibroblasts 21 1 2 uncloned 21 1 3 21 3 4 42 3 0.8 42 2 1
• the isoenzymes of GPI were used to assess the relative contribution of host and donor cells to the muscle fibres present in the regenerating implanted TA muscles as follows:
• the cryostat section was overlaid with a small piece of filter paper in order to absorb out the GPI isoenzymes.
• the filter paper with the GPI isoenzymes absorbed to it was loaded onto the surface of an agarose gel and electrofocused. Sites of GPI activity on the gel were revealed by using an agar overlay containing a reaction mixture of fructose-6-phosphate, NADP, glucose-6-phosphate dehydrogenase, phenazine methosulphate and nitroblue tetrazolium.
• Isoelectrofocusing separates three isoenzyme types of GPI in the mouse.
• the most anodally positioned isoenzyme consists of two sub-units coded for by the GPI- lb gene, and characterizes tissues derived from homozygous GPI-lb/GPI-lb mice, such as the 129/ReJ strains of the donor mice.
• the most cathodally positioned isoenzyme comprises two sub- units coded for by the GPI-la gene, and characterizes homozygous GPI-la/GPI/la mice, such as the C57Bl 10ScSn strain of the host (recipient) mice.'
• a "heterodimeric" isoenzyme form arises by association of one sub-unit coded for by the GPI- la gene, and one sub-unit coded for by the GPI-lb gene within the cytoplasm of the syncytial skeletal muscle fibre.
• a heterodimer occurs if mosaic muscle fibres have formed by the fusion of host and donor mono-nuclear precursor cells, resulting in the expression of both host and donor GPI genes with a common cytoplasm.
• EXAMPLE 2 Expression of muscle specific genes within the muscle of the mdx mouse implanted with mouse dermal fibroblasts
• the gene product chosen in this Example to show the expression of muscle-specific proteins within the mdx muscle implanted with mouse dermal fibroblasts was carbonic anydrase III which is expressed in early differentiating muscle.
• Four mdx nude mice were injected with 3 x 10*> cloned mouse dermal fibroblasts, prepared as in Example 1, part 1, into both irradiated and non-irradiated mdx tibialis anterior muscle.
• standard immunocytochemical staining of cryostat sections cut from the muscles was carried out.
• the primary antibody was rabbit anti- carbonic anydrase III (CA III) [obtained from Dr. Gary Coulton, Dept.
• This experiment was set up to investigate the effects of implanting dermal fibroblasts into muscle regenerating after injury and not due to disease, (e.g. mdx) muscle i.e., of normal C57B1/10 mice.
• the fluorescent marker PHK-26 from Sigma Chemical. Co. was used. This substance is a phospholipid which plugs itself into the cell membrane and remains there. It is said to remain in the membrane of the cells which are labelled for up to 100 days following the implantation of the cells in vivo.
• Cloned dermal fibroblasts were incubated in vitro with PHK-26, during which time the cell membrane was stably labelled with this compound. Such cells were then injected into the TA of normal C57B1/10 mice as previously described and left for 3 and 6 weeks.
• Muscles were removed and frozen. Sections were cut from the regenerating muscle. Assay for dystrophin was not undertaken, as the host mice are dystrophin-positive. After 24 days the fluorescent marker was seen associated with the muscle cell membrane, suggesting that these cells arose from dermal fibroblasts..
• dermal fibroblasts carrying the lac Z gene coding for ⁇ -galactosidase were used for implantation into regenerating tibialis anterior muscles of normal (C57B1/10) mice.
• Two sources of lac Z labelled cells were implanted:
• Example 1 part 5 was repeated on 23 mdx nude mice except that the third 8 ⁇ m section was taken for additional dystrophin immunostaining, instead of GPI analysis.
• the 8 ⁇ m sections and the further sections taken at 100 ⁇ m and 200 ⁇ m deeper levels were all stained as described, separately, with the P6 antibody and with another rabbit anti-dystrophin antibody HI 2, available from the same source. Whereas P6 recognises the carboxy terminus, HI 2 recognises the rod domain of the protein.
• the avidin-fluorescein conjugate of Example 1 was replaced by a streptavidin-' exas Red" conjugate for P6 and by a streptavidin-fluorescein conjugate for H12.
• the human dermal fibroblasts were derived from 3 sources: (a) from the facial skin of a 46 year old woman; (b) from the forearm of a 9 year old boy; and (c) from commercially available cells of neonatal foreskin.
• Example 1 At biopsy (under local anaesthesia) the skin sample from the 46 year old donor was placed in 10 ml of DMEM supplemented as in Example 1 except that Australian Fetal Calf Serum (Sigma Chemical Co.) was used. The skin was transferred to a sterile petri dish and then cut using crossed scalpel blades into 1 mm- pieces. The small pieces were removed singly into tissue culture flasks, each providing a 25 cm** growth area and each containing 1 ml of the above growth medium. The flasks were incubated as in Example 1 , except that they were inverted to encourage adhesion of the skin pieces to the substratum of the flask (hence skin pieces were made to hang on to the base of the flask which was placed upside down in the incubator). Additional growth medium was added as in Example 1 to ensure that the skin did not dry out.
• Example 1 The procedure then continued exactly as in Example 1 , part 1.
• the dermal cells from the 9 year old boy were received in a condition in which they had already been grown out from the skin.
• mice and injection of fibroblasts into the mice were as described in Example 1, parts 3 and 4.
• the slow myosin heavy chain is a human muscle-specific gene product. 8 ⁇ m sections were cut at 3, 4 or 5 different step levels through the muscle (varied from experiment to experiment). The cut sections were treated as follows:
• the sections were washed twice for 10 minutes and then for a further 20 minutes in PBS. 10. They were mounted in "Entellan" mounting medium and stored at 4°C before viewing under a fluorescent microscope. Others of the sections were immunostained with the P6 and HI 2 antibodies as described in Example 4. Using all three sources of cells, it was found that implantation into irradiated muscles of the mdx mouse resulted in the presence of small calibre, newly-formed dystrophin-positive fibres. Fewer were seen than when mouse cells were implanted into mouse tissue, but the numbers were still far greater than expected to be due to mouse revertant fibres. The best results were obtained using the neonatal foreskin cells as the donor cell for implantation.
• dystrophin-positive fibres were found, arranged in two groups - one of 33 fibres and the other of 35 fibres flanking the injection site. Greater numbers of dystrophin-positive fibres were found than have been reported by other workers following the injection of human myogenic cells into nude mouse muscles. The best result so far is the presence of 5% of dystrophin-positive fibres following implantation of 3 x 10- ⁇ cells into the mouse muscle. Other researchers have implanted significantly higher numbers of mvogenic cells into the TA muscle of nude mice.
• Mouse dermal fibroblasts for implantation into mdx mouse muscle were infected with the Mouse Moloney Leukemia Virus Retroviral Vector carrying the lac Z gene.
• This gene codes for ⁇ -galactosidase, which when expressed within cells stains a blue colour when the usual substrate for ⁇ -galactosidase is used in the staining procedure.
• the producer cells containing the retroviral vector were obtained from Dr. J. Price,
• Selection is carried out by growing cells in growth medium not supplemented with penicillin and streptomycin as previously described, but supplemented with 400 ⁇ g/ml of Geneticin, a neomycin analogue (Sigma Chemical Co). Only the cells which have become infected with the retroviral vector will survive in this selection medium.
• Mouse dermal fibroblasts were prepared as in Example 1, part 1 to sub-confluency.
• the culture medium was removed and replaced with 3 ml of medium which had been used to sustain the growth of the producer cell line which packages the retroviral vector.
• To the culture medium was added 8 mg/ml of "Polybrene", a reagent that helps the retrovirus to attach to the target cells, i.e. in this case dermal fibroblasts.
• the producer-cell medium was removed after 3 hours and replaced with normal growth medium. After 24 hours the growth medium was removed and replaced with medium containing 400 ⁇ g/ml of the neomycin Geneticin.
• Within 24 hours of infection of cultures with the retroviral vector those cells which have not been retro virally infected stop dividing and by one week they die.
• the remaining cells were therefore kept in the medium containing Geneticin and grown to increase their numbers. All these cells that survived contained the lac Z gene.
• the dermal fibroblasts infected with the retrovirus carrying the ⁇ -galactosidase gene, were implanted into the muscle of mdx nude mice. Three weeks after this implantation, the muscles were removed and prepared for sectioning as described in Example 1 section 5, but in this case sections were histologically examined and were also analysed for ⁇ -galactosidase activity. This involved cutting 12 ⁇ m cryostat sections (as opposed to 8 ⁇ m for histological and dystrophin analysis reported in Example 1). Sections were treated as follows:
• mice of the C57B1/10 strain were used. These mice express the dystrophin gene, but the introduction of the lac Z gene into the dermal fibroblasts enables the fibroblast origin of newly-formed muscle fibres to be verified.
• the suture of the EDL was tied around the distal tendons of the adjacent peroneal muscles to ensure that the EDL muscle was held under tension - as described in Watt et al , Nature 368, 406-407 (1994) [or, more comprehensively, D.J. Watt et al, J. Mus. Res. Cell. Motil. 14, 121-132 (1993)]. [If the muscle is not held under tension, the muscle atrophies and the experiment fails]. Three days after the tendon was sutured the dermal fibroblasts were introduced into the belly of the muscle by injection using a thin pipette, as for Example 1, part 4.
• This experiment was performed to show whether a dystrophin-negative mouse could be made to express the human dystrophin gene when this gene was introduced by implantation.
• Dermal fibroblasts were derived from skin of the mdx mice which were transgenic for the full length human dystrophin gene i.e. they had the mdx background, but were dystrophin-positive because of the transgene - N. Wells et al, Human Mol Gen. 1, 35-40
• the dermal fibroblasts from the mdx transgenic mouse were grown as in Example 1, part 1 and injected into irradiated mdx muscle as in Example 1, part 4. Three weeks after implantation, the injected muscles were removed and cryostat sections cut from the muscles and analysed for histology and for the presence of dystrophin-positive fibres. On histological examination, sections were characterised by the presence of high numbers of immature, newly formed muscle fibres. When stained with P6 antibody, up to 40% of the area of individual muscle sections contained dystrophin-positive fibres. Thus in the mdx mouse implanted with dermal fibroblasts where the dystrophin gene had been introduced by a transgenic route, high numbers of dystrophin-positive fibres were observed.
• the sole source of dystrophin in these experiments was from the implanted transgenic dermal fibroblasts carrying the human dystrophin gene.
• the P6 anti-dystrophki antibody reacts with human or mouse dystrophin.
• the sections will therefore be stained with an antibody which is specific for human dystrophin.

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