Neuromuac. Dlsord.. Vol. 2, No. 5/6, pp. 305-310, 1992 Printed in Great Britain
0960-8966/92 $5.00 + 0.00 Pergamon Press Ltd
EDITORIAL MYOBLAST
TRANSFER
IN MUSCULAR
DYSTROPHY:
PANACEA
OR
PIE IN THE SKY?
The spectacular advances in the molecular gen- transplantation of minced muscle between etics of muscular dystrophy, with the isolation of normal and dystrophic hamsters and mice, and the Duchenne dystrophy gene and the charac- showed that normal or dystrophic muscle was terization of its hitherto unknown protein, able to regenerate in a normal or dystrophic host dystrophin [1, 2], generated a wave of optimism, environment; and furthermore, with the use of in both sufferers and scientists, that this would isoenzymes of glucose phosphate isomerase as a open the way for effective treatment of this genetic marker in different strains of mice, that it was donor and not host muscle that was regenrelentlessly progressive and disabling disorder. Given the large size of dystrophin (over 400 erating [7]. Partridge and colleagues [8-10] established kDa) and its intimate connection with the muscle membrane, it seemed remotely unlikely that it that mononucleated muscle precursor cells, prewas going to be possible to replace the deficient pared by enzymatic disaggregation of neonatal protein or to find some biochemical means of mouse muscle, could be introduced into growing compensating for its function, which is still not muscle fibres of young hosts and that the resultfully understood. The alternative approach of ant mosaic muscle fibres expressed not only the gene therapy also posed major hurdles in relation glucose phosphate isomerase alleles of the host to handling such a gigantic gene, with over two and donor muscles but also a hybrid isoenzyme million base pairs, way beyond the capacity of dimer, which could only result from fusion of vectors such as retroviruses. On top of all this donor and host myoblasts in the same fibre. They there was still the problem of how to target the subsequently tried using this technique to correct gene, or its product, to the widely distributed the defect in a strain of mice with inherited phosphorylase kinase deficiency [9, 10]. Almusculature. Somatic ceiltherapy seemed to offer a possible though myonuclei of donor origin became ~nshortcut for delivering the normal gene, or its corporated into the regenerating muscle fibresin product, direct to the muscle. It entailed trans- eight of nine autografts, only three showed planting normal muscle cells directly into the phosphorylase kinase activity. Following indiseased muscle, with a view to obtaining fusion jection of normal muscle precursor cells into of the donor myoblasts with host myoblasts, the growing phosphorylase kinase-deficient skeletal so-called satellite cells, which are normally muscle, mosaic fibres with donor myonuclei were quiescent in muscle until activated to proliferate, detected in only 11 of 192 muscles examined from divide and fuse. This should then produce a 64 mice, and only 9 of these had phosphorylase mixture of dystrophin-positive and dystrophin- kinase activity. Law and colleagues conducted a series of negative fibres, comparable to the heterozygote experiments along similar lines, aimed directly at female carrier of the Duchenne gene. Muscle transplantation is not new. Following trying to improve the muscle structure and the pioneering work of Studitsky [3], who used function in mice with autosomal recessive minced muscle, which, after transplantation into muscular dystrophy [11]. As the normal genome a vacant muscle compartment, became fully is being incorporated into the dystrophic muscle restored into a muscle belly, complete with blood during myogenesis or regeneration, it would supply and innervation, this technique was theoretically not be necessary to know which extensively studied in a variety of animals [4]. gene is. responsible or what the nature of the In the early 1970s Neerunjun and Dubowitz defect is. Cultured myoblasts from healthy [5, 6] completed a number of experiments on mouse embryos were injected into the right 305
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soleus of 20-day-old healthy or dystrophic mice, the other leg serving as a control. Hosts and donors were immunocompatible but had different genotype markers for glucose phosphate isomerase. After 6 months, they found that the test dystrophic solei showed greater cross-sectional arca, total fibre number, wet weight, and twitch and tetanus tensions than the control solei,and also noted a more normal histological appearance and betterfibretype distinction.The presence of both the donor isozyme of glucose phosphate isomerasc plus the hybrid isozymc implied the survival and development of donor myoblasts into normal myofibres and the fusion of donor normal myoblasts with dystrophic host satellite cells to produce genetically mosaic myofibrcs. The recent discovery of several X-linked animal models, which wcrc genetic homologucs of Duchcnnc dystrophy, has provided an extra boost for thislineofresearch. Like the Duchcnnc patient,the mdx mouse also lacks dystrophin but has an essentiallynormal clinicalphcnotypc with no weakness and a normal lifespan[12-14].After an initialphase of necrosis at about 2 weeks of age, the muscle shows a continuing process of regeneration which presumably compensates for the disease. In contrast the xrnd dog lacks dystrophin and has scvcrc pathological changes and clinicalweakness comparable to the human disease [15, 16]. There is also an X-linked dystrophy in the cat [I7],and likethe mdx mouse it is deficient in dystrophin but has normal muscle power plus marked hypcrtrophy bf the musculature. If one only had more insight into how the mouse and the cat manage to compensate for the deficiency of dystrophin, it might open the way for possible treatment. The advent of antibodies to dystrophin providcd a tool for the direct asscssmcnt of the protein in the muscle of these muscular dystrophies connected with thd dystrophin gcnc. In a key experiment Partridge and colleagues [I8] were able to demonstrate that directinjection of myoblasts from dissociated normal neonatal muscle into the muscle of an mdx/nudc mouse produced dystrophin-positivc fibres. Mononucleated cellswere prepared by enzymatic dissociation of normal neonatal mouse muscle and between 5 × I05 and 4 x 106 cellsinjected into the extensor digitorum longus (EDL) muscle of 5-27-day-old mdx mice. After 20-99 days the E D L and adjacent tibialisanteriorand pcroncus longus muscles from the injectedand the contralateralcontrol leg were examined for the presence
of donor muscle cells, using glucose phosphate isomerase isozymes as markers. Of the 70 mice analysed, 39 had the heterodimer in one or more muscles, indicating the fusion of injected cells with host myofibres. The grafts were less frequently successful in the muscle of younger (5-7day-old) mice (3 out of 17), than in older (19-27day-old) mice (36 out of 53). In addition, fusion of donor myoblasts into host muscles was significantly more freqizent in the nude/mdx hosts (27 out of 34) than in the regular M9 hosts (9 out of 19), suggesting an immunological impediment to myoblast grafting, even in hosts that are MHC compatible and tolerant. In order to see if normal myoblasts incorporated into mdx myofibres could produce dystrophin, selected muscles (numerical details not provided), containing a high proportion of hcterodimer glucose phosphate isomerase, were tested for dystrophin by immunoblotting and immunofluorescence staining. In a single illustrative example, the immunoblot from the injected EDL muscles from two mdx mice indicated the presence of normal sized dystrophin at about 30-40% of normal level, and on immunostaining dystrophin was present in its normal sarcolemmal location 'in some 10-40% of muscle fibre profiles'. As only a small proportion (about 1 in 70) of these immunodeficient mice had this significant degree of fusion of donor and host myoblasts, it is difficult to assess the overall therapeutic potential of these experiments in producing dystrophin-positive myofibres in these mdx mice. In a similar approach, Karpati and his colleagues [19] prepared suspensions from clones of cultured normal human myoblasts, obtained from fresh biceps biopsies, at a concentration of approximately 25,000 cells per ~1, which were injected into the quadriceps of two groups ofmdx mice. The first group consisted of 6-10-day-old animals (n = 9), before the stage of muscle fibre necrosis; the second, 60-day-old animals (n = 5), whose muscle had been crushed percutaneously by a haemostat 4 days before the injection to promote regeneration. In the neonatal animals a total of 5-10/zl of the suspension was injected in three parallel tracts 1 mm apart, in the older animals 10-30/zl was used. As a control, the contralateral quadriceps was injected with the suspension fluid without myoblasts. Using an antibody to the carboxy terminal end of dystrophin they demonstrated the presence of dystrophin on immunoblot and immunofluorescence of sections in normal human and mouse muscle and
Editorial complete absence in the mdx mouse. In mdx muscle at 25-45 days after injection, there were clusters of fibres with strong sarcolemmal dystrophin immunostaining, comprising about 3-5% of fibres on transverse section in the young animals and about 5-7% in the older group. Despite not using any immunosuppression in the host animals, they found no evidence of major immunological rejection in the transplanted muscle. In an effort to define a strategy for potential myoblast therapy, the Muscular Dystrophy Association of America (MDA) organized a workshop meeting in New York in June 1989, aimed at providing a scientific basis for clinical trials of myoblast transfer therapy [20]. A number of participants struck a cautionary note. For example, Peter Ray [20, p. 107]: "May I add my voice here to those who have advocated caution in proceeding with human trials. Since so many questions regarding myoblast transfer remain unanswered there can be no expectation, but only a hope for real success. Most of these parameters could be systematically tested in either of the available animal models while they cannot be adequately addressed by human experimentation. There is the risk that human trials will lead to unfulfilled expectations in the general public resulting in distrust of and lack of support for the scientific community." From the clinical viewpoint Mike Brooke [20, p. 231] commented "But the treatment response that we all want has nothing to do with the dystrophin. The patient is ultimately interested in survival and strength and function. Therefore, we have to test this in any clinical trial." However, with the encouragement and financial support of the MDA, a number of units in the U.S.A. and Canada opted for direct experimental studies on human subjects, aimed at trying to assess the potential therapeutic benefit of myoblast transfer therapy. In the most comprehensive study to date, Karpati and colleagues in Montreal recruited into a double-blind study, eight young boys with Duchenne dystrophy, all of whom had a deletion in the gene and absence ofdystrophin in the muscle. Ten million cultured myoblasts from the father's muscle, of proven purity by cell sorting, were injected into multiple sites in one biceps, whereas a comparable injection, but without the myoblasts, was made into the other biceps as a control. Neither patient nor personnel were aware of which side was which. Immunosuppression was provided with cyclophosphamide. The power of the muscle was assessed by sequential myometry and the
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dystrophin status of the muscle on repeat biopsy at 3 and 12 months by Western blot and immunocytochemistry. All eight boys have now completed a 1 yr follow-up assessment and Karpati presented the preliminary data at a recent workshop in Rome [21]. There were no significant differences between the two sides in the individual cases, at either a clinical level or in the dystrophin status. In an uncontrolled study, Tremblay's group in Quebec performed myoblast transfer with repeated injections into several different muscles in four advanced, non-ambulant, cases of Duchenne dystrophy [22]. Meticulous attention was paid to histocompatibility of the donor and recipient for HLA classes I and II-DR. No immunosuppression was used. In one case the donor was a brother, in the other three sisters, including one Duchenne carrier. Three of the four patients were shown to form antibodies against the donor's myotubes. Muscle biopsies of the injected tibialis anterior showed some degree of dystrophin immunostaining in 80%, 75%, 25% and 0%, respectively, of the muscle fibres, and in the contralateral uninjected muscle in 16% of the first case and none in the other three. In another phase 1 study, Law and colleagues in Memphis injected into eight loci in the extensor digitorum brevis muscle of the foot in three boys with Duchenne dystrophy, eight million cultured myoblasts, obtained from a 1 g biopsy from the father or brother [23]. In the first patient the donor was the non-biological adoptive father. A comparable volume of carrier fluid without myoblasts was sham-injected into"the other side. In the second and third patients this was double blinded. After 3 months there was an increase in twitch tension, measured in the flexor hallucis longus, in the myoblast-injected side compared with a reduction on the sham-injected side. Bilateral open biopsy showed the presence of dystrophin by immunoblot and immunocytochemistry on the myoblast-injected side only. Eight further patients were also included in this study but were not analysed. Fired by the success of this limited series of experiments, Law established a Cell Therapy Research Foundation and proceeded to his phase 2 therapeutic trials of myoblast transfer into several major muscles in Duchenne dystrophy. He recently reported the results o f a 3 month follow-up on 18 of their 21 phase 2 cases, which included the 11 from the phase 1 study [24]. Five billion cultured, normal myoblasts were transferred via 48 intramuscular injections into 22
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major muscles of both lower limbs. Immunosuppression was provided by cyclosporin. Dynamometer measurements of the isometric tension in the knee flexors, knee extensors and plantar flexors before and 3 months after myoblast transfer in 18 subjects showed a mean increase of 41% in 30 of the total of 69 muscle groups measured, no change in 26, and a reduction of 23% in 13. Interpretation of these statistics is difficult, given the short period of follow-up and the uncontrolled nature of the study. When Law presented these results at the first meeting of the newly established Society of Cell Transplantation in June 1992, he came under considerable fire from his scientific colleagues about the validity of his data in such an uncontrolled study [25]. Blau and colleagues in Stanford studied the tibialis anterior muscle from eight cases of Duchenne dystrophy, with a documented deletion in the dystrophin gene, 1 month after the injection of 100 million cultured myoblasts into 80-100 injection sites [26]. In three of the eight they were able to show, by polymerase chain reaction, the expression ofdystrophin messenger RNA derived from the donor myoblast DNA. Given the extreme sensitivity of the PCR technique, this result presumably reflects the persistence, at 1 month, of donor D N A from a few of the implanted myoblasts. Immunocytochemically the number of dystrophin-positive fibres was only about 10 per 1000 fibres counted, which was no greater than the frequency of spontaneously occurring dystrophin-prsitive "revertant" fibres in some of the control side muscles. Although Nature hailed this as a "Transplant Success" in its contents page, from a clinical point of view it is no more than a fleabite in the ocean and an unequivocal therapeutic failure. Comparative studies ofmyoblast transfer in 11 dystrophic dogs have als~ proved negative on the basis of conversion to dystrophin-positive fibres (Dux, Sewry, Cooper and Dubowitz, unpublished observations). Similar negative or uncertain results have also been obtained by the Paris group in eight grafting experiments to date (M. Fardeau, personal communication ). On the other hand a detailed study of the regenerative potential of the dystrophic muscle in 6 monthold-dogs, following toxin-induced necrosis, has shown this to be comparable to that of normal muscle [27]. This at least suggests that Duchenne muscle should potentially be capable of regeneration.
Another recent approach to therapy has been to try and introduce the gene directly into muscle. In an interesting experiment, Wolff et al. [28] demonstrated that injection of plasmid D N A directly into rodent skeletal muscle expressed reporter genes such as fl-galactosidase or luciferase, suggesting one might be able to circumvent the need for viral or other vectors for the DNA. In a similar study Wolff's group have recently shown that non-human, primate muscle is also able to take up and express intramuscularly injected plasmid DNA, but the level of expression of luciferase was considerably lower than in rodent muscle [29]. This does not augur well for this approach in human muscle. Meanwhile, a number of laboratories have now succeeded in producing constructs of the 12 kb full-length human dystrophin complementary DNA gene, containing all the 70-odd exons necessary for protein production. This provided the possibility for attempting to introduce the dystrophin gene into the muscle cell. Lee et al. were able to introduce DNA constructs of mouse dystrophin into cultured COS cells (a kidney cell line) which then expressed dystrophin, thought to be membrane bound, in about 3-5% of cultured cells [30]. Acsadi et al. [31] were subsequently able to show that either a 12 kb full-length human dystrophin complementary DNA gene or a 6.3 kb minigene, derived from a Becker patient with a large deletion of the gene, could be expressed in cultured cells or in vivo. When human dystrophin expression plasmids were injected intramuscularly into dystrophin-deficient m d x mice, the human dystrophin was present in about 1% of myofibres. From a purely technical point of view this was a further important step in demonstrating that dystrophin could be expressed either in vivo or by transfection of cultured myoblasts in vitro. However, in spite of the wide exposure of this "breakthrough" in both the scientific and lay press, this is still a far cry from producing any clinical benefit and it is rather frustrating that none of these sophisticated studies have surpassed nature's spontaneous correction of the defect in individual muscle fibres in Duchenne dystrophy or the m d x mouse---the so-called "revertant" fibres. Furthermore the m d x mouse, with its normal clinical phenotype, cannot tell us whether the dystrophin expressed is functional and therapeutically viable, and comparable experiments will have to be done in the dystrophic dog or the dystrophic human, once a greater yield of dystrophin-positive fibres can be achieved.
Editorial A n u m b e r o f basic questions in relation to the transplant experiments still remain to be answered. These c o n c e r n the myogenicity o f the grafted cells and their potential for further division a n d fusion; the migration o f the grafted cells and their ability to cross the sarcolemmal m e m b r a n e o f host fibres; the c o n t r o l o f i m m u n e rejection o f the d o n o r cells; the stability with time o f converted muscle fibres; and the definition o f a threshold for functional restoration o f these corrected fibres. At a recent E M B O w o r k s h o p meeting on muscle regeneration, there was an active r o u n d table discussion on m y o b l a s t transfer and a general consensus that constraint was necessary at present on h u m a n experimental studies and m o r e basic studies were required [32]. A similar conclusion was d r a w n after a lively debate on m y o b l a s t transfer at the N a t i o n a l C o l l o q u i u m on N e u r o m u s c u l a r Diseases, organized by L ' A s s o ciation Fran~aise contre les M y o p a t h i e s ( A F M ) in June 1991 [33]. Meanwhile Peter Law, P h . D , has recently filed a United States patent for the use o f m y o b l a s t transfer in all 12 designated forms o f m u s c u l a r d y s t r o p h y and, for g o o d measure, also a n u m b e r o f other n e u r o m u s c u l a r disorders, such as a m y o t r o p h i c lateral sclerosis (ALS) and m y a s thenia gravis. Clinical science, clinical practice and clinical c o m m e r c e are currently in conflict and it is obviously o f p a r a m o u n t i m p o r t a n c e that the f u n d a m e n t a l scientific questions in relation to the potential value and viability o f m y o b l a s t transfer are speedily resolved. VICTOR DUBOWITZ Editor-in-Chief REFERENCES
1. Koenig M, Hoffman E P, Bertelson C H, et al. Complete cloning of Duchenne muscular dystrophy (DMD) eDNA and preliminary genomic organisation of the DMD gene in normal and affected individuals. Cell 1987; 50: 509-517. 2. Hoffman E P, Brown R H Jr, Kunkel L M. Dystrophin: the protein produced on the Duchenne muscular dystrophy locus. Cell 1987; 51:919-928. 3. Studitsky A N. Restoration of muscle by means of transplantation of minced muscle. Dokl Akad Nauk S S S R 1952;84: 389. 4. Carlson B M. The Regeneration o f Minced Muscles. Basel: Karger, I972. 5. Ncerunjun J S, Dubowitz V. Muscle transplantation and regeneration in the dystrophic hamster. Part !. Histological studies. J Neurol Sci 1974; 23: 505-519. 6. Neerunjun J S, Dubowitz V. Muscle transplantation between normal and dystrophic mice. 1. Histological studies. Neuropathol Appl Neurobiol 1975; 1:111-124.
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7. Neerunjun J S, Dubowitz V. Isoenzyme studies in the identification of transplanted muscle in the mouse. Clin Sci Mol Med 1974; 46: 555--558. 8. Partridge T A, Grounds M, Sloper J C. Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Nature 1978;273: 306-308. 9. Watt D J, Morgan J E, Partridge T A. Use of mononuclear precursor cells to insert allogeneic genes into growing mouse muscles. Muscle Nerve 1984; 7: 741-750. 10. Morgan J E, Watt D J, Sloper J C, Partridge T A. Partial correction of an inherited biochemical defect of skeletal muscle by grafts of normal muscle precursor cells. J Neurol Sci 1988;86: 137-147. 1I. Law P K, Goodwin T G, Wang M G. Normal myoblast injections provide genetic treatment for routine dystrophy. Muscle Nerve 1988; 11: 525-533. 12. Bulfield G, SiUer W G, Wight P A L, Moore K J.X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc NatlAcadSci USA 1984;81: 1189-1192. 13. Dangain J, Vrbova G. Muscle development in mdx mutant mice. Muscle Nerve 1984;7: 700-704. 14. Ryder-Cook A S, Sicinski P, Thomas K, et al. Localisati0n of the mdx mutation within the mouse dystrophin gene. EMBO J 1988; 7: 3017-3021. 15. Valentine B A, Cooper B J, Cummings J R, de Lahunta A. Progressive muscular dystrophy in a golden retriever dog: light microscope and ultrastructural features at 4 and 8 months. A cta Neuropathol 1986;7:30 I-310. 16. Cooper B J, Winand N J, Stedman H, et al. The homologue of the Duchenne locus is defective in Xlinked muscular dystrophy of dogs. Nature 1988; 334: 154-156.
17. Carpenter J L, Hoffman E P, Romanul F C A, et al. Feline muscular dystrophy with dystrophin deficiency. Am J Pathol 1989; 135: 909-919. 18. Partridge T A, Morgan J E, Coulton G R, Hoffman E P, Kunkel L M. Conversion of mdx myofibres from dystrophin-negative to positive by injection of normal myoblasts. Nature 1989;337: 176-179. 19. Karpati G, Pouliot Y, Zubrzycka-Gaarn E, et al. Dystrophin is expressed in mdx skeletal muscle fibres after normal myoblast implantation. Am J Pathol 1989; 134: 27-32. 20. Grigs R C, Karpati G. Myoblast transfer therapy. In: Advances in Experimental Medicine and Biology. New York: Plenum Press, 1990;Vol. 280. 21. Perry V. The Xp21 myopathies: current research and the prospect for treatment. Neuromusc Disord 1992; 2: 137-141. 22. Huard J, Bouchard J P, Roy R, et al. Human myoblast transplantation: preliminary results of 4 cases. Muscle Nerve 1992; 15: 550-560. 23. Law P K, Goodwin T G, Fang Q, et al. Pioneering development of myoblast transfer therapy. In: Angelini C, Danieli C A, Fontanari D, eds. Muscular Dystrophy Research: From Molecular Diagnosis Toward Therapy.
Amsterdam: Excerpta Medica, 1991. 24. Law P K, Goodwin T G, Fang Q, et al. Feasibility, safety, and efficacy of myoblast transfer therapy on Duchenne muscular dystrophy boys. Cell Transplant 1992; 1: 235-244. Thompson L. Cell transplant results under fire. Science 1992; 257: 472--474. 26. Gussoni E, Pavlath G K, Lanctot A M, et al. Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation. Nature 1992; 356: 435-438. 27. Sewry C A, Wilson L A, Dux L, Dubowitz V, Cooper B J. "Experimental regeneration in canine muscular dystrophy--l. Immunocytochemical evaluation of dystrophin and 08-spectrin. Neuromusc Disord 1992; 2: 331-342.
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28. Wolff J A, Malone R W, Williams P, et al. Direct gene transfer into muscle in vivo. Science 1990; 247: 14651468. 29. Jiao S, Williams P, Berg R K, et al. Direct gene transfer into nonhuman primate myofibres in vivo. Human Gene Therapy 1992; 3: 21-33. 30. Lee C C, Pearlman J A, Chamberlain J S, Caskey C T. Expression of recombinant dystrophin and its localisation to the cell membrane. Nature 1991; 349: 334-336.
3I.
Acsadi G, Dickson G, Love D R, et aL Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature 1991;35"2:815--818. 32. Amati P, Cossu G, Schiaflino S. Report on the EMBO workshop Molecular Biology and Pathology o f Skeletal and Cardiac Myogenesis, 25 September-i October 1992. Neuromusc Disord (in press). 33. Schwartz K. Meeting Report: The 4th National Colloquium on NeuromuscularDiseases (24-28 June 1991, Montpellier, France). Neuromusc Disord 1991; I: 299-304.
0960-8966/92 $5.00 + 0.00 Pergamon Press Ltd
EDITORIAL MYOBLAST
TRANSFER
IN MUSCULAR
DYSTROPHY:
PANACEA
OR
PIE IN THE SKY?
The spectacular advances in the molecular gen- transplantation of minced muscle between etics of muscular dystrophy, with the isolation of normal and dystrophic hamsters and mice, and the Duchenne dystrophy gene and the charac- showed that normal or dystrophic muscle was terization of its hitherto unknown protein, able to regenerate in a normal or dystrophic host dystrophin [1, 2], generated a wave of optimism, environment; and furthermore, with the use of in both sufferers and scientists, that this would isoenzymes of glucose phosphate isomerase as a open the way for effective treatment of this genetic marker in different strains of mice, that it was donor and not host muscle that was regenrelentlessly progressive and disabling disorder. Given the large size of dystrophin (over 400 erating [7]. Partridge and colleagues [8-10] established kDa) and its intimate connection with the muscle membrane, it seemed remotely unlikely that it that mononucleated muscle precursor cells, prewas going to be possible to replace the deficient pared by enzymatic disaggregation of neonatal protein or to find some biochemical means of mouse muscle, could be introduced into growing compensating for its function, which is still not muscle fibres of young hosts and that the resultfully understood. The alternative approach of ant mosaic muscle fibres expressed not only the gene therapy also posed major hurdles in relation glucose phosphate isomerase alleles of the host to handling such a gigantic gene, with over two and donor muscles but also a hybrid isoenzyme million base pairs, way beyond the capacity of dimer, which could only result from fusion of vectors such as retroviruses. On top of all this donor and host myoblasts in the same fibre. They there was still the problem of how to target the subsequently tried using this technique to correct gene, or its product, to the widely distributed the defect in a strain of mice with inherited phosphorylase kinase deficiency [9, 10]. Almusculature. Somatic ceiltherapy seemed to offer a possible though myonuclei of donor origin became ~nshortcut for delivering the normal gene, or its corporated into the regenerating muscle fibresin product, direct to the muscle. It entailed trans- eight of nine autografts, only three showed planting normal muscle cells directly into the phosphorylase kinase activity. Following indiseased muscle, with a view to obtaining fusion jection of normal muscle precursor cells into of the donor myoblasts with host myoblasts, the growing phosphorylase kinase-deficient skeletal so-called satellite cells, which are normally muscle, mosaic fibres with donor myonuclei were quiescent in muscle until activated to proliferate, detected in only 11 of 192 muscles examined from divide and fuse. This should then produce a 64 mice, and only 9 of these had phosphorylase mixture of dystrophin-positive and dystrophin- kinase activity. Law and colleagues conducted a series of negative fibres, comparable to the heterozygote experiments along similar lines, aimed directly at female carrier of the Duchenne gene. Muscle transplantation is not new. Following trying to improve the muscle structure and the pioneering work of Studitsky [3], who used function in mice with autosomal recessive minced muscle, which, after transplantation into muscular dystrophy [11]. As the normal genome a vacant muscle compartment, became fully is being incorporated into the dystrophic muscle restored into a muscle belly, complete with blood during myogenesis or regeneration, it would supply and innervation, this technique was theoretically not be necessary to know which extensively studied in a variety of animals [4]. gene is. responsible or what the nature of the In the early 1970s Neerunjun and Dubowitz defect is. Cultured myoblasts from healthy [5, 6] completed a number of experiments on mouse embryos were injected into the right 305
306
Editorial
soleus of 20-day-old healthy or dystrophic mice, the other leg serving as a control. Hosts and donors were immunocompatible but had different genotype markers for glucose phosphate isomerase. After 6 months, they found that the test dystrophic solei showed greater cross-sectional arca, total fibre number, wet weight, and twitch and tetanus tensions than the control solei,and also noted a more normal histological appearance and betterfibretype distinction.The presence of both the donor isozyme of glucose phosphate isomerasc plus the hybrid isozymc implied the survival and development of donor myoblasts into normal myofibres and the fusion of donor normal myoblasts with dystrophic host satellite cells to produce genetically mosaic myofibrcs. The recent discovery of several X-linked animal models, which wcrc genetic homologucs of Duchcnnc dystrophy, has provided an extra boost for thislineofresearch. Like the Duchcnnc patient,the mdx mouse also lacks dystrophin but has an essentiallynormal clinicalphcnotypc with no weakness and a normal lifespan[12-14].After an initialphase of necrosis at about 2 weeks of age, the muscle shows a continuing process of regeneration which presumably compensates for the disease. In contrast the xrnd dog lacks dystrophin and has scvcrc pathological changes and clinicalweakness comparable to the human disease [15, 16]. There is also an X-linked dystrophy in the cat [I7],and likethe mdx mouse it is deficient in dystrophin but has normal muscle power plus marked hypcrtrophy bf the musculature. If one only had more insight into how the mouse and the cat manage to compensate for the deficiency of dystrophin, it might open the way for possible treatment. The advent of antibodies to dystrophin providcd a tool for the direct asscssmcnt of the protein in the muscle of these muscular dystrophies connected with thd dystrophin gcnc. In a key experiment Partridge and colleagues [I8] were able to demonstrate that directinjection of myoblasts from dissociated normal neonatal muscle into the muscle of an mdx/nudc mouse produced dystrophin-positivc fibres. Mononucleated cellswere prepared by enzymatic dissociation of normal neonatal mouse muscle and between 5 × I05 and 4 x 106 cellsinjected into the extensor digitorum longus (EDL) muscle of 5-27-day-old mdx mice. After 20-99 days the E D L and adjacent tibialisanteriorand pcroncus longus muscles from the injectedand the contralateralcontrol leg were examined for the presence
of donor muscle cells, using glucose phosphate isomerase isozymes as markers. Of the 70 mice analysed, 39 had the heterodimer in one or more muscles, indicating the fusion of injected cells with host myofibres. The grafts were less frequently successful in the muscle of younger (5-7day-old) mice (3 out of 17), than in older (19-27day-old) mice (36 out of 53). In addition, fusion of donor myoblasts into host muscles was significantly more freqizent in the nude/mdx hosts (27 out of 34) than in the regular M9 hosts (9 out of 19), suggesting an immunological impediment to myoblast grafting, even in hosts that are MHC compatible and tolerant. In order to see if normal myoblasts incorporated into mdx myofibres could produce dystrophin, selected muscles (numerical details not provided), containing a high proportion of hcterodimer glucose phosphate isomerase, were tested for dystrophin by immunoblotting and immunofluorescence staining. In a single illustrative example, the immunoblot from the injected EDL muscles from two mdx mice indicated the presence of normal sized dystrophin at about 30-40% of normal level, and on immunostaining dystrophin was present in its normal sarcolemmal location 'in some 10-40% of muscle fibre profiles'. As only a small proportion (about 1 in 70) of these immunodeficient mice had this significant degree of fusion of donor and host myoblasts, it is difficult to assess the overall therapeutic potential of these experiments in producing dystrophin-positive myofibres in these mdx mice. In a similar approach, Karpati and his colleagues [19] prepared suspensions from clones of cultured normal human myoblasts, obtained from fresh biceps biopsies, at a concentration of approximately 25,000 cells per ~1, which were injected into the quadriceps of two groups ofmdx mice. The first group consisted of 6-10-day-old animals (n = 9), before the stage of muscle fibre necrosis; the second, 60-day-old animals (n = 5), whose muscle had been crushed percutaneously by a haemostat 4 days before the injection to promote regeneration. In the neonatal animals a total of 5-10/zl of the suspension was injected in three parallel tracts 1 mm apart, in the older animals 10-30/zl was used. As a control, the contralateral quadriceps was injected with the suspension fluid without myoblasts. Using an antibody to the carboxy terminal end of dystrophin they demonstrated the presence of dystrophin on immunoblot and immunofluorescence of sections in normal human and mouse muscle and
Editorial complete absence in the mdx mouse. In mdx muscle at 25-45 days after injection, there were clusters of fibres with strong sarcolemmal dystrophin immunostaining, comprising about 3-5% of fibres on transverse section in the young animals and about 5-7% in the older group. Despite not using any immunosuppression in the host animals, they found no evidence of major immunological rejection in the transplanted muscle. In an effort to define a strategy for potential myoblast therapy, the Muscular Dystrophy Association of America (MDA) organized a workshop meeting in New York in June 1989, aimed at providing a scientific basis for clinical trials of myoblast transfer therapy [20]. A number of participants struck a cautionary note. For example, Peter Ray [20, p. 107]: "May I add my voice here to those who have advocated caution in proceeding with human trials. Since so many questions regarding myoblast transfer remain unanswered there can be no expectation, but only a hope for real success. Most of these parameters could be systematically tested in either of the available animal models while they cannot be adequately addressed by human experimentation. There is the risk that human trials will lead to unfulfilled expectations in the general public resulting in distrust of and lack of support for the scientific community." From the clinical viewpoint Mike Brooke [20, p. 231] commented "But the treatment response that we all want has nothing to do with the dystrophin. The patient is ultimately interested in survival and strength and function. Therefore, we have to test this in any clinical trial." However, with the encouragement and financial support of the MDA, a number of units in the U.S.A. and Canada opted for direct experimental studies on human subjects, aimed at trying to assess the potential therapeutic benefit of myoblast transfer therapy. In the most comprehensive study to date, Karpati and colleagues in Montreal recruited into a double-blind study, eight young boys with Duchenne dystrophy, all of whom had a deletion in the gene and absence ofdystrophin in the muscle. Ten million cultured myoblasts from the father's muscle, of proven purity by cell sorting, were injected into multiple sites in one biceps, whereas a comparable injection, but without the myoblasts, was made into the other biceps as a control. Neither patient nor personnel were aware of which side was which. Immunosuppression was provided with cyclophosphamide. The power of the muscle was assessed by sequential myometry and the
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dystrophin status of the muscle on repeat biopsy at 3 and 12 months by Western blot and immunocytochemistry. All eight boys have now completed a 1 yr follow-up assessment and Karpati presented the preliminary data at a recent workshop in Rome [21]. There were no significant differences between the two sides in the individual cases, at either a clinical level or in the dystrophin status. In an uncontrolled study, Tremblay's group in Quebec performed myoblast transfer with repeated injections into several different muscles in four advanced, non-ambulant, cases of Duchenne dystrophy [22]. Meticulous attention was paid to histocompatibility of the donor and recipient for HLA classes I and II-DR. No immunosuppression was used. In one case the donor was a brother, in the other three sisters, including one Duchenne carrier. Three of the four patients were shown to form antibodies against the donor's myotubes. Muscle biopsies of the injected tibialis anterior showed some degree of dystrophin immunostaining in 80%, 75%, 25% and 0%, respectively, of the muscle fibres, and in the contralateral uninjected muscle in 16% of the first case and none in the other three. In another phase 1 study, Law and colleagues in Memphis injected into eight loci in the extensor digitorum brevis muscle of the foot in three boys with Duchenne dystrophy, eight million cultured myoblasts, obtained from a 1 g biopsy from the father or brother [23]. In the first patient the donor was the non-biological adoptive father. A comparable volume of carrier fluid without myoblasts was sham-injected into"the other side. In the second and third patients this was double blinded. After 3 months there was an increase in twitch tension, measured in the flexor hallucis longus, in the myoblast-injected side compared with a reduction on the sham-injected side. Bilateral open biopsy showed the presence of dystrophin by immunoblot and immunocytochemistry on the myoblast-injected side only. Eight further patients were also included in this study but were not analysed. Fired by the success of this limited series of experiments, Law established a Cell Therapy Research Foundation and proceeded to his phase 2 therapeutic trials of myoblast transfer into several major muscles in Duchenne dystrophy. He recently reported the results o f a 3 month follow-up on 18 of their 21 phase 2 cases, which included the 11 from the phase 1 study [24]. Five billion cultured, normal myoblasts were transferred via 48 intramuscular injections into 22
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major muscles of both lower limbs. Immunosuppression was provided by cyclosporin. Dynamometer measurements of the isometric tension in the knee flexors, knee extensors and plantar flexors before and 3 months after myoblast transfer in 18 subjects showed a mean increase of 41% in 30 of the total of 69 muscle groups measured, no change in 26, and a reduction of 23% in 13. Interpretation of these statistics is difficult, given the short period of follow-up and the uncontrolled nature of the study. When Law presented these results at the first meeting of the newly established Society of Cell Transplantation in June 1992, he came under considerable fire from his scientific colleagues about the validity of his data in such an uncontrolled study [25]. Blau and colleagues in Stanford studied the tibialis anterior muscle from eight cases of Duchenne dystrophy, with a documented deletion in the dystrophin gene, 1 month after the injection of 100 million cultured myoblasts into 80-100 injection sites [26]. In three of the eight they were able to show, by polymerase chain reaction, the expression ofdystrophin messenger RNA derived from the donor myoblast DNA. Given the extreme sensitivity of the PCR technique, this result presumably reflects the persistence, at 1 month, of donor D N A from a few of the implanted myoblasts. Immunocytochemically the number of dystrophin-positive fibres was only about 10 per 1000 fibres counted, which was no greater than the frequency of spontaneously occurring dystrophin-prsitive "revertant" fibres in some of the control side muscles. Although Nature hailed this as a "Transplant Success" in its contents page, from a clinical point of view it is no more than a fleabite in the ocean and an unequivocal therapeutic failure. Comparative studies ofmyoblast transfer in 11 dystrophic dogs have als~ proved negative on the basis of conversion to dystrophin-positive fibres (Dux, Sewry, Cooper and Dubowitz, unpublished observations). Similar negative or uncertain results have also been obtained by the Paris group in eight grafting experiments to date (M. Fardeau, personal communication ). On the other hand a detailed study of the regenerative potential of the dystrophic muscle in 6 monthold-dogs, following toxin-induced necrosis, has shown this to be comparable to that of normal muscle [27]. This at least suggests that Duchenne muscle should potentially be capable of regeneration.
Another recent approach to therapy has been to try and introduce the gene directly into muscle. In an interesting experiment, Wolff et al. [28] demonstrated that injection of plasmid D N A directly into rodent skeletal muscle expressed reporter genes such as fl-galactosidase or luciferase, suggesting one might be able to circumvent the need for viral or other vectors for the DNA. In a similar study Wolff's group have recently shown that non-human, primate muscle is also able to take up and express intramuscularly injected plasmid DNA, but the level of expression of luciferase was considerably lower than in rodent muscle [29]. This does not augur well for this approach in human muscle. Meanwhile, a number of laboratories have now succeeded in producing constructs of the 12 kb full-length human dystrophin complementary DNA gene, containing all the 70-odd exons necessary for protein production. This provided the possibility for attempting to introduce the dystrophin gene into the muscle cell. Lee et al. were able to introduce DNA constructs of mouse dystrophin into cultured COS cells (a kidney cell line) which then expressed dystrophin, thought to be membrane bound, in about 3-5% of cultured cells [30]. Acsadi et al. [31] were subsequently able to show that either a 12 kb full-length human dystrophin complementary DNA gene or a 6.3 kb minigene, derived from a Becker patient with a large deletion of the gene, could be expressed in cultured cells or in vivo. When human dystrophin expression plasmids were injected intramuscularly into dystrophin-deficient m d x mice, the human dystrophin was present in about 1% of myofibres. From a purely technical point of view this was a further important step in demonstrating that dystrophin could be expressed either in vivo or by transfection of cultured myoblasts in vitro. However, in spite of the wide exposure of this "breakthrough" in both the scientific and lay press, this is still a far cry from producing any clinical benefit and it is rather frustrating that none of these sophisticated studies have surpassed nature's spontaneous correction of the defect in individual muscle fibres in Duchenne dystrophy or the m d x mouse---the so-called "revertant" fibres. Furthermore the m d x mouse, with its normal clinical phenotype, cannot tell us whether the dystrophin expressed is functional and therapeutically viable, and comparable experiments will have to be done in the dystrophic dog or the dystrophic human, once a greater yield of dystrophin-positive fibres can be achieved.
Editorial A n u m b e r o f basic questions in relation to the transplant experiments still remain to be answered. These c o n c e r n the myogenicity o f the grafted cells and their potential for further division a n d fusion; the migration o f the grafted cells and their ability to cross the sarcolemmal m e m b r a n e o f host fibres; the c o n t r o l o f i m m u n e rejection o f the d o n o r cells; the stability with time o f converted muscle fibres; and the definition o f a threshold for functional restoration o f these corrected fibres. At a recent E M B O w o r k s h o p meeting on muscle regeneration, there was an active r o u n d table discussion on m y o b l a s t transfer and a general consensus that constraint was necessary at present on h u m a n experimental studies and m o r e basic studies were required [32]. A similar conclusion was d r a w n after a lively debate on m y o b l a s t transfer at the N a t i o n a l C o l l o q u i u m on N e u r o m u s c u l a r Diseases, organized by L ' A s s o ciation Fran~aise contre les M y o p a t h i e s ( A F M ) in June 1991 [33]. Meanwhile Peter Law, P h . D , has recently filed a United States patent for the use o f m y o b l a s t transfer in all 12 designated forms o f m u s c u l a r d y s t r o p h y and, for g o o d measure, also a n u m b e r o f other n e u r o m u s c u l a r disorders, such as a m y o t r o p h i c lateral sclerosis (ALS) and m y a s thenia gravis. Clinical science, clinical practice and clinical c o m m e r c e are currently in conflict and it is obviously o f p a r a m o u n t i m p o r t a n c e that the f u n d a m e n t a l scientific questions in relation to the potential value and viability o f m y o b l a s t transfer are speedily resolved. VICTOR DUBOWITZ Editor-in-Chief REFERENCES
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