- Email: [email protected]
Becker muscular dystrophies
Neuromuscular Disorders 12 (2002) S49–S51 www.elsevier.com/locate/nmd
Current protocol of a research phase I clinical trial of full-length dystrophin plasmid DNA in Duchenne/Becker muscular dystrophies Part I: rationale Christine Thioudellet a, Ste´phane Blot b, Patrick Squiban a, Michel Fardeau c, Serge Braun a,* a
b
Transgene S.A., 11 rue de Molsheim 67082 Strasbourg Cedex, France Unite´ Pe´dagogique et de Recherche de Me´decine, Ecole Nationale Ve´te´rinaire d’Alfort, 94700 Maisons-Alfort, France c Institut de Myologie – INSERM U523, CHU-Pitie´-Salpeˆtrie`re, 75651 Paris Cedex 13, France
Abstract Since the identification of abnormalities in the dystrophin gene as primary cause of Duchenne muscular dystrophy, gene therapy has been seen as an obvious option among various approaches to treat the disease. It is also considered to be especially challenging, as in this context, one must achieve massive transfer of the gene with a sustained lifelong correction of the muscle phenotype. Our goal is to allow large scale transfection of skeletal muscle fibers of Duchenne muscular dystrophy patients with the full-length 11-kb human dystrophin cDNA. Extensive in vitro and in vivo studies, together with safety considerations and the prospects of a very efficient intraarterial delivery procedure, led us progressively to focus our efforts on plasmid DNA administration. We are now conducting a phase I safety clinical trial which will pave the way for future therapeutic gene therapy trials for Duchenne muscular dystrophy. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Duchenne muscular dystrophy; Dystrophin; Gene therapy
1. Introduction Soon after the identification of the cause of Duchenne muscular dystrophy (DMD), namely mutations in the dystrophin gene [1,2], it appeared that due to its high mutation rate, individuals affected by DMD will continue to be present in large proportion by de novo mutations, and the search for direct therapies remains a high priority. The consequences of the mutations have to be eliminated or at least reduced in order to treat the disease. The first studies involving engrafting of healthy myoblast stem cells showed that this technique which looked promising in mice was not effective in Duchenne boys (e.g. [3–5]). An important parameter among potential explanations for this lack of efficiency is the scale-up gap between rodents and human skeletal muscles. Gene therapy is therefore an alternative. Studies to determine the type, amount, and distribution of dystrophin needed for therapy, have been identified by generating transgenic animals that express various forms of dystrophin on an otherwise dystrophindeficient background, and by physical gene transfer using
* Corresponding author. Tel.: 133-3-88-27-91-50; fax: 133-3-88-22-5807. E-mail address: [email protected] (S. Braun).
various viral and non-viral vectors (for review see Refs. [6,7]). These studies suggest that dystrophin must be uniformly expressed, along the entire length of the muscle fiber, at least at 20% of normal endogenous level or muscle fibers to allow some functional preservation or improvement. Expression must be sustained and might have to evade the potential immune response to previously unseen segments of the exogenous protein. These two major obstacles (need for a life-time gene correction and massive amounts of tissue to be treated) can only be overcome with particularly safe and efficient gene transfer systems. 2. Choice of plasmid DNA as vector and of full-length dystrophin as therapeutic gene DMD gene correction of dystrophin deficiency may involve either the full-length dystrophin 11-kb coding sequence [8] or the Becker muscular dystrophy minidystrophin 6.3-kb cDNA, deleted of much of the central part of the gene, that was cloned from a nearly asymptomatic Becker patient [9]. The recourse to micro- or mini-dystrophins remains, however, questionable as the strict correlation between the nature of the dystrophin protein and the clinical impact is not established, and may vary among patients. Our preferred
0960-8966/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0960-896 6(02)00082-2
S50
C. Thioudellet et al. / Neuromuscular Disorders 12 (2002) S49–S51
approach was therefore based on the delivery of the fulllength dystrophin cDNA. Our in vivo investigations were carried out in both dystrophin-deficient mice [mdx (C57BL/10ScSn-DMD mdx) or mdx 5Cv (C57BL/6Ros-DMD mdx-5Cv) strains] and dogs (GRMD). These animal models for DMD all contain a point mutation in the dystrophin gene leading to the absence of dystrophin protein synthesis [10–12]. Our studies were all conceived and directed towards the ultimate goal being the clinical development of a gene therapy product for DMD. For instance, all our gene transfer experiments were carried out in adult or young adult, therefore, immunocompetent dystrophic mice and dogs, and not in newborn, more immunotolerant animals. We compared non-viral (non-condensed plasmid DNA or lipoplexes) and viral (adenoviruses, retroviruses) vectors administered directly into skeletal muscles. The choice of plasmid DNA as dystrophin vector was based on several parameters and observations: Plasmid DNA allows a good efficacy/safety ratio, especially given the amount of vector to be used in DMD patients and the relative efficiency of viral and non-viral vectors in immunocompetent adult mdx mice and GRMD dogs. It is also known since the pioneering work by Jon Wolff [13] that plasmid DNA, once within the skeletal muscle fibers, remains episomal and allows sustained transgene expression in vivo.
Vectors will have to be eventually re-administered because of elimination of dystrophin expressing muscle fibers over time, loss of the vector, and/or downregulation of the promoter. In this respect, most viral vectors cannot be envisaged as viral proteins generate immune responses, lead to the destruction of infected muscle cells, and may prevent re-administration. Moreover, viral vector proteins induce inflammation which may have a strong negative impact in the context of already inflamed dystrophic muscles. We have assessed dystrophin expression (protein 1 mRNA) in vitro using human, mouse and canine muscle primary cultures, and in vivo in mouse and dog, showing proper sarcolemmal dystrophin localization (Fig. 1) and dystrophin-associated glycoproteins recovery. The newly expressed dystrophin was also shown in vitro to decrease the intracellular calcium concentration and prolong cell survival [14]. In a coming publication, Marchand et al. show that gene transfer of the full-length dystrophin cDNA in vitro model seems to allow better functional preservation of the muscle cells than the minigene. Our candidate gene therapy product is therefore based on plasmid DNA carrying the full-length dystrophin cDNA. Studies in mdx and mdx 5Cv mice (the latter display very few revertant fibers as compared with mdx mice) showed in many cases the rapid disappearance of the muscle fibers expressing the exogenous human dystrophin or minidystrophin with
Fig. 1. Expression of human dystrophin in mdx 5Cv mice and in GRMD dogs following intramuscular injection of pCMV-human full-length dystrophin plasmid. (A) Untreated mdx 5Cv mouse tibialis anterior muscle in which no dystrophin is detected. (B) Normal C57BL/6 mouse muscle showing 100% dystrophinpositive fibers. (C) mdx 5Cv tibialis anterior muscle 7 days after administration of 25 mg pCMV-human full-length dystrophin plasmid (in this particular experiment, 20–25% of fibers were dystrophin-positive). (D) GRMD extensor digitori communis muscle 2 weeks after administration of 100 mg of pCMVhuman full-length dystrophin plasmid. Immunodetection was obtained using the MANDRA1 monoclonal antibody that recognizes epitopes of the C-terminal portion of both the human and murine dystrophins but does not label the canine protein.
C. Thioudellet et al. / Neuromuscular Disorders 12 (2002) S49–S51
lack of dystrophin re-expression upon vector re-administration. This correlates with the occurrence of specific humoral and cellular immune responses to the xenogenic dystrophins [15,16]. However, these observations do not preclude the human situation. Nevertheless, they point out the need for a very cautious, conservative clinical development approach. Based on our preclinical studies, the decision was made to initiate a phase I clinical trial of direct intramuscular injection of a plasmid carrying the human full-length dystrophin cDNA [8] under control of the CMV IE1 promoter. Our goal here is to provide evidence on safety of a dystrophin plasmid vector in DMD patients (see Part II). The risk of inducing an autoimmunity is, however, extremely limited. There is no indication from previous plasmid DNA clinical trials of appearance of anti-DNA antibodies. As far as dystrophin is concerned, only myoblast graft trials have been undertaken so far. None of them reported any detrimental side effects despite the occurrence of anti-dystrophin antibodies as reported in some of the myoblast graft trials (for review see Ref. [15]). Myoblast graft represents an unfavorable context from the immunological point of view, since in addition to the exogenous dystrophin itself, the cell vector as well as multiple culture medium protein factors, which bear many antigenic determinants, may trigger immune rejection. The use of more defined preparations such as gene therapy vectors is in this respect of potential advantage. This clinical trial (with results expected in the last quarter 2002) would not have taken place without prospects for an efficient dystrophin gene transfer in human skeletal muscles. In our view, intra-arterial delivery of plasmid DNA represents a major breakthrough in the field. A recent work reported by Jon Wolff’s group showed that intra-arterial locoregional delivery of plasmids results in high levels of foreign gene expression throughout the limb skeletal muscles in rhesus monkeys ranging from less than 1% to more than 30% in various muscles [17]. This indicates that one can potentially transfect sufficient amounts of skeletal muscle fibers to expect some functional improvement at a body scale close to young human patients. Together with Wolff’s team, we are now adapting this technique in GRMD dogs to demonstrate its clinical feasibility. The current human clinical trial, together with the ongoing dog studies, will pave the way for a future therapeutic trial in Duchenne patients. Acknowledgements The authors thank Bruno Cavallini, Fre´ de´ ric Perraud, Marie-Christine Claudepierre, Denise Stuber, Patricia Kleinpeter, Nathalie Accart, Vincent Romanet, Virginie Nourtier, Thierry Huss, Isabelle Renardet, Catherine Brua, Patrick Rodriguez, Dalila Ali-Hadji, Ronald Rooke, Celine Halluard, Patrick Salou, Jean-Marc Balloul, Michel Geist, Karine Dott, Doris Schmitt, Miloud Benchaı¨bi, Martine
S51
N’guyen, Jacqueline Reymund, Re´ my Gloeckler, Daniel Schubnel, Brigitte Mourot, (Transgene), Kirsten Gnirs, Catherine Escriou, Ste´ phane Blot (Ecole Nationale Ve´ te´ rinaire d’Alfort, Maisons-Alfort, France), Dominic Wells (Imperial College School of Medicine, London), George Dickson (Royal Holloway College, London) for their contribution to this programme. This work was supported by the Association Franc¸ aise contre les Myopathies (AFM).
References [1] Hoffman EP, Brown Jr RH, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51:919–928. [2] Koenig M, Monaco AP, Kunkel LM. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 1988;53:219–228. [3] Mendell JR, Kissel JT, Amato AA, et al. Myoblast transfer in the treatment of Duchenne’s muscular dystrophy. N Engl J Med 1995;333:832–838. [4] Tremblay JP, Malouin F, Roy R, et al. Results of a triple blind clinical study of myoblast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy. Cell Transplant 1993;2:99–112. [5] Gussoni E, Blau HM, Kunkel LM. The fate of individual myoblasts after transplantation into muscles of DMD patients. Nat Med 1997;3:970–977. [6] Inui K, Okada S, Dickson G. Gene therapy in Duchenne muscular dystrophy. Brain Dev 1996;18:357–361. [7] Fassati A, Murphy S, Dickson G. Gene therapy of Duchenne muscular dystrophy. Adv Genet 1997;35:117–153. [8] Dickson G, Love DR, Davies KE, Wells KE, Piper TA, Walsh FS. Human dystrophin gene transfer: production and expression of a functional recombinant DNA-based gene. Hum Genet 1991;88:53–58. [9] Acsadi G, Dickson G, Love DR, et al. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature 1991;352:815–818. [10] Sicinski P, Geng Y, Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 1989;244:1578–1580. [11] Im WB, Phelps SF, Copen EH, Adams EG, Slightom JL, Chamberlain JS. Differential expression of dystrophin isoforms in strains of mdx mice with different mutations. Hum Mol Genet 1996;5:1149–1153. [12] Sharp NJH, Kornegay JN, Van Camp SD, et al. An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy. Genomics 1992;13:115– 121. [13] Wolff JA, Malone RW, Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science 1990;247:1465–1468. [14] Marchand E, Constantin B, Vandebrouck C, Raymond G, Cognard C. Calcium homeostasis and cell death in Sol8 dystrophin-deficient cell line in culture. Cell Calcium 2001;29:85–96. [15] Braun S, Thioudellet C, Rodriguez P, et al. Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice. Gene Ther 2000;7:1447–1457. [16] Ferrer A, Wells KE, Wells DJ. Immune responses to dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Gene Ther 2000;7:1439–1446. [17] Zang G, Budker V, Williams P, Subbotin V, Wolff JA. Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates. Hum Gene Ther 2001;12:427–438.
Current protocol of a research phase I clinical trial of full-length dystrophin plasmid DNA in Duchenne/Becker muscular dystrophies Part I: rationale Christine Thioudellet a, Ste´phane Blot b, Patrick Squiban a, Michel Fardeau c, Serge Braun a,* a
b
Transgene S.A., 11 rue de Molsheim 67082 Strasbourg Cedex, France Unite´ Pe´dagogique et de Recherche de Me´decine, Ecole Nationale Ve´te´rinaire d’Alfort, 94700 Maisons-Alfort, France c Institut de Myologie – INSERM U523, CHU-Pitie´-Salpeˆtrie`re, 75651 Paris Cedex 13, France
Abstract Since the identification of abnormalities in the dystrophin gene as primary cause of Duchenne muscular dystrophy, gene therapy has been seen as an obvious option among various approaches to treat the disease. It is also considered to be especially challenging, as in this context, one must achieve massive transfer of the gene with a sustained lifelong correction of the muscle phenotype. Our goal is to allow large scale transfection of skeletal muscle fibers of Duchenne muscular dystrophy patients with the full-length 11-kb human dystrophin cDNA. Extensive in vitro and in vivo studies, together with safety considerations and the prospects of a very efficient intraarterial delivery procedure, led us progressively to focus our efforts on plasmid DNA administration. We are now conducting a phase I safety clinical trial which will pave the way for future therapeutic gene therapy trials for Duchenne muscular dystrophy. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Duchenne muscular dystrophy; Dystrophin; Gene therapy
1. Introduction Soon after the identification of the cause of Duchenne muscular dystrophy (DMD), namely mutations in the dystrophin gene [1,2], it appeared that due to its high mutation rate, individuals affected by DMD will continue to be present in large proportion by de novo mutations, and the search for direct therapies remains a high priority. The consequences of the mutations have to be eliminated or at least reduced in order to treat the disease. The first studies involving engrafting of healthy myoblast stem cells showed that this technique which looked promising in mice was not effective in Duchenne boys (e.g. [3–5]). An important parameter among potential explanations for this lack of efficiency is the scale-up gap between rodents and human skeletal muscles. Gene therapy is therefore an alternative. Studies to determine the type, amount, and distribution of dystrophin needed for therapy, have been identified by generating transgenic animals that express various forms of dystrophin on an otherwise dystrophindeficient background, and by physical gene transfer using
* Corresponding author. Tel.: 133-3-88-27-91-50; fax: 133-3-88-22-5807. E-mail address: [email protected] (S. Braun).
various viral and non-viral vectors (for review see Refs. [6,7]). These studies suggest that dystrophin must be uniformly expressed, along the entire length of the muscle fiber, at least at 20% of normal endogenous level or muscle fibers to allow some functional preservation or improvement. Expression must be sustained and might have to evade the potential immune response to previously unseen segments of the exogenous protein. These two major obstacles (need for a life-time gene correction and massive amounts of tissue to be treated) can only be overcome with particularly safe and efficient gene transfer systems. 2. Choice of plasmid DNA as vector and of full-length dystrophin as therapeutic gene DMD gene correction of dystrophin deficiency may involve either the full-length dystrophin 11-kb coding sequence [8] or the Becker muscular dystrophy minidystrophin 6.3-kb cDNA, deleted of much of the central part of the gene, that was cloned from a nearly asymptomatic Becker patient [9]. The recourse to micro- or mini-dystrophins remains, however, questionable as the strict correlation between the nature of the dystrophin protein and the clinical impact is not established, and may vary among patients. Our preferred
0960-8966/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0960-896 6(02)00082-2
S50
C. Thioudellet et al. / Neuromuscular Disorders 12 (2002) S49–S51
approach was therefore based on the delivery of the fulllength dystrophin cDNA. Our in vivo investigations were carried out in both dystrophin-deficient mice [mdx (C57BL/10ScSn-DMD mdx) or mdx 5Cv (C57BL/6Ros-DMD mdx-5Cv) strains] and dogs (GRMD). These animal models for DMD all contain a point mutation in the dystrophin gene leading to the absence of dystrophin protein synthesis [10–12]. Our studies were all conceived and directed towards the ultimate goal being the clinical development of a gene therapy product for DMD. For instance, all our gene transfer experiments were carried out in adult or young adult, therefore, immunocompetent dystrophic mice and dogs, and not in newborn, more immunotolerant animals. We compared non-viral (non-condensed plasmid DNA or lipoplexes) and viral (adenoviruses, retroviruses) vectors administered directly into skeletal muscles. The choice of plasmid DNA as dystrophin vector was based on several parameters and observations: Plasmid DNA allows a good efficacy/safety ratio, especially given the amount of vector to be used in DMD patients and the relative efficiency of viral and non-viral vectors in immunocompetent adult mdx mice and GRMD dogs. It is also known since the pioneering work by Jon Wolff [13] that plasmid DNA, once within the skeletal muscle fibers, remains episomal and allows sustained transgene expression in vivo.
Vectors will have to be eventually re-administered because of elimination of dystrophin expressing muscle fibers over time, loss of the vector, and/or downregulation of the promoter. In this respect, most viral vectors cannot be envisaged as viral proteins generate immune responses, lead to the destruction of infected muscle cells, and may prevent re-administration. Moreover, viral vector proteins induce inflammation which may have a strong negative impact in the context of already inflamed dystrophic muscles. We have assessed dystrophin expression (protein 1 mRNA) in vitro using human, mouse and canine muscle primary cultures, and in vivo in mouse and dog, showing proper sarcolemmal dystrophin localization (Fig. 1) and dystrophin-associated glycoproteins recovery. The newly expressed dystrophin was also shown in vitro to decrease the intracellular calcium concentration and prolong cell survival [14]. In a coming publication, Marchand et al. show that gene transfer of the full-length dystrophin cDNA in vitro model seems to allow better functional preservation of the muscle cells than the minigene. Our candidate gene therapy product is therefore based on plasmid DNA carrying the full-length dystrophin cDNA. Studies in mdx and mdx 5Cv mice (the latter display very few revertant fibers as compared with mdx mice) showed in many cases the rapid disappearance of the muscle fibers expressing the exogenous human dystrophin or minidystrophin with
Fig. 1. Expression of human dystrophin in mdx 5Cv mice and in GRMD dogs following intramuscular injection of pCMV-human full-length dystrophin plasmid. (A) Untreated mdx 5Cv mouse tibialis anterior muscle in which no dystrophin is detected. (B) Normal C57BL/6 mouse muscle showing 100% dystrophinpositive fibers. (C) mdx 5Cv tibialis anterior muscle 7 days after administration of 25 mg pCMV-human full-length dystrophin plasmid (in this particular experiment, 20–25% of fibers were dystrophin-positive). (D) GRMD extensor digitori communis muscle 2 weeks after administration of 100 mg of pCMVhuman full-length dystrophin plasmid. Immunodetection was obtained using the MANDRA1 monoclonal antibody that recognizes epitopes of the C-terminal portion of both the human and murine dystrophins but does not label the canine protein.
C. Thioudellet et al. / Neuromuscular Disorders 12 (2002) S49–S51
lack of dystrophin re-expression upon vector re-administration. This correlates with the occurrence of specific humoral and cellular immune responses to the xenogenic dystrophins [15,16]. However, these observations do not preclude the human situation. Nevertheless, they point out the need for a very cautious, conservative clinical development approach. Based on our preclinical studies, the decision was made to initiate a phase I clinical trial of direct intramuscular injection of a plasmid carrying the human full-length dystrophin cDNA [8] under control of the CMV IE1 promoter. Our goal here is to provide evidence on safety of a dystrophin plasmid vector in DMD patients (see Part II). The risk of inducing an autoimmunity is, however, extremely limited. There is no indication from previous plasmid DNA clinical trials of appearance of anti-DNA antibodies. As far as dystrophin is concerned, only myoblast graft trials have been undertaken so far. None of them reported any detrimental side effects despite the occurrence of anti-dystrophin antibodies as reported in some of the myoblast graft trials (for review see Ref. [15]). Myoblast graft represents an unfavorable context from the immunological point of view, since in addition to the exogenous dystrophin itself, the cell vector as well as multiple culture medium protein factors, which bear many antigenic determinants, may trigger immune rejection. The use of more defined preparations such as gene therapy vectors is in this respect of potential advantage. This clinical trial (with results expected in the last quarter 2002) would not have taken place without prospects for an efficient dystrophin gene transfer in human skeletal muscles. In our view, intra-arterial delivery of plasmid DNA represents a major breakthrough in the field. A recent work reported by Jon Wolff’s group showed that intra-arterial locoregional delivery of plasmids results in high levels of foreign gene expression throughout the limb skeletal muscles in rhesus monkeys ranging from less than 1% to more than 30% in various muscles [17]. This indicates that one can potentially transfect sufficient amounts of skeletal muscle fibers to expect some functional improvement at a body scale close to young human patients. Together with Wolff’s team, we are now adapting this technique in GRMD dogs to demonstrate its clinical feasibility. The current human clinical trial, together with the ongoing dog studies, will pave the way for a future therapeutic trial in Duchenne patients. Acknowledgements The authors thank Bruno Cavallini, Fre´ de´ ric Perraud, Marie-Christine Claudepierre, Denise Stuber, Patricia Kleinpeter, Nathalie Accart, Vincent Romanet, Virginie Nourtier, Thierry Huss, Isabelle Renardet, Catherine Brua, Patrick Rodriguez, Dalila Ali-Hadji, Ronald Rooke, Celine Halluard, Patrick Salou, Jean-Marc Balloul, Michel Geist, Karine Dott, Doris Schmitt, Miloud Benchaı¨bi, Martine
S51
N’guyen, Jacqueline Reymund, Re´ my Gloeckler, Daniel Schubnel, Brigitte Mourot, (Transgene), Kirsten Gnirs, Catherine Escriou, Ste´ phane Blot (Ecole Nationale Ve´ te´ rinaire d’Alfort, Maisons-Alfort, France), Dominic Wells (Imperial College School of Medicine, London), George Dickson (Royal Holloway College, London) for their contribution to this programme. This work was supported by the Association Franc¸ aise contre les Myopathies (AFM).
References [1] Hoffman EP, Brown Jr RH, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51:919–928. [2] Koenig M, Monaco AP, Kunkel LM. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 1988;53:219–228. [3] Mendell JR, Kissel JT, Amato AA, et al. Myoblast transfer in the treatment of Duchenne’s muscular dystrophy. N Engl J Med 1995;333:832–838. [4] Tremblay JP, Malouin F, Roy R, et al. Results of a triple blind clinical study of myoblast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy. Cell Transplant 1993;2:99–112. [5] Gussoni E, Blau HM, Kunkel LM. The fate of individual myoblasts after transplantation into muscles of DMD patients. Nat Med 1997;3:970–977. [6] Inui K, Okada S, Dickson G. Gene therapy in Duchenne muscular dystrophy. Brain Dev 1996;18:357–361. [7] Fassati A, Murphy S, Dickson G. Gene therapy of Duchenne muscular dystrophy. Adv Genet 1997;35:117–153. [8] Dickson G, Love DR, Davies KE, Wells KE, Piper TA, Walsh FS. Human dystrophin gene transfer: production and expression of a functional recombinant DNA-based gene. Hum Genet 1991;88:53–58. [9] Acsadi G, Dickson G, Love DR, et al. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature 1991;352:815–818. [10] Sicinski P, Geng Y, Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 1989;244:1578–1580. [11] Im WB, Phelps SF, Copen EH, Adams EG, Slightom JL, Chamberlain JS. Differential expression of dystrophin isoforms in strains of mdx mice with different mutations. Hum Mol Genet 1996;5:1149–1153. [12] Sharp NJH, Kornegay JN, Van Camp SD, et al. An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy. Genomics 1992;13:115– 121. [13] Wolff JA, Malone RW, Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science 1990;247:1465–1468. [14] Marchand E, Constantin B, Vandebrouck C, Raymond G, Cognard C. Calcium homeostasis and cell death in Sol8 dystrophin-deficient cell line in culture. Cell Calcium 2001;29:85–96. [15] Braun S, Thioudellet C, Rodriguez P, et al. Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice. Gene Ther 2000;7:1447–1457. [16] Ferrer A, Wells KE, Wells DJ. Immune responses to dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Gene Ther 2000;7:1439–1446. [17] Zang G, Budker V, Williams P, Subbotin V, Wolff JA. Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates. Hum Gene Ther 2001;12:427–438.