Skeletal muscle as an artificial endocrine tissue

Best Practice & Research Clinical Endocrinology and Metabolism Vol. 17, No. 2, pp. 211 –222, 2003 doi:10.1053/ybeem.2003.248, www.elsevier.com/locate/jnlabr/ybeem

3 Skeletal muscle as an artificial endocrine tissue Geoffrey Goldspink* Professor Basic Medical Sciences and Department of Surgery, Royal Free and University College Medical School, Royal Free Campus, University of London, London, UK

Muscle has the ability to take up and express engineered genes and, because it is a post-mitotic tissue, their half-life of expression is prolonged. Although muscle is not regarded as a secretory tissue, in many cases, the gene products enter the systemic circulation. The possibility exists, therefore, of using this approach to alter levels of endocrine and paracrine factors. As a therapeutic procedure, this method has an advantage over the administration of the peptide/protein, which has a relatively short half-life and requires repeated injections. Engineered genes in plasmid or viral vectors under the control of a muscle-specific regulatory sequence may be introduced by intramuscular injection or by the introduction of transfected myoblasts. The latter is also being used in bioreactors to produce medicinal proteins/peptides in vitro as these offer some advantages over bacterial expression systems. However, for gene therapy purposes, there are still safety issues to be addressed. Key words: gene transfer; paracrine; growth factors; hormones; muscle-specific expression.

The use of skeletal muscle as an in vivo expression system is based on the relatively recent appreciation that this tissue, although not traditionally regarded as a secretory tissue, is an endocrine organ capable of expressing several local and systemic factors. A secondary important aspect is that muscle fibres will take up and express gene constructs when introduced by simple intramuscular injection. A third important aspect is that muscle is a post-mitotic tissue in which there is no cell replacement and in which extra-chromosomal DNA has a prolonged life span compared with that in other tissues which undergo continuous cell division throughout life. There are several ways in which muscle can be utilized to express gene products intracellularly or systemically. These have, in some cases, been developed for gene therapy purposes and are not the subject of this chapter. Here, the emphasis is—as the title suggests—on the use of muscle as an endocrine organ. Nevertheless, the technology of gene transfer is essentially the same and therefore some overlap is inevitable. Therefore, the systems used are reviewed under two sections: intramuscular injection of gene constructs and transfer of transfected cells. Gene transfer into muscle tissue is currently being developed as a method for the production, secretion and delivery of therapeutic proteins. This methodology has been used to produce a variety of physiologically active proteins and may ultimately be * Corresponding author. Tel.: þ44-20-7830-2410; Fax: þ44-20-7830-2917. E-mail address: [email protected] (G. Goldspink). 1521-690X/03/$ - see front matter Q 2003 Elsevier Science Ltd. All rights reserved.

212 G. Goldspink

applied to the treatment of several diseases. In this chapter, we consider several applications of this methodology and discuss approaches for modulating therapeutic protein production and secretion from muscle, using growth hormone (GH) as an example. In addition, factors limiting the effectiveness of muscle gene transfer are also discussed, as these determine the efficacy of muscle gene transfer when applied to humans.

REQUIREMENTS FOR GENE TRANSFER TO BE EFFECTIVE Introduction of gene constructs into muscle may not require high levels of expression or the regulation of expression and this greatly influences the choice of vector. Vaccines using antigen cDNA need to be expressed at only low levels. For this, muscle is a good expression system as a single intramuscular injection sufficient to transfect a small percentage of muscle fibres that is adequate to result in small amounts of the protein appearing in the circulation. This is then reacted upon by the professional antibodyproducing cells. DNA vaccines offer several advantages over conventional vaccines, particularly for the prevention of disease in underdeveloped countries as DNA is stable and does not require refrigeration. These methods also have the potential to be applied to the treatment of non-contagious diseases—for example, cancer and endocrine disorders. There is also a range of applications in which the production of low levels of a protein, which is normally deficient or incorrect, can be very beneficial. Overproduction is a not a problem for the treatment of several conditions but regulation of the expression of the introduced gene is required for ‘systems’ in which there is a feedback mechanism such as insulin in diabetes mellitus and GH in pituitary dwarfism. The method of switching on and switching off introduced genes requires that a response element be included in the construct. As yet, no really suitable regulatory sequences are available except for experimental use in animal as the transactivation/repressors are not safe. Muscle, however, offers the possibility that when the gene construct includes certain regulatory sequences, expression can be regulated by muscle activity. One strategy is to include antibiotic-inducible elements in the gene construct, for example, tetracycline-controllable systems (expression or induction) or rapamycininducible systems. Another strategy for regulating the expression of the introduced cDNA is similar to that used in transgenic biology. This involves two components of which one is a transcription factor that is induced by a low-molecular-weight compound. The second is a regulatory sequence spliced into the cDNA that has a binding site for this transcription factor. This approach is being extended to the production of synthetic regulatory sequences that include multiple copies of receptors such as nuclear steroid receptor sites. Thus, the engineered gene could be activated using the appropriate steroid. No doubt much more research will be carried out in this area of regulation of expression of introduced genes. A problem encountered in most attempts at gene transfer for gene therapy purposes has been low expression. For this reason, a number of viral vectors have been used which, in themselves, introduce additional problems—particularly immunological reactions. These include retroviral, herpes, adenovirus and adenovirus-associated viral (AAV) vectors. As well as transfecting muscle cells, these vectors also tend to introduce the cDNA into other types of cell—which raises yet another safety issue. Viral vectors are also very much more immunogenic than plasmid (naked DNA) vectors; however, they are much more efficient in transfecting cells—including muscle cells. Adenoviral vectors in which some of the replication and immunogenic sequences have been

Skeletal muscle as an artificial endocrine tissue 213

deleted have been used in several clinical trials. One of these resulted in the death of a patient due to liver toxicity as the viral vectors tend to accumulate in the liver. AAV vectors are less toxic and less immunogenic; however, they have a relatively small capacity so that only relatively small cDNAs can be introduced by this means. It is not, however, the purpose of this chapter to list the pros and cons of the different vectors as this is dealt with elsewhere and they are mentioned when appropriate in the different ways of using muscle as an expression system.

INTRAMUSCULAR INJECTION OF GENE CONSTRUCTS The injection of gene constructs into skeletal muscle for producing therapeutic proteins is a rapidly developing area of gene therapy. In the past decade, this approach has progressed from the expression of reporter genes1, to neurotrophic factors2, and to the production of autocrine and systemic growth factor hormones.3 The rather unique property of muscle—that of taking up gene constructs and expressing different types of protein (some produced systemically)—makes muscle gene transfer appear promising. However, as yet, little is known about medium- to long-term safety. In addition, it is important to develop techniques for regulating therapeutic gene expression in muscle tissue, as a controlled release of protein is required for many clinical applications. Although several of the proteins described in the following sections are not classical hormones, their production in muscle serves as a useful paradigm for secreted proteins in general. As mentioned, several factors make skeletal muscle an attractive site for therapeutic gene expression. First, it is an abundant tissue, making up about 40% of the average adult’s body mass, and second, it is accessible to most of the delivery methods currently used in gene therapy. There is no significant cell replacement in skeletal muscle tissue4 and the introduced genes are not constantly lost following mitosis. This results in durable therapeutic protein production in muscle.3,5,6 In addition to skeletal muscle, smooth muscle7 and cardiac muscle tissue8 are also being investigated as potential sites for therapeutic gene expression, where local delivery of protein is often desirable. Therapeutic genes have been delivered by injection into muscle tissue using several different types of vector, the choice of which depends on the specific application.9,10 Recombinant viral vectors derived from human adenoviruses (AV) and adenoassociated viruses (AAV) are frequently used because of their capacity for transfecting large numbers of cells, although, for the purpose of safety, viral vectors are modified by removal of immunogenic and regulatory replication sequences.11 – 13 The danger is that transfection is not restricted to muscle. Insertional mutagenesis of viral DNA has proved to be a problem and has caused cancer in two patients treated for severe combined immune deficiency (SCID).14 Although this treatment did not involve intramuscular injection of the engineered genes, the promiscuity of the viral constructs does represent a problem, particularly if the gene cells become transfected. Plasmid DNA-based vectors are often used for muscle gene transfer as these also transfect significant numbers of muscle fibres following intramuscular injection.15 The efficiency of plasmid uptake by muscle cells can be further increased by combining these DNA molecules with synthetic polymers such as liposomes16 or polyvinyl derivatives17, and electrical stimulation methods such as in vivo electroporation have been shown to increase both the number of muscle fibres taking up DNA and the plasmid copy number within these fibres.18 In common with viral vectors, the expression of plasmid-encoded cDNA is under the control of introduced

214 G. Goldspink

regulating elements19,20 that can not only increase expression but also result in muscle-specific expression.

CELL TRANSFER Another method for introducing genes into muscle tissue uses genetically modified myoblasts or fibroblasts. Myoblasts are muscle progenitor cells which differentiate to form the multinucleate myotubes found in muscle tissue and these will stably produce a therapeutic protein once incorporated into new muscle fibres. In addition, cells can be genetically modified to express a variety of different genes and this enables a more precise control of therapeutic gene expression.21,22 However, implanted myoblasts and fibroblasts have to be autologous, or the recipient must be given immunosuppressants to prevent rejection of these cells.23 Myoblasts can also differentiate into myotubes in tissue culture, and these multinucleated cells express many of the genes which are active in mature muscle fibres. Thus, effective combinations of transcription for driving therapeutic gene expression can be determined first in vitro.20 This methodology is also used to confirm that the expressed protein has an appropriate biological activity prior to any in vitro study.3,24 This approach is being extended to introducing groups of transfected myoblasts encapsulated in a biocompatible membrane. These are referred to as organoids and can be implanted into other tissues as well as skeletal muscle. These organoids can, therefore, deliver growth factors and hormones required locally, for example tendon repair.

SOME APPLICATIONS OF GENE TRANSFER INTO MUSCLE The following are some applications of gene transfer using direct intramuscular injection or cell transfer, for medical conditions which involve deficiency of a single gene. Gene therapy for growth hormone deficiency In humans, as in rodents, GH is released from the pituitary gland in an episodic pulse which is controlled by GH-releasing hormone (GHRH), somatostatin and possibly other GH secretagogues.25 Many of the features associated with an endogenous GH deficiency, such as low growth rate and serum IGF-I levels, can be corrected in murine models for GH deficiency following injection of GH-secreting modified myoblasts26 or adenovirus-encoded GH cDNA.27 Plasmid vectors have also been used to produce biologically active GH in differentiated muscle cells, using combinations of viral and muscle-specific transcription for generating optimal levels of this hormone.24 Moreover, serum IGF-I levels and growth rate can be increased following intramuscular injection of plasmids containing either GHRH or GH coding sequence controlled by a muscle-specific promoter.28,29 Although muscle transcription elements can restrict gene expression to specific types of muscle fibre30, the continuous GH typified by the above methods can cause organ hyperplasia.27,31 Therefore, a gene therapy strategy for GH deficiency should ideally

Skeletal muscle as an artificial endocrine tissue 215

possess an inbuilt regulatory element in an effort to minimize the physiological secretion pattern of GH. Insulin-like growth factors It is known that the IGF-I gene is spliced to give rise to several forms of IGF-I. Our groups discovered an RNA transcript of IGF-I in exercised muscle that could not be detected in the resting control muscles. This has exons which differ from those in systemic or liver-type IGF-IEa and it was therefore called mechano growth factor (MGF). There is a 52-base insert in the rat and a 49-base insert in the human that result in a reading frame-shift and a different carboxy peptide sequence. However, as with all the IGF-I isoforms, MGF has the same mature IGF-I sequence encoded by exons 3 and 4. The cDNA of MGF has been introduced into murine muscle using a plasmid vector with a myosin regulatory sequence and it was found that the injected muscle increased in mass by 20% within 2 weeks. This was associated with a 25% increase in average muscle fibre cross-sectional area. Therefore, it is clear that this autocrine form of IGF-I is very potent in initiating muscle hypertrophy.32 The liver type of IGF-I(IGF-IEa) has been introduced using an adenoviral vector33, and although this resulted in hypertrophy, it took 4 months for the muscle mass to increase by 20%. Recent work has indicated that the downstream sequences of MGF activate the muscle satellite (stem) cells. These provide the extra stem cells required for hypertrophy and therefore it appears that MGF ‘kick starts’ the process of muscle hypertrophy or repair. A synthetic peptide of MGF which includes the downstream sequences has the same effect in that it activates mononucleated myoblasts but prevents them fusing to form myotubes.34 However, the MGF peptide has a short half-life so the native peptide is not suitable for in vivo administration. Using its cDNA in an engineered gene means that this aspect is not a problem as the gene product is produced continuously—but this, and the fact that it is very potent, could present other problems. This illustrates, however, the advantages of delivering hormones and growth factors over protein therapy as the short half-life of the peptide/protein means that, in this treatment, frequent injections would have to be given. Erythropoietin insufficiency The production of the haemopoietic factor, erythropoietin (Epo), in muscle tissue has been suggested as an alternative to the frequent doses of recombinant Epo required to treat severe anaemia resulting from renal failure in humans.3 In mice, both serum Epo levels and haematocrit can be raised by muscle gene transfer of Epo coding sequence, and the dose of Epo produced in muscle tissue is dependent on the amount of vector used. It is also significant that serum Epo levels remain elevated for several months, indicating that the delivery vector is maintained in the muscle tissue for this time. However, the Epo levels observed in these studies were higher than those normally observed under physiological conditions, and a persistently high level of haematocrit would not be desirable in humans. Therefore, the production of Epo must be regulated, as under normal physiological conditions, an oxygen deficit increases Epo mRNA levels, with subsequent increases in serum Epo levels stimulating erythrocyte production.35 A rapamycin-inducible plasmid system was used to introduce EPO into mice36 with good indications that its level of expression could be controlled with the vector system and the administration of rapamycin.

216 G. Goldspink

Diabetic neuropathy Recent studies in both animals and humans demonstrate that neurotrophic factors, such as nerve growth factor (NGF)37 and insulin-like growth factor (IGF)38,39, can reverse the peripheral neuropathy that is associated with diabetes. Unfortunately, it is difficult to access some of these data as they are commercially sensitive. Peripheral vascular disease Muscle gene transfer could have a significant clinical impact in treating the peripheral vascular diseases which can cause limb ischaemia.40 Several studies using animal models for limb ischaemia have demonstrated a positive response to treatment with plasmidencoded vascular endothelial growth factor (pVEGF), a factor which stimulates angiogenesis in muscle tissue.41,42 Muscle gene transfer of pVEGF into the ischaemic limb caused an increase in capillary density and blood flow43, suggesting a potential for its application in humans with an advanced disease, where conventional vascularization is not possible. However, VEGF is also produced by some types of tumour cell and it inhibits the maintenance of dendritic cells.44 Inadequate tumour antigen presentation by dendritic cells has been suggested as a potential mechanism for tumour cell immune evasion.45 Therefore, the role of this factor in the pathogenesis of cancer must be considered as a potential side-effect if VEGF is to be applied to the treatment of peripheral vascular disease.

REGULATION OF GENE EXPRESSION IN MUSCLE The gene transfer protocols discussed above are designed for continuous release of protein after muscle gene transfer and, although this may be potentially useful in treating systemic disorders such as Fabry’s disease, which require a regular supply of agalactosldase20, this type of delivery may cause side-effects in other applications where production of the deficient protein is normally tightly regulated. Here we consider why regulation of therapeutic gene expression is important and, using gene therapy for GH deficiency as an example, then discuss how regulation can be achieved in muscle tissue. Regulated production of growth hormone in muscle A controlled release of GH from striated muscle has been achieved following implantation of engineered human fibrosarcoma cells into nude mice.21 These cells express two hybrid proteins consisting of the ligand-binding domains of FKBP12 and FRP fused with the zinc finger DNA-binding domain of ZEBP1 and the transcriptional activation domain of NF-kB p65 proteins, respectively. The human proteins FKBP12 and FRP form a high-affinity complex with the immunosuppressant, rapomycin. Thus, following administration of this drug, the fusion derivatives of FKBP12 and FRP expressed in implanted fibroblasts form a tripartite complex, activating GH expression from a ZFHD1-dependent promoter. A dose-dependent rise in serum GH is subsequently observed in response to rapomycin administration.21 A similar system, using implanted myoblasts, has been used to modulate serum Epo levels following administration of antibiotic22 and, as a component of antibiotic regulation is currently

Skeletal muscle as an artificial endocrine tissue 217

being incorporated into the design of viral46 and plasmid47 vectors, this methodology has the potential to deliver an even wider spectrum of biologically active proteins.

FACTORS INFLUENCING THE EFFICACY AND SAFETY OF GENE TRANSFER Muscle gene transfer for producing several different proteins, and methods for regulating therapeutic expression within muscle tissue, have clear advantages. However, there are several other important factors that will ultimately determine the success rate of muscle gene transfer when applied to humans. Efficiency of gene transfer Where muscle gene transfer has progressed to human trials, comparison with the corresponding animal studies has not been favourable—as illustrated by recent developments in gene therapy for Duchenne muscular dystrophy (DMD). Implantation of myoblasts expressing a dystrophin-coding sequence into skeletal muscle of DMD patients was not clinically beneficial to these subjects48, in contrast to a similar application in a murine model for DMD which resulted in long-term expression of a dystrophin cDNA.49 The reasons for the failure of dystrophin gene transfer in humans are not clear at present. However, there are obvious size and anatomical differences between humans and rodents in terms of muscle composition, activity and blood/ lymphatic microcirculation. These are factors which contribute to the efficiency of gene transfer and ultimately to the effectiveness of these methods in humans. In addition, the age of the muscle used seems to play an important role in efficiency of gene transfer.50,51 Immuogenicity of vectors An immune and/or inflammatory response directed against viral, cell and plasmid-based delivery vectors can greatly limit the efficacy of muscle gene transfer, as with other methods of gene transfer.9 Early studies using an adenovirus-based gene therapy to treat cystic fibrosis patients were unsuccessful owing to an inflammatory response directed against the delivery vector in some individuals.52 Adenoviral vectors have been engineered—so, hopefully, they contain no antigenic protein-coding sequences; therefore, any observed immune response should not result from de novo viral protein synthesis.53,54 However, over 40 different adenoviral serotypes have been identified in humans55 and pre-existing antibodies in any individual may limit the efficacy of even a ‘gutted’ adenoviral vector, as this still requires pre-packaging in a viral protein coat to facilitate entry into the cell.53 This is especially relevant where repeated administration of a delivery vector is necessary for effective treatment. AAV vectors are derived from human paraviruses and are intrinsically less immunogenic than AV.12 However, AAV vectors could present a problem similar to that of AV, as a significant proportion of the population will have been exposed to this pathogen55 and this could also obstruct re-administration of an AAV vector. Moreover, it may be possible to suppress the specific immune responses which limit the re-administration of viral vectors. For instance, activity of the Th2 subset of T helper cells, which block a second administration of recombinant adenovirus virus, can be diminished by coadministration with interferon-gamma or interleukin-12.56 Plasmid-based vectors have

218 G. Goldspink

also been shown to be immunogenic, as these contain immunostimulatory DNA sequences (ISS) which stimulate both macrophage- and T-lymphocyte-based immune responses.57,58 These ISS also have an adjuvant property which enhances the effect of DNA vaccines.59 However, an immune response directed against these ISS will be undesirable in most of the applications of muscle gene transfer because a significant proportion of the population have antibodies to bacterial DNA.60 Therefore, it must also be established whether a patient’s immune system will tolerate plasmid-based gene transfer into muscle. Immunogenicity of expressed transgenes Although an immune response is the desired consequence of using a DNA vaccine, for the purpose of gene therapy applications, the opposite is true. A common observation following muscle gene transfer of a sequence encoding a heterogeneous protein is a peak of expression at 14 days, followed by a decline and then a loss of expression.61 Work in this laboratory has shown that an immune response is generated against human factor VII after gene transfer into mouse muscle tissue.62 The subsequent loss of expression in muscle tissue results from an elimination of muscle fibres through a cytotoxic T-cell response directed against the immunologically foreign protein.61 The expression of divergent proteins in muscle can also break immune tolerance to the corresponding endogenous protein as, for instance, production of human Epo in mouse muscle activates an IgG immunoresponse to endogenous Epo.63 Therefore, the primary sequence of a protein must match that of the recipient to prevent elimination of those transfected muscle fibres which express the protein. Moreover, where a protein is completely absent, as occurs in DMD64, patients are immunologically naı¨ve with respect to the native protein (i.e. dystrophin). This factor could limit the efficacy of gene therapy for DMD. Non-muscle expression The safety of muscle gene transfer also depends on expression of the introduced gene being restricted to the site where the vector was administered. Recent studies demonstrate that both viral and plasmid vectors can be transported retrogradely to motor neurones innervating the injection site, with subsequent expression of the introduced gene in the neuronal cell body.2,65 Although vector uptake by motor neurones might be beneficial in treating motor neurone disease, it could also result in further damage occurring to the peripheral and central nervous systems and, therefore, must be investigated further. The targeting of gene expression to muscle tissue is an important issue and this can be achieved in several ways. First, the use of muscle-specific transcription elements in delivery vectors can restrict expression to muscle cells. For instance, the combination of myosin heavy-chain promoter and lightchain enhancer element used to produce human factor VII in muscle tissue62 directs gene expression to muscle fibres in both the fast tibialis anterior and slow soleus muscles in mice.30 Muscle-specific expression can also be accomplished by myoblast transfer, as myoblasts will form new muscle fibres at the site of introduction.23 In addition, an even more stringent level for controlling therapeutic gene expression in muscle can be achieved by combining both of the above methods with an element of antibiotic regulation. This method has been utilized in the antibiotic-regulated expression of Epo in muscle, as a human desmin promoter with muscle-specific

Skeletal muscle as an artificial endocrine tissue 219

activity drives expression of an antibiotic-binding protein and activates expression of Epo within implanted myoblasts.22

MUSCLE ORGANOIDS FOR THE IN VITRO EXPRESSION OF TRANSFERRED GENES As mentioned above, myoblast transfer is one means of transferring genes, and these muscle cells can be encapsulated to form organoids. As part of a continuing search for new expression systems in which the products of engineered genes are not only expressed but glycosylated and folded correctly, muscle cells have been cultured in bioreactors. These bioreactors are designed for the continuous perfusion of the myotubes/muscle fibres with culture medium and they maintain pH and O2/CO2 levels. The culture system includes an extracellular matrix and the means of subjecting the muscle cells to mechanical strain. To achieve the latter, specially designed muscle cell bioreactors were sent into space.66 Devices for holding the muscle tissue organoids at a basal tension level67, or simply stretching them, have been shown to be very effective in maintaining the muscle mass in culture.68

SUMMARY The examples of muscle gene transfer discussed in this chapter demonstrate the potential clinical applications of this methodology. However, as with any new drug therapy, the success of muscle gene transfer depends on both the efficacy and safety of the methodology used to deliver therapeutic genes into muscle tissue will take time. The clinical trials for muscular dystrophy reveal the difficulties inherent in applying muscle gene transfer to humans. These include immune responses to delivery vectors, combined with the tendency of viral vectors to be taken up by other tissues, including liver and neural tissue. These factors currently limit the efficiency and safety of muscle gene transfer. However, the progress currently being made in understanding the cellular mechanisms which underlie vector immunology and transport kinetics are expected to result in much safer methods. The combination of tissue-specific transcription elements with antibiotic regulation can now be used to ensure appropriate levels of muscle-specific expression. Therefore, considering the complex safety and efficacy aspects which must still be resolved, muscle gene transfer is likely to be applied to the most clinically difficult situations first, i.e. those where no effective treatment exists. These include muscular dystrophy, motor neurone disease and Fabry’s disease, and this methodology may also be applied to limb ischaemia resulting from peripheral vascular disease. These and other applications of muscle gene transfer have now progressed to the clinical trial stage, and the results of these studies will give a clearer indication of where muscle gene transfer will be most effective.

ACKNOWLEDGEMENTS While this chapter was being written, the author was in receipt of research grants from the Wellcome Trust, BBSRC and the International Olympic Games WADA Committee.

220 G. Goldspink

REFERENCES 1. Wolfe JA, Malone RW, Williams P et al. Direct-gene transfer into mouse muscle in vivo. Science 1990; 247: 1466–1468. 2. Hasse G, Kennel P, Vigne E et al. Gene therapy of murine motor neuron disease using adenoviral vectors for neurotrophic factors. Nature Medicine 1996; 3: 429– 436. 3. Tripathy SK, Goldwasser E, Lu MM et al. Stable delivery of physiological levels of recombinant erythropoietin to the systemic circulation by intramuscular injection of replication-deficient adenovirous. Proceedings of the National Academy of Sciences of the USA 1994; 91: 11 557 –11 561. 4. Fischman D. Development of striated muscle. In Bourne GH (ed.) The Structure and Function of Muscle, 2nd edn. New York: Academic Press, 1972, pp 75–142. 5. Yao SN, Wilson JM, Nabel EG et al. Expression of human factor IX in rat capillary endothelial cells; toward somatic gene therapy for haemophilia B. Proceedings of the National Academy of Sciences of the USA 1991; 88: 8101–8105. 6. Danko I & Wolff JA. Direct gene-transfer into muscle. Vaccine 1994; 12: 1499–1502. 7. Feldman LJ, Tahlil O & Steg G. Perspectives of arterial gene-therapy for the prevention of restenosis. Cardiovascular Research 1996; 32: 194– 207. 8. Rothman T, Katus HA, Hartong R et al. Heart muscle-specific gene-expression using replication-defective recombinant adenovirus. Gene Therapy 1996; 3: 919– 926. 9. Gorecki DC & MacDermott KD. Gene therapy: panacea or placebo? Main application of gene therapy. Archivum Immunologiae et Therapiae Experimentalis 1997; 45: 367–374. 10. Vile RG, Sunassee K & Diaz RM. Strategies for achieving multiple layers of selectivity in gene therapy. Molecular Medicine 1998; 4: 84 –92. 11. Yang Y, Haeker SE, Su Q & Wilson JM. Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle. Human Molecular Genetics 1996; 5: 1703–1712. 12. Fisher KJ, Jooss K, Alston J et al. Recombinant adeno-associated virus for muscle directed gene therapy. Nature Medicine 1997; 3: 306–312. 13. Yeh P & Perricaudet M. Advances in adenoviral vectors from genetic engineering to their biology. FASEB Journal 1997; 11: 615–623. * 14. Check E. Gene therapy: a tragic setback. Nature 2002; 420: 116 –118. * 15. Davis HL, Demeneix BA, Quantin B et al. Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle. Human Gene Therapy 1993; 4: 733 –740. 16. Gregoriadis G, Saffie R & deSouza JB. Liposome-mediated DNA vaccination. FEBS Letters 1997; 402: 107–110. 17. Mumper RJ, Wang JJ, Klakamp SL et al. Protective interactive noncondensing (PINC) polymers for enhanced plasmid distribution and expression in rat skeletal muscle. Journal of Controlled Release 1998; 52: 191–203. 18. Aihara H & Miyazaki J. Gene transfer into muscle by electroporation in vivo. Nature Biotechnology 1998; 16: 867–870. 19. Wang J-M, Zheng H, Sugahara Y et al. Construction of human factor IX expression vectors in retroviral vector frames optimized for muscle cells. Human Gene Therapy 1996; 7: 1743–1756. 20. Novo FJ, Gorecki DC, Goldspink G & MacDermott KD. Gene transfer and expression of human alphagalactosidase from mouse muscle in vitro and in vivo. Gene Therapy 1997; 4: 488 –492. 21. Rivera VM, Clackson T, Natesan S et al. A humanized system for pharmacological control of gene expression. Nature Medicine 1996; 2: 1028–1032. 22. Bohl D, Naffiakh N & Heard JM. Long term control of erythropoietin secretion by doxycycline in mice transplanted with engineered primary myoblasts. Nature Medicine 1997; 3: 299 –305. 23. Pin CL & Merrifield PA. Developmental potential of rat L6 myoblasts in vivo following injection into regenerating muscles. Developmental Biology 1997; 188: 147– 166. 24. MacColl G, Novo J, Marshall N et al. A method for producing biologically active growth hormone in muscle cells. Journal of Endocrinology 1998; 156: OC20. 25. Blue-pajot MT, Epelbaum J, Gourj D et al. Hypothalamic and hypophyseal regulation of growth hormone secretion. Cellular and Molecular Neurobiology 1998; 18: 101 –123. 26. Al-Hendy A, Hortelano G, Tannenbaum GS & Chang PL. Correction of the growth defect in dwarf mice with nonautologous microencapsulated myoblasts—an alternative approach to somatic gene-therapy. Human Gene Therapy 1995; 6: 165–175. 27. Hahn TM, Copeland KC & Woo SLC. Phenotypic correction of dwarfism by constitutive expression of growth hormone. Endocrinology 1996; 137: 4988–4993. 28. Draghia-Akli R, Li X & Schwartz RJ. Enhanced growth by ectopic expression of growth hormone releasing hormone using an injectable myogenic vector. Nature Biotechnology 1997; 15: 1285–1289.

Skeletal muscle as an artificial endocrine tissue 221 29. Anwer K, Shi M, French MF et al. Systemic effect of human growth hormone after intramuscular injection of a single dose of muscle-specific gene medicine. Human Gene Therapy 1998; 9: 959–970. 30. Skarli M, Kiri A, Vrbova G et al. Myosin regulatory elements as vectors for gene transfer by intramuscular injection. Gene Therapy 1998; 5: 514–520. 31. Ng ST, Zhou JA, Adesanya OO et al. Growth hormone treatment induces mammary gland hyperplasia in ageing. Nature Medicine 1997; 3: 1141–1149. 32. Goldspink G. Methods of treating muscular disorders. United States Patent No: US 6,211,842 B1 Apr 24 2001. 33. Barton-Davies ER, Shoturma DI, Musaro A et al. Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function. Proceedings of the National Academy of Sciences of the USA 1998; 95: 15 603 –15 607. * 34. Yang SY & Goldspink G. Different roles of the IGF-I Ec peptide (MGF) and mature IGF-I in myoblast proliferation and differentiation. FEBS Letters 2002; 522: 156–160. 35. Ye X, Rivera VM, Zoltick P et al. Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer. Science 1999; 283: 88– 91. 36. McGary EC, Rondon IJ & Beckman BS. Post-transcriptional regulation of erythropoietin mRNA stability by erythropoietin mRNA-binding protein. Journal of Biological Chemistry 1997; 272: 8628–8634. 37. Rask CA, Carlsen RC & Elias KA. Site of action of recombinant human nerve growth factor (rhNGF) in diabetic neuropathy appears to be small sensory neurons in the peripheral nervous system; phase II data support clinical results. Journal of Endocrinology 1998; 156(supplement): P91. 38. Ishii DN & Lupien SB. Insulin-like growth factors protect against diabetic neuropathy—effects on sensory nerve regeneration in rats. Journal of Neuroscience 1995; 40: 138–144. 39. Apfel SC & Kessler JA. Neurotrophic factors in the treatment of peripheral neuropathy. CIBA Foundation Symposia 1996; 196: 98– 108. 40. Lepantalo M & Tukianen E. Combined vascular reconstruction and microvascular muscle flap transfer for salvage of ischemic legs with major tissue loss and wound complications. European Journal of Vascular and Endovascular Surgery 1996; 12: 65 –69. 41. Cherwek DH, Hopkins MB, Thompson MJ et al. Fiber type-specific differential expression of angiogenic factors in response to chronic hindlimb ischemia. American Journal of Physiology 2000; 279: H932–H938. 42. Battegay EJ. Angiogenesis-mechanistic insights, neovascular diseases, and therapeutic prospects. Journal of Molecular Medicine 1995; 73: 333–346. 43. Takeshit S, Pu LQ, Stein LA et al. Intramuscular administration of vascular endothelial growth factor induces dose-dependent collateral artery augmentation in a rabbit model of limb ischaemia. Circulation 1994; 90: 228– 243. 44. Gabrilovich DI, Chen HL, Cunningham HT et al. Production of vascular endothelial growth-factor by human tumours inhibits the functional maturation of dendritic cells. Nature Medicine 1996; 2: 1096–1103. 45. Oyama T, Ran S, Ishida Tet al. Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-kappa B activation in hemopoietic cells. Journal of Immunology 1998; 160: 1224– 1232. 46. Hu SX, Ji W, Zhou YL et al. Development of an adenovirus vector with tetracycline-regulatable human tumor necrosis factor alpha gene expression. Cancer Research 1997; 57: 3339–3343. 47. Liang X, Hartika J, Sukhu L et al. Novel high expressing and antibiotic controlled plasmid vectors designed for use in gene therapy. Gene Therapy 1996; 5: 350–356. 48. Miller RG, Sharma KR, Pavlath GK et al. Myoblast implantation in Duchenne muscular dystrophy: the San Francisco study. Muscle and Nerve 1997; 20: 469–478. 49. Floyd SS, Booth DK, van Deukekom JCT et al. Autologous myoblast transfer: a combination of myoblast transplantation and gene therapy. Basic and Applied Myology 1997; 7: 241– 250. * 50. Wells DJ & Goldspink G. Age and sex influence expression of plasmid DNA directly injected into mouse skeletal muscle. FEBS Letters 1992; 306: 203 –205. 51. Danko I, Williams P, Herweijer H et al. High expression of naked plasmid DNA in muscles of young rodents. Human Molecular Genetics 1997; 6: 1435–1443. 52. Zabner J, Couture LA, Gregory RJ et al. Adenovirus-mediated gene-transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell 1993; 75: 207–216. * 53. Kochanek S, Clemens PR, Mitani K et al. A new adenoviral vector: replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and betagalactosidase. Proceedings of the National Academy of Sciences of the USA 1996; 93: 5731–5736. 54. Morsy MA, Gu MC, Motzel S et al. An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of leptin transgene. Proceedings of the National Academy of Sciences of the USA 1998; 95: 7866– 7871. 55. Scadding JW & Gibbs J. Neurological disease. In Souhami RL & Moxham J (eds) Textbook of Medicine, 2nd edn. Edinburgh: Churchill Livingstone, 1994, pp 858–982.

222 G. Goldspink 56. Yang YP, Trinchieri G & Wilson JM. Recombinant IL-12 prevents formation of blocking IgA antibodies to recombinant adenovirus and allows repeated gene-therapy to mouse lung. Nature Medicine 1995; 1: 890–893. 57. Sato Y, Roman M, Tighe H et al. Immunostimulatory DNA sequences necessary for effective intradermal gene immunizations. Science 1996; 272: 352–354. 58. Stacy KJ, Sweet MJ & Hume DA. Macrophages ingest and are activated by bacterial DNA. Journal of Immunology 1996; 157: 2116–2122. 59. Roman M, Martin-Orozco E, Goodman JS et al. Immunostimulatory DNA sequences function as T helper1-promoting adjuvants. Nature Medicine 1997; 3: 849 –854. 60. DeVlam K, de Keyser F, Verbruggen G et al. Detection and identification of antinuclear autoantibodies in the serum of normal blood donors. Clinical and Experimental Rheumatology 1993; 11: 393–397. 61. Wells KE, Maule J, Kingston R et al. Immune responses, not promoter inactivation, are responsible for decreased long-term expression following plasmid gene transfer into skeletal muscle. FEBS Letters 1997; 407: 164 –168. * 62. Miller G, Steinbrecher RA, Murdock PJ et al. Expression of factor VII by muscle cells in vitro and in vivo following direct gene transfer—modelling gene therapy for haemophilia. Gene Therapy 1995; 2: 736–742. 63. Tripathy SK, Black HB, Goldwasser E & Leiden M. Immune responses to transgene encoded proteins limit the stability of gene expression after injection of replication defective adenovirus vectors. Nature Medicine 1996; 2: 545–550. 64. Petrof B. The molecular basis of activity-induced muscle injury in Duchenne muscular dystrophy. Molecular and Cellular Biochemistry 1998; 179: 111 –123. 65. Johnson IP, Skarli M & Goldspink G. Comparison of plasmid and viral DNA for the transfer of genes to motor neurones. Society for Neuroscience Abstracts 1999; 24: 610.6. 66. Chromiak A, Shansky J, Perrone C & Vandenburgh HH. Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids. In Vitro Cellular & Developmental Biology Animal 1998; 34: 694–703. 67. Kosnik PE, Faulkner JA & Dennis RG. Functional development of engineered skeletal muscle from adult and neonatal rats. Tissue Engineering 2001; 7: 573–584. * 68. Powell CA, Smiley BL, Mills J & Vandenburgh HH. Mechanical stimulation improves tissue-engineered human skeletal muscle. American Journal of Cell Physiology 2002; 283: C1557–C1565.