JPH08501210A - Utilization of myoblasts for sustained release of gene products - Google Patents

Abstract

  (57) [Summary] Myoblasts containing a construct that comprises a fragment encoding the gene product of interest under the control of a muscle-specific promoter / enhancer are useful for sustained release of the gene product to a subject.

Description

DETAILED DESCRIPTION OF THE INVENTION Utilization of Myoblasts for Sustained Release of Gene Products Acknowledgments This invention was made with Government support under Grant No. CA44360 awarded by the National Institutes of Health. The government has certain rights in this invention. FIELD OF THE INVENTION The present invention relates to the use of myoblasts for the sustained release of gene products to a subject. BACKGROUND OF THE INVENTION Myoblasts are mesodermal progenitor cells that are destined for myogenesis. Established myoblasts recognize other myoblasts and can spontaneously fuse with them to produce differentiated myotubes. Polynuclear myotubes no longer undergo division or DNA synthesis, but produce large amounts of muscle proteins. These proteins include components of contractile organs and specific cell surface components essential for neuromuscular transmission. Eventually, the differentiated muscle cells show characteristic striations and rhythmic contractions. Further down this path is maturation; contractile organs and muscles at different developmental stages contain isoforms of differently-characterized muscle proteins, such as myosin and actin, encoded by different multigene families. Developed myoblast production from fetal and adult tissues. For example, Blau and Webster, Proc. Natl. Acad. USA Vol. 78 : 5623-5627 (1981) describes the isolation and cloning of myocytes for the expansion or differentiation of individual clones. The success of these methods suggests that large numbers of myoblasts, well depleted of other cells, can be made from living muscle tissue. Myoblasts have the potential to be used for various purposes. For example, myoblasts may serve as a vehicle for cell therapy, in which case one or more genes will be introduced into myoblasts to obtain the product in question. However, for their widespread use in therapeutic applications, it is necessary to develop methods for the sustained production of the product in question by myoblasts. BRIEF DESCRIPTION OF THE INVENTION Under the control of muscle-specific gene promoter / enhancer sequences or elements, primary myoblasts containing a construct consisting of a sequence encoding the gene product of interest are serum free for use in cell therapy. Made in medium or low serum medium. The transplanted myoblasts can migrate, fuse with already existing fibers, and serve as a carrier for the transgene in question. Migration of myoblasts through the basal lamina provides a method for efficient release of the desired gene product. In addition, migration of myoblasts through the basal lamina allows for treatment of disease with reduced injection frequency. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a schematic diagram of various configurations used in the examples described herein. FIG. 2 is a summary of canine factor IX expression levels in nude mice infused with myoblasts infected with the promoter / enhancer / factor IX construct shown in FIG. DETAILED DESCRIPTION OF THE INVENTION Presented are methods and compositions for use in cell therapy for the treatment of disease. The method involves preparing large amounts of clonally pure or substantially enriched myoblasts in a nutrient medium in the absence of serum or in the substantial absence of serum. The resulting cells can be used to treat various diseases associated with muscle tissue or other tissues containing soluble factors. In accordance with the present invention, a method of providing sustained release of gene product (s) in a host subject is described. The method of the invention comprises: (a) obtaining myoblasts from said subject, (b) introducing a DNA sequence into said myoblasts, wherein the DNA sequences are operatively related to Consisting of at least one muscle-specific gene enhancer sequence or element, at least one promoter capable of inducing expression, and a gene encoding the product in question and then (c) modified Myoblasts are introduced into the muscle tissue of the subject. In accordance with another aspect of the invention, another method of sustained release of gene product (s) in a host subject is disclosed. This alternative method consists of: (a) directing a DNA sequence into primary myoblasts obtained from the subject, the DNA sequence consisting of the following operatively related to each other: Together with one muscle-sustaining gene enhancer sequence or element, at least one promoter capable of inducing expression, and a gene encoding the product in question, and then (b) a modified myoblast. It is introduced into the muscle tissue of the subject. According to yet another aspect of the invention, yet another method of providing sustained release of gene product (s) in a host subject is described. This alternative method comprises introducing modified myoblasts into the muscle tissue of the subject, wherein the myoblasts are obtained from the subject and comprise the following operatively related DNA sequences: At least one muscle-specific gene enhancer sequence or element, at least one promoter capable of inducing expression, and a gene encoding the product in question. In accordance with yet another aspect of the present invention, there is provided a method of tissue-specific expression of gene product (s) in muscle tissue, the method comprising: muscular tissue operatively associated with an inducible promoter capable of expression. Placing the gene encoding the product (s) under the control of a specific gene enhancer sequence or element: In accordance with a further aspect of the invention, presenting a DNA construct consisting of the following DNA sequences operatively related to each other: To: At least one muscle-specific gene enhancer sequence or element, at least one promoter capable of inducing expression, and a gene encoding the product in question. A presently preferred DNA construct according to the invention consists of the following: a creatine kinase enhancer as the muscle-specific gene enhancer or element, a cytomegalovirus promoter / enhancer as the promoter, and a gene encoding the product of interest. According to a further aspect of the invention there is provided a primary myoblast comprising a DNA construct (s) as described above. According to yet another aspect of the present invention, there is provided a method of treating a disease state in which the normally secreted gene product is insufficiently produced, said method introducing modified myoblasts into a muscle tissue of a subject. Where the modified myoblast comprises a DNA construct (s) as described above. The promoters contemplated for use in the practice of the present invention are inducible promoters capable of high level expression. Such promoters include muscle-specific gene promoters such as the muscle creatine kinase gene promoter, as well as cytomegalovirus (CMV) enhancer / promoter, long terminal repeat (LTR) promoter / enhancer, α-globin promoter, and the like. Included. According to the present invention, the above-mentioned promoters are further associated with tissue-specific enhancers that direct muscle-specific expression to the gene of interest. Muscle-specific enhancers contemplated for use in the practice of the present invention include muscle creatine kinase enhancers, myo D enhancers, myosin enhancers, actin enhancers, and the like. The structural gene used will encode an intracellular product (ie, retained in the cytoplasm or in the organelle (eg, nucleus)). Otherwise, the product may be delivered to or secreted from the membrane (intracellular or cell membrane) to give a native signal sequence or a signal sequence not naturally present as a structural gene. In situations in which the protein in question is a soluble fragment of a larger protein, it may be necessary to have a signal sequence with such a protein that facilitates secretion and efficient processing of the expressed peptide. . Genes contemplated for use in the practice of the present invention include soluble products such as Factor VIII (or other clot-forming factors), insulin, adenosine deaminase (ADA), purine nucleoside phosphorylase, and the like. Is included. Further, genes encoding therapeutically effective products such as growth hormone, α, -antitrypsin, dystrophin, and the like are suitable for use in the practice of the invention. Sources of such genes can be obtained from a variety of sources, such as synthetically, from mammalian species, and the like. Myoblasts fuse with other myoblasts to give rise to myotubes, which eventually grow into smooth muscle fibers. The term as used herein refers to all cells that act as immediate precursors to smooth muscle fibers. The myoblasts used in the practice of the present invention are obtained from tissue samples, including undifferentiated tissues obtained from fetal, neonatal or aged humans. These cells are either fresh and either removed from the subject and immediately frozen, or they are frozen and stored for use in clonal cultures grown as a population that has been divided about 5 to 30 times. The cells are grown under optimal conditions in a suitable culture medium in a cell culture incubator (37 ° C., 5% CO _{2 in} air, saturated humidity). Other conditions may be used if desired. The selected medium allows growth without significant differentiation. Thus, the medium retains the concentration of mature myoblasts, and if necessary, may be introduced into an environment in which myoblasts differentiate and mature. The medium consists of sources of essential amino acids, inorganic salts, trace elements and vitamins, as well as other organic components. The following table shows what is generally considered to be optimal, but as is known in the art, various changes to individual components without adversely affecting myoblast growth. It is possible to give Additional factors are added to the basal medium. The first factor is 0.3 to 0.5 μg / ml of dexamethasone, preferably 0.39 to 0.40 μg / ml. Serum albumin, especially bovine serum albumin, is used at 0.25 to 0.75 mg / ml, preferably about 0.50 mg / ml. About 5 to 15 mg of epidermal growth factor is used, preferably about 10 mg / ml. Fetuin is used at about 0.25 to 0.75 mg / ml, preferably 0.5 mg / ml. Finally, insulin, preferably bovine insulin, is used at 150-200 μg / ml, preferably about 180 μg / ml. To develop myoblasts, the inoculum is introduced into the above medium and the cells are grown under the conditions described. After addition of the inoculum, gentle agitation is performed to ensure a uniform distribution of cells for further growth towards confluence. Cells may be collected in any convenient way. For example, the tissue is dissociated for a total of 40-60 minutes by two or three consecutive treatments with 0.05% trypsin-EDTA at 37 ° C in a Wheatin enriched trypsin treated flask with constant agitation. After each trypsin treatment, the cells collected in the supernatant are pooled and cooled to 4 ° C on ice. To stop further protease activity, add horse serum to a final concentration of 10% (vol / vol). The separated cells are then centrifuged (2 minutes, 25 ° C.): the cell pellet is then resuspended in conditioned medium and plated in medium or frozen in liquid nitrogen at a tissue density of approximately 0.1 cm ³ / ml. To do. During culture, myoblasts will transform into any of a wide variety of variables including, for example, fusion, transfection, infection, electroporation, ballistics, or the like. The particular method used to introduce foreign DNA into myoblasts is not critical to the practice of the invention. Depending on the purpose of the introduction of the foreign DNA, the interest there is to carry out directed homology, ie, canonical integration by recombination or illegitimate recombination. As an example, Smit hieset al., Nature Vol. 317: 230-235 (1985); Thomas and Capecchl, Cell Vol. 51: 503-512 (1987) and Mansour et al., Natu re Vol. 336: 348-352 (1988). For directed integration, flank each gene by at least 50 bp of DNA homologous to the site of integration for the gene of interest. Usually, it is flanked by at least 100 bp of DNA homologous to the site of integration target for the gene of interest. The homologous DNA used for integration is usually no larger than about 5 Kbp, whereas the homologous DNA used for directed integration is 10 Kbp in size. The flanking regions are preferably about the same size. Sequences for integration may include DNA associated with particular muscle defects. Thus, myoblasts are removed from the host, transformed by homologous recombination, and the cells cloned and screened for homologous recombination at the defective site. Otherwise, if the gene of interest is a naturally occurring gene, it would not be normally expressed in myoblasts or mature muscle tissue and would be ready for homologous recombination. , Where transcription initiation regulatory sequences (eg, promoters with muscle-specific enhancers) have been modified to provide different basis for inducible or constitutive transcription. Thus, myoblasts will then be used for the expression of endogenous (host-specific) or heterologous genes that are not normally expressed in muscle tissue. For example, one would want to prepare for the expression of products such as cytokines, growth factors, colony scavenging factors, interferons, surface membrane receptors, insulin, or the like. Modification of the transcription initiation regulatory region will allow myoblasts to constitutively produce the expression product. Instead (or in combination with the modifications mentioned above), a gene encoding a receptor will be introduced which induces transcription of proteins in the cell, eg cytoplasm, nucleus, etc. By activating the receptor, myoblasts will be induced to produce the desired expression product under the induction of the relevant ligand. A variety of mediator or vector constructs are used for transformation of myoblasts. Of particular interest for transfection and infection are replication-defective viral vectors, DNA viruses or retroviral vectors that will be introduced into cells. The vector normally does not contain any prokaryotic DNA and, as mentioned above, consists of several different functional sequences. There are markers for selection of cells that contain the transmitter composition. Normally, a marker can make a positive selection by protecting it from one or more cytotoxic agents. For example, using kanamycin resistance, cells would be selected on G418; dihydrofolate reductase is used for resistance to methotrexate and the like. Markers are inducible or non-inducible genes, so that selection will occur under inducing conditions or in the absence of inducing conditions. The vector also contains the origin of replication and other genes necessary for replication in the host. A system consisting of some gene that encodes a protein associated with origin and replication is included as part of the organization. An exemplary replication system contains the Epstein-over virus. Instead, replication-defective transporters will be used, particularly replication-defective retroviral vectors. These vectors are described below: by Price et al., Proc.Natl.Acad.Sci.Vol. 84 156-1650 (1987) and Sares et al., EMBOJ. Vol. 5 : 3133-3 142 (1986). The final mediator composition will have one or more genes of interest. The cDNA gene or chromosomal gene may be used. Of particular interest is the provision for at least one intron, which may be in the 5'-noncoding or coding region. The presence of introns is known to enhance the stability of messenger RNA. Instead, cells are transformed in vivo by injection with infectious replication-defective virus. The vector is introduced into the retroviral producer cells for ecotropic packaging. The cells are then harvested, filtered, concentrated by centrifugation and the virus stock is injected into the site in vivo . Since myoblasts are known to migrate and spread to adjacent regions, relatively little injection into myofibers is required. Administration of the vector is conveniently accomplished by injection in a physiologically acceptable medium, such as water, saline, phosphate buffered saline and the like. Viral concentrations are generally from 10 ⁵ cfu (colony forming units per ml). Other adducts present include polybrene. Usually, about 10 ⁵ cells are injected per cm ^{3 of} muscle fiber. Trauma to the tissue will be substantially minimized by minimizing the number of injections in the area of interest. Clearly, it is desirable to have a low number of injections in a particular area, especially where the patient may require extensive treatment. Now I would like to explain the invention in more detail by citing the following non-limiting examples. Construction CMV-IX Examples Example 1 Retroviral vectors [i.e., Proc. USA 87: 5173-51 77 (1990) LNcdF9L] contains 580 bp of human cytomegalovirus immediate-early gene enhancer / promoter (CMV). A schematic diagram of CMU-IX is shown in FIG. MECMV-IX is a mouse muscle creatine kinase (MCK) enhancer [Jaynes et al., In Mol., In the Sal I site 250 bp downstream from the CMV enhancer / promoter. Cell.Biol. 8: 62-70 (1988)]. A schematic diagram of MECMV-IX (or EMCMV-IX, where the MCK enhancer is incorporated in the reverse configuration) is shown in FIG. MEaGPcap-IX is a Sal I-Bam HI fragment (containing CMV enhancer / promoter) of CMV-IX vector, a fragment containing 220 bp MCK enhancer, a 650 bp fragment containing human α-globin promoter, and Xenopus Xenopus. ) Was made by substituting a 55 bp fragment from the β-globin 5'-untranslated region [see Malone et al., In Proc. Natl. Acad. Sci. USA 86 : 6077 (1989)]. A schematic of MEaGPcap-IX (or EMaGPcap-IX with the MCK enhancer incorporated in the reverse configuration) is shown in FIG. Ecotropic packaging cell line ψ _cre cell [Danos, D. and Mulligan, R. et al. C. By Proc.Natl.Acad.Sci. USA 85 : 6460-6464 (1988)] by the calcium phosphate precipitation method with about 20 μg DNA of each of the three retroviral vectors described above [Chen, C and Okayama, H. et al. in Mol. Cell. Biol. 7 (8): 2745-2752 (1987)]. G418-resistant colonies were pooled, approximately 3 ml of supernatant from these transfected cells were filtered through a 0.45 μm microfilter, and in the presence of 8 μg / ml polybrene, an amphotropic packing cell line. To AM12 cells [Markowitz, D. et al. . et al, the Virology 1 67: according to 400-406 (1988)]. G418-resistant colonies of infected AM12 cells were isolated and assayed for retroviral titer. The highest titer colonies were used to produce the virus for infecting primary myoblasts. Example II Preparation of primary myoblasts Hindlimb muscles were removed from 3-4 day old Swiss Webster or Balb / C mice and minced and then containing 0.259-6 trypsin (Gibco) and 100 μg / ml collagenase. The cells were separated three times in an aqueous solution (about 10-20 minutes each time). Dissociated cells were seeded on gelatin-coated dishes in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum and 1% avian embryo extract (inactivated at 65 ° C for 10 minutes). Myoblasts were enriched by pre-plating for 20 minutes at 37 ° C. on non-gelatin-coated dishes to which fibroblasts preferentially attach. Example III Infusion of Myoblasts and Assay for Canine Factor IX Expression Early stage myoblasts were infected with amphoteric virus from AM12 packaging cells in the presence of 4 μg / ml polyprene and 200 μg / ml G418. (Activity, Gibco). G418-resistant myoblasts were pooled and expanded. Myoblasts were harvested and resuspended in phosphate buffer at a density of about 0.5-2 × 10 ⁸ cells / ml and then placed in the hind foot muscle of 6-8 week old nude or Swiss Webster mice. Injected (5-10 μl / site, 100 μl / paw). Plasma was collected from tail vein blood at the time points shown in Figure 2 and an ELISA assay was performed for the presence of dog Factor IX in mouse plasma [Axelrod et al. By Proc.Natl.Acad.Sci. USA 8 7: according to 5173-5177 (1990)]. The results are shown in FIG. A closer look at this figure clearly shows that all of the tested constructs initially showed prominent Factor IX production and disappeared after a few days. Factor IX production (ie, using the muscle creatine kinase enhancer in combination with the cytomegalovirus promoter) induced by the constitutive MECMV of the present invention is reduced to only about a few days, but then dramatically. Increased to the initial expression level, which lasts at least 120 days. In contrast, the control configuration (B MaGP and CMV only) produced very low levels of factor IX after the first overhanging production or decline (ie within a few days). Two consecutive transplantations were performed at 4-5 day intervals in an attempt to increase the concentration of Factor IX produced and secreted into the circulation. Circulating Factor IX concentration decreased 4 days after the first transplant but increased with the second transplant. As with the first transplant, the concentration of factor IX decreased after 8 days and then increased for at least 120 days. Factor IX up to 20 ng / ml can be detected, which is about twice the amount detected after the first transplant. Therefore, the concentration of Factor IX expression is proportional to the number of transplanted myoblasts. The orientation of the enhancer with respect to the CMV promoter, if any, was reversed to measure the effect of the relative orientation of the promoter and enhancer. Higher factor IX expression was observed when the enhancer was oriented the same as the promoter. Since the present invention has been described in detail with reference to some preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of the invention as described and claimed herein.

[Procedure Amendment] Patent Act Article 184-8 [Submission Date] July 22, 1994 [Correction content] The scope of the claims   1. The following integrated, recombinant DNA vector sequences operatively related to each other are included: In a host subject, comprising introducing modified myoblasts into the muscle tissue of the subject. Methods of causing gene product release:     At least one muscle creatine kinase (MCK) sequence,     At least one cytomegalovirus promoter, and at least one At least one gene encoding a product of interest.   2. Introducing the recombinant DNA vector sequence into primary myoblasts obtained from the subject The method according to claim 1, wherein the modified myoblast is prepared.   3. The promoter has a blood concentration of the gene product of at least 1 ng / ml The method according to claim 1, which is capable of inducing such a high level of expression.   4. A method of causing release of a gene product in a host subject, the method comprising:   (a) a recombinant DNA vector sequence comprising the following operatively related to each other: The recombinant DNA vector sequence is incorporated into the primary myoblast so that it is integrated into the primary myoblast genome. Introducing into cells:         At least one muscle creatine kinase (MCK) enhancer sequence,         At least one cytomegalovirus (CMV) promoter, and And         At least one gene encoding at least one of the products in question, And then   (b) introducing the modified myoblasts prepared according to step (a) into the muscle tissue of the subject The above method comprising:   5. The method of claim 4, wherein the primary myoblasts are obtained from the subject.   6. The promoter has a blood level of gene product of at least 1 ng / ml The method according to claim 4, which is capable of inducing such a high level of expression.   7. A method of causing release of a gene product in a host subject, the method comprising:   (a) obtaining myoblasts from the subject,   (b) a recombinant DNA vector sequence that operatively relates to each other, including: The recombinant DNA vector sequence is integrated in the myoblast so that it integrates into the myoblast genome. To be introduced in:         At least one muscle creatine kinase (MCK) enhancer sequence,         At least one cytomegalovirus (CMV) promoter, and And         At least one gene encoding at least one of the products in question, And then   (c) introducing modified myoblasts prepared according to step (a) into the muscle tissue of the subject The above method comprising:   8. The promoter has a blood concentration of the gene product of at least 1 ng / ml 8. The method of claim 7, which is capable of inducing high levels of expression, such as.   9. High level expressible CMV promotion for making recombinant DNA vectors Gene under the control of a muscle creatine kinase enhancer that is operatively associated with At least one gene encoding a product is placed, the DNA vector is A method for tissue-specific expression of a gene product in muscle tissue, which comprises introducing into the tissue.   Ten. An isolated recombinant DNA comprising the following DNA sequences operatively related to each other: Vector constructs:         At least one muscle creatine kinase (MCK) enhancer sequence,         At least one cytomegalovirus (CMV) promoter, and And   At least one gene encoding at least one product of interest.   11. The following integrated, recombinant DNA vector sequences operatively related to each other were Isolated primary myoblasts containing:         At least one muscle creatine kinase (MCK) enhancer sequence,         At least one cytomegalovirus (CMV) promoter, and And         At least one gene encoding at least one product of interest.   12. The following integrated, recombinant DNA vector sequences operatively related to each other: Secreted at normal times, including introducing modified myoblasts containing Methods for treating disease states associated with inadequate production of the following gene products:         At least one muscle creatine kinase (MCK) enhancer sequence,         At least one cytomegalovirus (CMV) promoter, and And         At least one gene encoding at least one product of interest.

Claims (1)

[Claims]   1. Modified myoblasts containing the following DNA sequences operatively related to each other The host provides sustained release of the gene product in the subject, including introduction into muscle tissue. How to tell:     At least one muscle-specific gene enhancer sequence or element, expression At least one promoter capable of inducing Gene to be read.   2. By introducing the DNA sequence into primary myoblasts obtained from the subject. The method according to claim 1, wherein the modified myoblast is prepared.   3. The muscle-specific gene enhancer sequence or element is added to muscle creatine. Kinase enhancer, myo D enhancer, myosin enhancer, or The method according to claim 1, wherein the method is selected from Kuching enhancers.   4. The method according to claim 1, wherein the promoter is capable of high level expression. Law.   5. The method according to claim 1, wherein the promoter is a muscle-specific gene promoter. Law.   6. The promoter is a cytomegalovirus (CMV) enhancer / pro Motor, muscle creatine kinase promoter, long terminal repeat (LTR) promoter A promoter / enhancer or an α-globin promoter. The method described.   7. A method of providing a host subject with sustained release of a gene product, the method comprising:   (a) Primary myoblasts are provided with DNA sequences containing the following operatively related: What to introduce:        At least one muscle-specific gene enhancer sequence or element, At least one promoter capable of inducing expression, and the product in question The gene that encodes And then   (b) introducing modified myoblasts into the muscle tissue of the subject.   8. The method according to claim 7, wherein the primary myoblasts are obtained from the subject.   9. The muscle-specific gene enhancer sequence or element is added to muscle creatinine Enzyme enhancer, myo D enhancer, myosin enhancer, or activator The method according to claim 7, wherein the method is selected from among enhancers.   Ten. The method according to claim 7, wherein the promoter is capable of high level expression. Law.   11. The method according to claim 7, wherein the promoter is a muscle-specific gene promoter. Law.   12. The promoter is a cytomegalovirus (CMV) enhancer / promo , Muscle creatine kinase promoter, long terminal repeat (LTR) promoter Selected from a promoter / enhancer or an α-globin promoter. How to list.   13. A method of providing sustained release of a gene product in a host subject, the method comprising:   (a) obtaining myoblasts from the subject,   (b) adding to said myoblast a DNA sequence comprising the following operatively related to each other: What to introduce:        At least one muscle-specific gene enhancer sequence or element, At least one promoter capable of inducing expression, and the product in question The gene that encodes And then   (c) introducing modified myoblasts into the muscle tissue of the subject.   14. The muscle-specific gene enhancer sequence or element is Nase enhancer, myo D enhancer, myosin enhancer, or actin 14. The method of claim 13 selected from tin enhancers.   15． 14. The method of claim 13, wherein the promoter is capable of inducing high levels of expression.   16. 14. The promoter according to claim 13, wherein the promoter is a muscle-specific gene promoter. Method.   17． The promoter is a cytomegalovirus (CMV) enhancer / pro Motor, muscle creatine kinase promoter, long terminal repeat (LTR) promoter Ter / enhancer, or α-globin promoter. The method described in.   18. A gene encoding a gene product is induced by a promoter that enables high-level expression. Of a muscle creatine kinase enhancer that is operatively associated with A method for tissue-specific expression of a gene product in muscle tissue, comprising:   19. A DNA construct comprising the following DNA sequences operatively related to each other:         At least one muscle-specific gene enhancer sequence or element, At least one promoter capable of inducing expression, and the product in question Gene that encodes.   20. The muscle-specific gene enhancer sequence or element is And a cytomegalovirus promoter 20. The DNA construct according to claim 19, which is / enhancer.   twenty one. Primary myoblasts containing the following DNA sequences operatively related to each other:         At least one muscle-specific gene enhancer or element, expression At least one promoter capable of inducing Gene to be read.   twenty two. Modified myoblasts containing the following DNA sequences operatively related to each other Insufficient production of gene products that are secreted under normal conditions, including introduction into muscle tissue Methods of treating disease states related to nothing:         At least one muscle-specific gene enhancer sequence or element, At least I promoter capable of inducing expression, and the product in question Gene that encodes.