Human myoblast cell lines and their uses

Details Human myoblast cell lines and their uses Human myoblast cell lines and their uses

2000-04-27

2003-02-04

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Reexamination Certificate

active

06514756

ABSTRACT:

The present invention relates generally to the field of human muscular cell lines, and more particular to human myoblast cell lines produced by a new process and to uses of these cell lines, especially in gene therapy or in understanding and approach of the etiology of diseases affecting muscle tissue.
Cell lines are widely used as in vitro models for studying the events involved during in vivo cellular or tissular development. For example, muscular development events can be reproduced during the differentiation of muscle cell lines. Accordingly, permanent mammalian cell lines, especially human myogenic cell lines, would be of considerable value for providing useful tools for dissecting the molecular and biochemical cellular events, for identifying and testing new drugs for muscular diseases, such as dystrophies, for the study of myogenesis, etc . . . .
In vivo, myoblasts are precursor cells of the mesoderm that are destined for myogenesis. The determined myoblasts are capable of recognizing and spontaneously fusing with other myoblasts leading to the production of a differentiated myotube. The multinucleated myotube no longer divides nor synthesizes DNA, but it produces muscle proteins in large quantity which are constituents of the contractile apparatus and specialized cell-surface components essential to neuromuscular transmission.
Some myogenic spontaneous cell lines have been isolated from primary muscle cultures obtained by enzymatic disaggregation of rodent skeletal muscle (Mulle et al., 1988, P.N.A.S., USA, 85, 5728-5732). However, primary myogenic clones obtained from human muscle do not give rise to such cell lines and show a finite life span which diminishes with increasing donor age. Moreover, the proliferation capacity of myoblasts from patients with Duchenne muscular dystrophy (DMD) is particularly restricted in vitro (Webster et Blau, 1990, Som. Cell. Mol. Genet., 16, 557-565) preventing for obtaining a satisfactory testing or studying model for this disease.
Furthermore, there is substantial interest in developing ways in which myoblasts, produced as stable cell line, may be used for therapeutic purposes. For example, the myoblasts may serve as cell therapy vector for the treatment of various diseases associated with genetic or non-genetic defect, for vaccination protocols, involving muscle tissue as targeted tissue or administration site. In the specific case where myoblasts are used as carriers for gene therapy, one or more genes are introduced into said myoblasts which are selected among muscle or non-muscle genes and which encode a polypeptide useful for the treatment of muscle or non-muscle diseases, or for providing novel or enhanced genetic capabilities or new vaccination tool.
Moreover, it has been previously shown that in vivo the myoblasts are capable of migrating to distant sites, particularly to sites of injury, and of fusing into pre-existing fibers. In cellular gene therapy perspectives, this myoblast migration across basal lamina may allow with only a few administration of myoblasts serving as carriers for genes, to treat a relatively large area. Myoblasts may be administered by injecting directly into the damaged site or at adjacent tissue or may be introduced into the blood stream, particularly in a vessel feeding the-damaged site and upstream from such site.
In more widely therapeutic applications, the myoblasts may be used as non-modified cells or modified to express compounds of interest, to increase or decrease the expression or availability of surface membrane molecules, MHC antigens, etc . . . which may correct genetic defects, supply surface membrane proteins or secreted products such as immunogenic peptides at a site which may be located at a distant site of the administration site.
Nevertheless, the above described applications require first to establish cell lines in culture that are capable to proliferate, to differentiate and to express properties characteristic of the cells in the tissue from which they were derived. The ability to establish particular cell lines has been widely described for many, but not all, cell types. Some of them maintain their original characteristics although many lose their differentiated phenotype upon continuous passage in culture. Finally, unlike rat and mouse for which several cell lines exist, there are few or no established human muscular cell lines available for applications yet.
Fogel et Defendi, 1967, Proc. Nati. Acad. Sci., 58, 967-973, have demonstrated that human myoblasts were susceptible to infection with wild-type SV40 and that permanent cell lines could be generated following infection. However, these cell lines rapidly lost the ability to differentiate. Several immortalized myogenic cell lines have been isolated from primary muscle culture of rodent skeletal muscle (Yaffe, D. “Retention of differentiation potentialities during prolonged cultivation of myogenic cells” Proc Natl Acad Sci USA (1968) 61: 477-483 and Yaffe, D. and Saxel, O. “Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle” Nature (1977) 270: 725-727). However, clones obtained from human primary cultures do not give rise to spontaneous cell lines and have a finite life. This phenomenon could be related to the observation that muscle tumors are very rare. More recently, Simon et al, 1996, Exp. Cell Res., 224, 264-271 have for the first time obtained long-term myogenic cultures from DMD muscle by infection of explant cultures with a recombinant retrovirus containing a mutated, temperature-sensitive form of the SV40 large T oncogene. Nevertheless, use of retrovirus is not satisfactory in terms of safety because they are integrating virus, said integration could interfere with the expression of vital genes or result in viral protein expression which can lead to CTL response in the treated patient.
Accordingly, the prior art is deficient in providing a satisfactory human muscular cell lines which can proliferate and differentiate, especially originating from muscular biopsies obtained from Duchenne muscular dystrophy (DMD) patients as well as from normal, dystrophin-positive individuals. The present invention fulfills this longstanding need and desire in the art.
The present invention provides human muscular cell lines obtained according to an improved process for establishing long-term mammalian cell line. Said human muscular cell lines show a great proliferative capacity, are able to differentiate and may prove valuable for in vitro investigations related to the cellular and molecular muscular metabolisms, to new drug screening or to methods assessing for cellular toxicity or cellular damages and providing a cellular model of choice for studies aimed to correct the molecular pathology of diseases, especially inherited disorders such as DMD.
Thus, the present invention first concerns a human muscular cell line which is able to proliferate wherein said human muscular cell line is generated from primary human muscular cells by a process comprising the step of:
a) pre-treating a culture of said primary human muscular cells or a suspension thereof with at least one glucocorticoid,
b) optional step comprising obtaining a suspension of said pre-treated culture of step a),
c) transferring into the pre-treated cells of the suspension of step a) or b) at least one nucleic acid vector which is not of retroviral origin and which is competent to immortalize said pre-treated cells and
d) culturing the transferred cells of step c).
In accordance with the present invention, immortalized human myogenic cell lines from skeletal muscle biopsies have been established using the calcium phosphate transfection with a SV40 large T Antigen (TAg) plasmid further carrying a phleomycine selection gene. After transfection and selection, clones were derived. They express TAg and the myoblast natural marker desmin. In appropriate culture conditions, cells of said cell lines aligned and fused to form multinucleated myotubes, indicating they still proliferate, differentiate and express properties characteristic of musc

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