Grace K. Pavlath

Myoblast implantation in Duchenne muscular dystrophy: The San Francisco study

We evaluated myoblast implantation in 10 boys with Duchenne muscular dystrophy (DMD) and absent dystrophin (age 5–10 years) who were implanted with 100 million myoblasts in the anterior tibial muscle of one leg and placebo in the other. Cyclosporine (5 mg/kg/day) was administered for 7 months. Pre‐ and postimplantation (after 1 and 6 months) muscle biopsies were analyzed. Force generation (tetanic tension and maximum voluntary contraction) was measured monthly in a double‐blind design. There was increased force generation in both legs of all boys, probably due to cyclosporine. Using the polymerase chain reaction, evidence of myoblast survival and dystrophin mRNA expression was obtained in 3 patients after 1 month and in 1 patient after 6 months. These studies suggest a salutary effect of cyclosporine upon muscular force generation in Duchenne muscular dystrophy; however, myoblast implantation was not effective in replacing clinically significant amounts of dystrophin in DMD muscle.

Isolation of human myoblasts with the fluorescence-activated cell sorter

We have established procedures for the rapid and efficient purification of human myoblasts using the fluorescence-activated cell sorter. Our approach capitalizes on the specific reaction of monoclonal antibody 5.1H11 with a human muscle cell surface antigen. For each of the five samples analyzed, an enrichment of myoblasts to greater than 99% of the cell population was immediately achieved. Following 3 to 4 weeks of additional growth in vitro, sorted myoblast cultures remained 97% pure. Differentiation of the sorted myoblast cultures, assessed by creatine kinase activity and isozyme content, was comparable to that of pure myoblast cultures obtained by cloning, and was significantly greater than that of mixed fibroblast and myoblast cultures. An average of 10(4) viable myoblasts can be obtained per 0.1 g tissue, each with the potential to undergo approximately 40 cell divisions. Accordingly, if only two-thirds of this proliferative capacity is utilized, the potential yield approximates 10(12) myoblasts, equivalent to 1 kg of cells. Human myogenesis in vitro is no longer limited by cell number and is now amenable to molecular and biochemical analysis on a large scale.+

Myoblasts in pattern formation and gene therapy

The tissues of a multicellular animal are composed of diverse cell types arranged in a precisely organized pattern. Features unique to muscle allow an analysis of pattern formation and maintenance in mammals. The progeny of single cells can be taken full cycle from the animal to the culture dish and back to the animal where they fuse into mature myofibers of the host. These features not only facilitate the use of genetically engineered myoblasts in studies of pattern formation, but also in cell-mediated gene therapy: a novel mode of drug delivery for the treatment of muscle and nonmuscle diseases such as hemophilia, cardiac disease and cancer.

Evidence for Defective Myoblasts in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a degenerative disorder associated with progressive muscle weakness. Affected children eventually die from respiratory or cardiac failure and rarely survive to adulthood. It is a genetic disease due to a defect at a single locus on the X-chromosome and is therefore transmitted by female carriers to their sons. The disease is relatively common; one in 4,800 males or a total of approximately 20,000 boys in the United States has DMD (239).

Muscle Gene Expression in Heterokaryons

One approach to the study of how gene regulation occurs during muscle differentiation and development is to put a non-muscle cell into an environment where it is reprogrammed to synthesize muscle proteins, which it would never normally express. One way to do this is to fuse two cells together. In this talk I will show you how this kind of fusion system can be used to ascertain the requirements for gene expression and to address certain questions about the determination and differentiation.

Purification and Proliferation of Human Myoblasts Isolated with Fluorescence Activated Cell Sorting

Myoblast therapy, or the transfer of normal myoblasts into dystrophic muscle, requires both a large number and high purity of human myoblasts. Solutions to both of these problems are presented below.

Localization of Muscle Gene Products in Nuclear Domains: Does this Constitute a Problem for Myoblast Therapy?

A question of major interest in considering myoblast therapy is whether the gene product dystrophin, provided by the introduced myoblasts, can contribute to the function of the myofiber into which the cells fuse. As shown in several papers in this volume, there is now ample evidence that injected myoblasts can fuse with myofibers and produce dystrophin, but the question of whether that dystrophin can restore muscle function still remains. In this regard, a major consideration is whether dystrophin can gain access to distant sites within the myofiber or remains localized in the vicinity of the nucleus that encoded it.

In vivo aging of human fibroblasts does not alter nuclear plasticity in heterokaryons

In vivo aging of human fibroblasts altered proliferative properties but not the potential for novel gene expression in response to muscle trans-acting factors. Heterokaryons produced by fusing fibroblasts with muscle cells permitted a dissociation of the effects of aging on cell division and other cell functions. Skin fibroblasts derived from fetal and adult stages of development were distinct cell types based on their doubling time, protein content, cell size, and specific binding of insulin and insulin-like growth factor I. Despite these differences in growth parameters, the two cell types were indistinguishable in heterokaryons. Muscle gene activation occurred in the absence of changes in chromatin structure requiring DNA replication. In addition, the time course, maximal efficiency, and effect of gene dosage on the expression of muscle gene products were similar for heterokaryons containing fetal and adult fibroblasts but distinct for heterokaryons containing keratinocytes. The difference between fibroblasts and keratinocytes in the time course of muscle gene expression is likely to reflect mechanisms of gene activation at the transcriptional level, since the kinetics of muscle protein accumulation paralleled that of muscle transcripts. These results indicate that nuclear plasticity is not altered in fibroblasts by in vivo aging.

Myoblast Mediated Gene Therapy

A novel approach to drug delivery in the treatment of disease involves using cells to introduce genes into the body that express therapeutic proteins continuously (Friedman, 1989; Miller, 1990; Anderson, 1992; Miller, 1992). Myoblasts appear to be well suited for this purpose due to their unique biological properties. By contrast with other cell types, myoblasts become an integral part of the tissues into which they are injected. As a result, myoblasts are currently primary candidates as cellular vehicles for gene delivery in the treatment of both muscle and nonmuscle disorders. Transplanted myoblasts may be used to correct defects in well characterized inherited myopathies such as Duchenne muscular dystrophy (DMD) (Gussoni et al., 1992). In addition, myoblasts may provide genes to correct myopathies that are not understood at the molecular level. Finally, and perhaps most exciting, is the finding that genetically engineered myoblasts can be used to deliver nonmuscie recombinant proteins to the circulation (Dhawan et al., 1991; Barr and Leiden, 1991). Candidates for delivery include hormones, coagulation factors, and antitumor agents that could have broad applications ranging from the treatment of inherited hormone deficiencies to symptoms of aging, hemophilia, and cancer.