Future Prospects For Myoblastmediated Therapies

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A. Inherited Myopathies

As noted earlier, a problem in using the ex vivo delivery approach to treat inherited myopathies is that for such therapies to be effective, a large proportion of skeletal muscle must be targeted. Direct intramuscular implantation of myoblasts leads to fusion of the injected myoblasts to a majority of fibers in the region of the injection site; the number of fibers to which genes are delivered by this approach decreases with increasing distance from the site (70). Thus, for a sufficiently large percentage of fibers to be treated, many closely spaced injections

Figure 7 Tetracycline-inducible expression using tTA and rtTA. (A) Schematic of a binary retroviral system allowing tetracycline-inducible expression of both human growth hormone (hGH) and green fluorescent protein (GFP). The expression of both proteins is ensured by the use of an internal ribosomal entry site (IRES), allowing both genes to be encoded within the same mRNA transcript. The reporter virus contains a self-inactivating (SIN) retroviral backbone to avoid interference of the viral long terminal repeat (LTR) with the tet-responsive promoter (O7-CMVm). The diagram represents the system after integration into the chromosome; hence, the SIN LTR exists in both the 5' and 3' positions. Tet-sensitive transactivators (either tTA or rtTA) are provided constitutively from a second retrovirus. (B) Dose response of the binary tet-inducible system shown in (A). RTAb( -) cells transduced with tet reporter virus and tTA virus, and RTAb( +) cells transduced with tet reporter virus and rtTA virus, were assessed for their dox dose response of hGH (o) expression and GFP (A) expression. Both systems exhibit concentration dependence over several orders of magnitude. (C) Histogram plots of GFP expression obtained from FACS analysis are shown in overlay at three selected doses [RTAb( -): 0.1 ^g/mL (light gray), 0.001 ^g/mL (dark gray), and 0 ^g/mL (black); RTAb( + ): 0 ^g/mL (light gray), 0.1 ^g/mL (dark gray), and 5 ^g/mL (black)]. These plots show that with changing concentrations of dox, populations uniformly shift to intermediate and high levels of expression. (Adapted and reprinted from Ref. 128 with permission of the Proceedings of the National Academy of Sciences USA, 2101 Constitution Ave., NW, Washington, DC 20418. Reproduced by permission of the publisher via Copyright Clearance Center, Inc.)

Figure 7 Tetracycline-inducible expression using tTA and rtTA. (A) Schematic of a binary retroviral system allowing tetracycline-inducible expression of both human growth hormone (hGH) and green fluorescent protein (GFP). The expression of both proteins is ensured by the use of an internal ribosomal entry site (IRES), allowing both genes to be encoded within the same mRNA transcript. The reporter virus contains a self-inactivating (SIN) retroviral backbone to avoid interference of the viral long terminal repeat (LTR) with the tet-responsive promoter (O7-CMVm). The diagram represents the system after integration into the chromosome; hence, the SIN LTR exists in both the 5' and 3' positions. Tet-sensitive transactivators (either tTA or rtTA) are provided constitutively from a second retrovirus. (B) Dose response of the binary tet-inducible system shown in (A). RTAb( -) cells transduced with tet reporter virus and tTA virus, and RTAb( +) cells transduced with tet reporter virus and rtTA virus, were assessed for their dox dose response of hGH (o) expression and GFP (A) expression. Both systems exhibit concentration dependence over several orders of magnitude. (C) Histogram plots of GFP expression obtained from FACS analysis are shown in overlay at three selected doses [RTAb( -): 0.1 ^g/mL (light gray), 0.001 ^g/mL (dark gray), and 0 ^g/mL (black); RTAb( + ): 0 ^g/mL (light gray), 0.1 ^g/mL (dark gray), and 5 ^g/mL (black)]. These plots show that with changing concentrations of dox, populations uniformly shift to intermediate and high levels of expression. (Adapted and reprinted from Ref. 128 with permission of the Proceedings of the National Academy of Sciences USA, 2101 Constitution Ave., NW, Washington, DC 20418. Reproduced by permission of the publisher via Copyright Clearance Center, Inc.)

Figure 8 The need for tet modulators with distinct dimerization domains. Coexpression of tetR fusion proteins with different functional domains such as repressor domains (represented in the top row by the ''do not enter'' sign), and activator domains (represented by the ''go'' sign), or DNA-binding domains with distinct specificity (symbolized in the middle row by the light gray and dark gray ''feet''), leads to formation of both functional homodimers and nonfunctional heterodimers. Such nonfunctional heterodimers can be eliminated by engineering distinct dimeriza-tion domains into the tetR portion of the tet modulators (symbolized by the dark gray and light gray shaded midsections in the bottom row). (Reprinted from Ref. 138 with permission of the Proceedings of the National Academy of Sciences USA, 2101 Constitution Ave., NW, Washington, DC 20418. Reproduced by permission of the publisher via Copyright Clearance Center, Inc.)

Figure 8 The need for tet modulators with distinct dimerization domains. Coexpression of tetR fusion proteins with different functional domains such as repressor domains (represented in the top row by the ''do not enter'' sign), and activator domains (represented by the ''go'' sign), or DNA-binding domains with distinct specificity (symbolized in the middle row by the light gray and dark gray ''feet''), leads to formation of both functional homodimers and nonfunctional heterodimers. Such nonfunctional heterodimers can be eliminated by engineering distinct dimeriza-tion domains into the tetR portion of the tet modulators (symbolized by the dark gray and light gray shaded midsections in the bottom row). (Reprinted from Ref. 138 with permission of the Proceedings of the National Academy of Sciences USA, 2101 Constitution Ave., NW, Washington, DC 20418. Reproduced by permission of the publisher via Copyright Clearance Center, Inc.)

would be necessitated. This requirement imposes a major limitation to the utility of myoblast-mediated gene transfer in treating human muscular dystrophies, which often affect cardiac and diaphragm muscles as well as skeletal muscles. Unless a myoblast population is isolated that can efficiently migrate to damaged or degenerated muscle, this approach seems too inefficient to be useful.

Recent observations that cells originating from bone marrow can become incorporated into regions of induced muscle regeneration has been met with much interest (144-146). This finding was significant because it elucidated the possibility that such bone marrow-derived cells can travel through the circulation and enter into skeletal muscle tissue (147). A possi ble solution to the problem of targeting skeletal muscles throughout the body may lie in introducing genetically engineered muscle precursor cells to the circulation, where they can reach muscles throughout the entire body. One study has examined the feasibility of intra-arterial delivery of genetically labeled, immortalized L6 myoblasts to skeletal muscle (71). After infusion of these cells into the arterial circulation, a small number of labeled fibers were observed in skeletal leg muscle, showing that the circulation may be capable of delivering muscle precursor cells to differentiated myofibers, although some were also found in the lung. Alternatively, if muscle stem cells of the bone marrow could be isolated, genetically engineered ex vivo and injected back into the patient, they could serve as a continual pool of circulating therapeutic effectors for the treatment of myopathies.

The existence of a muscle stem cell has been suggested from several pieces of evidence. Populations of cells that are capable of self-renewal and that give rise to differentiated cells have been identified both in the myogenic C2 cell line (148), and in clones of human myoblasts (149). In addition, a recent paper showed, using 2 genetic markers with different modes of inheritance to examine the fate of myoblasts transplanted into skeletal muscle, that only a discrete minority of transplanted myoblasts participate in regeneration of host muscle (150). This minority population of cells appears to divide slowly in vitro, but proliferates rapidly in vivo upon transplantation into regenerating muscle (150). If methods for characterizing and isolating this muscle stem cell population could be devised, such cells could be genetically engineered ex vivo and then introduced to patients, either by infusion into the circulation or through introduction to the bone marrow.

B. Circulating Therapeutic Proteins

The utility of myoblast-mediated gene delivery has broadened to include disorders that benefit from long-term secretion of recombinant proteins into the circulation, including treatment of lysosomal storage deficiencies, hemophilia B, anemias, and possibly cancer. For application of myoblasts in delivering genes encoding recombinant secreted proteins, a hurdle limiting its utility in the therapeutic realm is the necessity of using syngeneic cells to avoid immunological rejection of transplanted cells (151). Although myoblasts may be both isolated from and implanted back into the same individual, such procedures are both time consuming and costly. An alternative strategy would be to encapsulate myoblasts in an immunoisolated environment prior to implantation. Using this approach, myoblasts are enclosed within a matrix, for example, an alginate matrix (although other materials may be used), that allows secreted proteins to leave the capsules. The recipient's immune cells are prevented from coming into contact with the myoblasts, obviating the need for a genetically identical donor. This technology has been shown to be effective in delivering myoblasts engineered to secrete mouse growth hormone (27) and human factor IX (28) intraperitoneally. Encapsulated myoblasts were shown to be retrievable as long as 213

Figure 9 The RetroTet-ART system. (A) By coexpressing in the same cells a repressor and an activator that respond oppositely to dox and that do not heterodimerize because of different dimerization domains, the basal expression level of genes under tet control can be reduced without affecting the fully induced level. The net result is an increase in the dynamic range of the tet system (Reprinted from Ref. 138 with permission of the Proceedings of the National Academy of Sciences USA, 2101 Constitution Ave., NW, Washington, DC 20418. Reproduced by permission of the publisher via Copyright Clearance Center, Inc.). (B) Proof of concept of the RetroTet-ART system is demonstrated by FACS (left) and Northern blot (right) analysis. 10T1/2 fibroblasts were transduced with the GFP reporter retrovirus. Subsequently, cells were transduced with the transactivator retrovirus (b), the transrepressor virus (c), or with both transactivator and transrepressor virus (d). With the addition of both transactivator and transrepressor, the dynamic range of gene expression is increased. Gene expression can be fully extinguished, and induced to maximal levels, as shown in (d). (From Ref. 142 with permission)

Figure 9 The RetroTet-ART system. (A) By coexpressing in the same cells a repressor and an activator that respond oppositely to dox and that do not heterodimerize because of different dimerization domains, the basal expression level of genes under tet control can be reduced without affecting the fully induced level. The net result is an increase in the dynamic range of the tet system (Reprinted from Ref. 138 with permission of the Proceedings of the National Academy of Sciences USA, 2101 Constitution Ave., NW, Washington, DC 20418. Reproduced by permission of the publisher via Copyright Clearance Center, Inc.). (B) Proof of concept of the RetroTet-ART system is demonstrated by FACS (left) and Northern blot (right) analysis. 10T1/2 fibroblasts were transduced with the GFP reporter retrovirus. Subsequently, cells were transduced with the transactivator retrovirus (b), the transrepressor virus (c), or with both transactivator and transrepressor virus (d). With the addition of both transactivator and transrepressor, the dynamic range of gene expression is increased. Gene expression can be fully extinguished, and induced to maximal levels, as shown in (d). (From Ref. 142 with permission)

days postimplantation. These cells were found to be fully viable and capable of secreting recombinant proteins ex vivo at undiminished rates even at this late time point (28). More recently, encapsulated primary myoblasts were used to deliver VEGF to mice subcutaneously and intraperitoneally, causing an angiogenic response (29). This type of technology provides a promising method of attaining nonautologous gene therapy, in which universal donor cells can be created simply by encapsulation in a benign, immunoprotective environment.

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