Gene Transfer Using HACs: A Key Step Closer to Ex Vivo Gene Therapy Using Autologous Gene–Corrected Cells to Treat Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutations in an X-linked gene encoding for the muscle-cell structural protein dystrophin.1Bushby K Finkel R Birnkrant DJ Case LE Clemens PR Cripe L et al.Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management.Lancet Neurol. 2010; 9: 77-93Abstract Full Text Full Text PDF PubMed Scopus (1340) Google Scholar,2Bushby K Finkel R Birnkrant DJ Case LE Clemens PR Cripe L et al.Diagnosis and management of Duchenne muscular dystrophy, part 2: implementation of multidisciplinary care.Lancet Neurol. 2010; 9: 177-189Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar Gene therapy strategies to treat DMD face the daunting challenge of how to deliver the very large dystrophin gene to the entire musculature of patients. Cell replacement therapy, also being investigated as a treatment for DMD, aims to deliver the patient's own ex vivo gene-corrected cells to replace the diseased muscle and/or stimulate its growth and repair. Of course, such a strategy faces the same challenge of how to deliver such a large gene to the cells and then how to distribute the corrected cells throughout the muscles of the patient. However, a recent study by Tedesco et al.3Tedesco FS Hoshiya H D’Antona G Gerli MF Messina G Antonini S et al.Stem cell–mediated transfer of a human artificial chromosome ameliorates muscular dystrophy.Sci Transl Med. 2011; 3: 96ra78Crossref PubMed Scopus (118) Google Scholar takes us one step closer to a possible solution to these problems by making use of a human artificial chromosome (HAC) to deliver the entire genomic dystrophin gene to a particular type of blood vessel stem cell—a mesoangioblast—for the treatment of the mdx mouse model of DMD. Although a great many technical hurdles remain before the strategy can be developed clinically, the study provides a promising new approach to safely treating both muscle and nonmuscle hereditary diseases. Mesoangioblasts are unique in their ability to cross blood vessel walls and can differentiate into a variety of mesodermal cell types, including muscle cells. Cossu's research group had previously reported that wild-type mesoangioblasts could be delivered systemically to repair the muscle fibers in dystrophic animal models.4Sampaolesi M Blot S D’Antona G Granger N Tonlorenzi R Innocenzi A et al.Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs.Nature. 2006; 444: 574-579Crossref PubMed Scopus (604) Google Scholar,5Sampaolesi M Torrente Y Innocenzi A Tonlorenzi R D’Antona G Pellegrino MA et al.Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts.Science. 2003; 301: 487-492Crossref PubMed Scopus (540) Google Scholar The new twist is that the authors used an HAC vector developed by Oshimura's team6Kazuki Y Oshimura M Human artificial chromosomes for gene delivery and the development of animal models.Mol Ther. 2011; 19: 1591-1601Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar to deliver the complete dystrophin locus (2.4 Mb) to the cells before transplantation. The HAC vector was developed by removing the entire coding regions of human chromosome 21, such that it retained only the centromere, the two telomeres, and the noncoding regions. A gene or genes of choice can subsequently be cloned into the HAC using the Cre-loxP or multi-integrase system in Chinese hamster ovary cells or using homologous recombination in DT40 chicken pre-B cells.6Kazuki Y Oshimura M Human artificial chromosomes for gene delivery and the development of animal models.Mol Ther. 2011; 19: 1591-1601Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar The engineered HAC is transferred to the target cells—the mesoangioblasts in the study by Tedesco et al.—by a process called microcell-mediated chromosome transfer, also developed in Oshimura's laboratory. This process involves fragmentation of the donor cells carrying the engineered HAC by cytochalasin B treatment and centrifugation to produce microcells, each of which contains a single chromosome. These microcells are then fused with the mesoangioblasts using polyethylene glycol. Mesoangioblasts containing the HAC are then expanded following selection for the dominant marker also carried on the HAC. A further twist in the new study3Tedesco FS Hoshiya H D’Antona G Gerli MF Messina G Antonini S et al.Stem cell–mediated transfer of a human artificial chromosome ameliorates muscular dystrophy.Sci Transl Med. 2011; 3: 96ra78Crossref PubMed Scopus (118) Google Scholar was that the engineered mesoangioblasts were also transduced with a lentiviral vector encoding MyoD, so as to potentiate their myogenic differentiation following intramuscular or intra-arterial delivery. These engineered mesoangioblasts were delivered to immunodeficient mdx mice (so as to prevent an immune reaction to the human protein they carried). The authors showed that the transplanted mesoangioblasts could engraft in dystrophic muscles, expressed normal dystrophin, and produced functional muscle fibers with amelioration of the dystrophic pathology. Immunohistochemical and western blot analyses of the transplanted muscles revealed reconstitution of the dystrophin protein complex as shown by expression and correct membrane localization of α-sarcoglycan. Moreover, transplanted dystrophic mice showed a statistically significant reduction in fiber fragility, increased force generation, and enhanced motor capacity after treatment. Unexpectedly, the detection of donor-derived satellite cells showed that MyoD overexpression did not interfere with self-renewal or with their recruitment into the muscle satellite cell niche, a phenomenon previously observed following the transplantation of myoblasts.7Skuk D Paradis M Goulet M Chapdelaine P Rothstein DM Tremblay JP Intramuscular transplantation of human postnatal myoblasts generates functional donor-derived satellite cells.Mol Ther. 2010; 18: 1689-1697Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar The autologous transplantation of cells genetically modified with an HAC could have enormous advantages for ex vivo gene therapy for a variety of hereditary diseases compared with other gene and cell therapy strategies. First, this therapeutic approach may avoid the need for the long-term immunosuppression required by allogeneic strategies. Of course, immunosuppression might still be required if the corrective gene encoded an immunogenic protein, as a result of its previous absence from the patient's proteome. Second, there is essentially no limit to the size and the number of genes or complementary DNAs that could be accommodated in the HAC. Indeed, the HAC used by Tedesco et al.3Tedesco FS Hoshiya H D’Antona G Gerli MF Messina G Antonini S et al.Stem cell–mediated transfer of a human artificial chromosome ameliorates muscular dystrophy.Sci Transl Med. 2011; 3: 96ra78Crossref PubMed Scopus (118) Google Scholar encoded not only dystrophin but also a suicide protein (herpes simplex virus–thymidine kinase) to enable the workers to kill the transplanted cells if necessary—for example, if they have led to tumor formation or the induction of an immune reaction. Third, episomal HACs are likely to have a better safety profile as compared with integrating vectors. Moreover, Oshimura's group has recently developed a new HAC that can be removed from the cells by inactivation of the centromere.6Kazuki Y Oshimura M Human artificial chromosomes for gene delivery and the development of animal models.Mol Ther. 2011; 19: 1591-1601Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar This conditional centromere includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. The reverse transcriptional transactivator binds to tet-O sequences in the presence of doxycycline, causing centromere inactivation and HAC destabilization.8Iida Y Kim JH Kazuki Y Hoshiya H Takiguchi M Hayashi M et al.Human artificial chromosome with a conditional centromere for gene delivery and gene expression.DNA Res. 2010; 17: 293-301Crossref PubMed Scopus (49) Google Scholar As noted earlier, Tedesco et al. delivered the HAC carrying the dystrophin gene into mesoangioblasts that had also been engineered to express higher levels of the MyoD gene, so as to increase their myogenic differentiation. In an eventual clinical application of the combination HAC–mesoangioblast technology, the MyoD gene could also be placed under a drug-inducible promoter in the HAC containing the dystrophin gene. This would avoid the possible random integration of the MyoD transgene and ensure that all the cells containing the HAC can be induced to differentiate into myogenic cells. The systemic delivery of autologous mesoangioblasts should be applicable to all DMD patients independent of the type of dystrophin mutation they carry9Aartsma-Rus A Fokkema I Verschuuren J Ginjaar I van Deutekom J van Ommen GJ et al.Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations.Hum Mutat. 2009; 30: 293-299Crossref PubMed Scopus (414) Google Scholar because the presence of the entire dystrophin gene locus should ensure correctly regulated expression of the dystrophin gene, including all exons. Moreover, the combination of HAC and mesoangioblast ex vivo gene therapy may be applicable to other muscular dystrophies, especially those in which the mutated gene is too large to be accommodated within the smaller genomes of integrating viral vectors (e.g., dysferlin). HAC technology may find application in the treatment of other, nonmuscle diseases such as the immunodeficiencies, using HAC-engineered hematopoietic stem cells. As such, this vector may obviate the concerns over the possibility of tumorigenicity that arose when randomly integrating vectors were used. HAC technology may also be used to induce the reprogramming of induced pluripotent stem cells (iPSCs) and at the same time correct any genetic defects they carry. The ability of iPSCs to proliferate indefinitely could resolve the problem of eventual senescence that would occur with most other cell types. Moreover, because the iPSCs could in principle be induced to differentiate into any type of cell, this opens the possibility of treating diseases affecting a great variety of tissues. However, HAC transfer into mammalian cells remains inefficient (1/105 cells or less) and requires selection. This is not a problem when using mouse cells that easily immortalize in culture, but it may lead to senescence of several types of human cells, including mesoangioblasts. Therefore, additional steps, such as the induction of reversible immortalization, accompanied by the relevant safety controls, will probably be necessary before this strategy could be translated to clinical experimentation. Moreover, it will be necessary to either remove or inactivate the selective gene before transplantation of the corrected cells to patients or to use a selective gene of human origin. These caveats notwithstanding, the new work by Tedesco et al. has clearly validated the utility of another promising tool for the continuing development of gene and cell therapy applications.

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