Hidetoshi Hoshiya

Transplantation of Genetically Corrected Human iPSC-Derived Progenitors in Mice with Limb-Girdle Muscular Dystrophy

Genetically corrected mesoangioblasts from human iPSCs derived from limb-girdle muscular dystrophy patients produce muscle fibers expressing the therapeutic gene in a mouse model of the disease. Muscle Progenitors Find Their Way Home Muscular dystrophies are genetic disorders primarily affecting skeletal muscle that result in greatly impaired mobility and, in severe cases, respiratory and cardiac dysfunction. There is no effective treatment, although several new approaches are entering clinical testing including cell therapy. Cell therapy aims to replace lost muscle fibers by transplanting healthy donor muscle progenitor cells or cells from dystrophic patients that have been genetically corrected in vitro. Mesoangioblasts are progenitor cells from blood vessel walls that have shown potential as a cell therapy in animal models of muscular dystrophy. In a new study, Tedesco et al. explore whether genetically corrected mesoangioblasts from patients with limb-girdle muscular dystrophy 2D (LGMD2D) have potential as an autologous cell therapy to treat this disease. The authors quickly found that they could not derive a sufficient number of mesoangioblasts from LGMD2D patients because the muscles of the patients were depleted of these progenitor cells. To overcome this problem, the authors reprogrammed fibroblasts or myoblasts from the LGMD2D patients to obtain human induced pluripotent stem cells (iPSCs) and induced them to differentiate into mesoangioblast-like cells that were then genetically corrected in vitro using a viral vector expressing the defective gene SGCA, which encodes α-sarcoglycan. After intramuscular or intra-arterial injection of these genetically corrected, iPSC-derived mesoangioblasts into mice with LGMD2D (immune-deficient Sgca-null mice), the cells homed to damaged mouse skeletal muscle, engrafted, and formed muscle fibers expressing α-sarcoglycan. Using mouse iPSC-derived mesoangioblasts, the researchers showed that the transplanted engrafted cells imbued muscle with greater strength and enabled the dystrophic mice to run for longer on a treadmill than dystrophic mice that did not receive the cells. This strategy offers the advantage of being able to produce unlimited numbers of genetically corrected progenitor cells, which perhaps could be used in the future as cell therapy for treating LGMD2D and other forms of muscular dystrophy. Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne’s muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan–null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan–null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.

Complete Genetic Correction of iPS Cells From Duchenne Muscular Dystrophy

Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Induced pluripotent stem (iPS) cells have great potential for gene therapy, as such cells can be generated from the individuals own tissues, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we show herein the complete correction of a genetic deficiency in iPS cells derived from Duchenne muscular dystrophy (DMD) model (mdx) mice and a human DMD patient using a HAC with a complete genomic dystrophin sequence (DYS-HAC). Deletion or mutation of dystrophin in iPS cells was corrected by transferring the DYS-HAC via microcell-mediated chromosome transfer (MMCT). DMD patient- and mdx-specific iPS cells with the DYS-HAC gave rise to differentiation of three germ layers in the teratoma, and human dystrophin expression was detected in muscle-like tissues. Furthermore, chimeric mice from mdx-iPS (DYS-HAC) cells were produced and DYS-HAC was detected in all tissues examined, with tissue-specific expression of dystrophin. Therefore, the combination of patient-specific iPS cells and HAC-containing defective genes represents a powerful tool for gene and cell therapies.

Stem Cell–Mediated Transfer of a Human Artificial Chromosome Ameliorates Muscular Dystrophy

Combining gene delivery using a human artificial chromosome with stem cell transplantation ameliorates muscular dystrophy in a mouse model. Stem Cells Muscle in on the Action The progressive muscle loss that is the hallmark of Duchenne muscular dystrophy (DMD) has proved very difficult to halt or reverse. Although the causative mutations of DMD were identified in the X-linked gene encoding dystrophin (a structural muscle protein) several decades ago, translating this genetic discovery into new treatments has been challenging. Most therapeutic strategies aim to use gene therapy to deliver the normal dystrophin gene to the dystrophic muscles of DMD patients. However, the dystrophin gene is too large to be carried by the viral vectors usually used in gene therapy and all muscles in the body would have to be injected with the vector and replacement gene. Now, Tedesco and colleagues combine stem cell therapy with a human artificial chromosome vector to overcome these two challenges in the mdx mouse model of DMD. This team had previously identified a blood vessel stem cell called “mesoangioblast” that has the dual talents of being able to cross blood vessel walls and to differentiate into a variety of mesodermal cell types including muscle cells. Would these stem cells be able to deliver a replacement dystrophin gene to dystrophic muscles in the mdx mouse? Predicting that they would, Tedesco and colleagues used a human artificial chromosome vector engineered to carry the entire normal human dystrophin gene including the regulatory regions. They transferred the vector and its large cargo into mesoangioblasts isolated from mdx mice; then they injected the corrected mesoangioblasts directly into the dystrophic skeletal muscles of recipient immune-deficient mdx mice (to prevent reaction against the human protein). The authors showed that the transplanted mesoangioblasts were able to engraft in dystrophic muscles, express normal dystrophin, and produce functional muscle fibers with amelioration of dystrophic pathology. They also found that the transplanted mesoangioblasts contributed to the muscle satellite cell pool, which produces new muscle cells under normal conditions. Next, the authors showed that if they injected the corrected mesoangioblasts into the arterial circulation of mdx mice, the cells were able to cross blood vessel walls, home to dystrophic muscles and graft contribute to the formation of new dystrophin-expressing myofibers. The authors then showed that mice receiving the mesoangioblast transplants showed reduced fiber fragility, increased force, and greater motor capacity on treadmill and freewheel tests. Although there are still technical and regulatory hurdles to be overcome before this strategy can be used in DMD patients, stem cell–mediated transfer of the normal dystrophin gene using a human artificial chromosome shows promise as a treatment for this tragic and ultimately fatal disease. In contrast to conventional gene therapy vectors, human artificial chromosomes (HACs) are episomal vectors that can carry large regions of the genome containing regulatory elements. So far, HACs have not been used as vectors in gene therapy for treating genetic disorders. Here, we report the amelioration of the dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD) using a combination of HAC-mediated gene replacement and transplantation with blood vessel–associated stem cells (mesoangioblasts). We first genetically corrected mesoangioblasts from dystrophic mdx mice with a HAC vector containing the entire (2.4 Mb) human dystrophin genetic locus. Genetically corrected mesoangioblasts engrafted robustly and gave rise to many dystrophin-positive muscle fibers and muscle satellite cells in dystrophic mice, leading to morphological and functional amelioration of the phenotype that lasted for up to 8 months after transplantation. Thus, HAC-mediated gene transfer shows efficacy in a preclinical model of DMD and offers potential for future clinical translation.

Frequent loss of imprinting of IGF2 and MEST in lung adenocarcinoma

Genomic imprinting is a parental origin–specific chromosomal modification that causes differential expression of maternal and paternal alleles of a gene. Accumulating evidence suggests that deregulation of imprinted genes, including loss of imprinting (LOI), plays a role in oncogenesis. In the present study, we investigated allelic expression of six imprinted genes in human lung adenocarcinomas as well as in matched normal lung tissue. Informative cases showing heterozygosity for the gene of interest were selected from 35 patients. LOI of the insulin‐like growth factor 2 gene (IGF2) and mesoderm‐specific transcript (MEST, also known as paternally expressed gene 1) was noted in 47% (seven of 15) and 85% (11 of 13) of informative cases, respectively. Monoallelic expression was maintained in all the matched normal tissues examined. LOI of IGF2 was seen more frequently in moderately to poorly differentiated adenocarcinomas. In contrast, H19, small nuclear ribonucleoprotein–associated polypeptide N gene (SNRPN), necdin gene (NDN), and long QT intronic transcript 1 (LIT1) exhibited consistent monoallelic expression in all the informative samples. These findings indicated that independent deregulation took place in imprinted genes and suggested that aberrant imprinting of IGF2 and MEST was involved in the development of lung adenocarcinoma.

A highly Stable and Nonintegrated Human Artificial Chromosome (HAC) Containing the 2.4 Mb Entire Human Dystrophin Gene

Episomal vector with the capacity to deliver a large gene containing all the critical regulatory elements is ideal for gene therapy. Human artificial chromosomes (HACs) have the capacity to deliver an extremely large genetic region to host cells without integration into the host genome, thus preventing possible insertional mutagenesis and genomic instability. Duchenne muscular dystrophy (DMD) is caused by mutation in the extremely large dystrophin gene (2.4 Mb). We herein report the development of a HAC vector containing the entire human dystrophin gene (DYS-HAC) that is stably maintained in mice and human immortalized mesenchymal stem cells (hiMSCs). The DYS-HAC was transferred to mouse embryonic stem (ES) cells, and isoforms of the DYS-HAC-derived human dystrophin in the chimeric mice generated from the ES cells were correctly expressed in tissue-specific manner. Thus, this HAC vector containing the entire dystrophin gene with its native regulatory elements is expected to be extremely useful for future gene and cell therapies of DMD.

Refined human artificial chromosome vectors for gene therapy and animal transgenesis

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.

A new imprinted cluster on the human chromosome 7q21-q31, identified by human-mouse monochromosomal hybrids

We have previously established a series of human monochromosomal hybrids containing a single human chromosome of defined parental origin as an in vitro resource for the investigation of human imprinted loci. Using the hybrids with a paternal or maternal human chromosome 7, we determined the allelic expression profiles of 76 ESTs mapped to the human chromosome 7q21-q31. Seven genes/transcripts, including PEG10 which has previously been reported to be imprinted, showed parent-of-origin-specific expression in monochromosomal hybrids. One of the 6 candidate genes/transcripts, i.e., DLX5 was confirmed to be imprinted in normal human lymphoblasts and brain tissues by a polymorphic analysis. Thus, an imprinted domain has been newly defined in the region of human chromosome 7q21-q31 using human-mouse monochromosomal hybrids.

Predominant maternal expression of the mouse Atp10c in hippocampus and olfactory bulb

AbstractThe human chromosome 15q11-q13 region is one of the most intriguing imprinted domains, and the abnormalities inherited are associated with neurological disorders including Prader-Willi syndrome (PWS), Angelman syndrome (AS) and autism. Recently we have identified a novel maternally expressed gene, ATP10C, that encodes a putative aminophospholipid translocase within this critical region, 200 kb distal to UBE3A in an imprinted domain on human chromosome 15. ATP10C, with UBE3A, displayed tissue-specific imprinting with predominant expression of the maternal allele in the brain. In this study, we demonstrated that the mouse homologue, Atp10c/pfatp showed tissue-specific maternal expression in the hippocampus and olfactory bulb, which overlapped the region of imprinted Ube3a expression. These data suggest that the imprinted transcript of Atp10c in the specific region of CNS may be associated with neurological disorders including AS and autism.

Human Artificial Chromosome with a Conditional Centromere for Gene Delivery and Gene Expression

Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoidtet-O (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy.

A Novel Human Artificial Chromosome Vector Provides Effective Cell Lineage–Specific Transgene Expression in Human Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) hold promise for use in adult stem cell–mediated gene therapy. One of the major aims of stem cell–mediated gene therapy is to develop vectors that will allow appropriate levels of expression of therapeutic genes along differentiation under physiological regulation of the specialized cells. Human artificial chromosomes (HACs) are stably maintained as independent chromosomes in host cells and should be free from potential insertional mutagenesis problems of conventional transgenes. Therefore, HACs have been proposed as alternative implements to cell‐mediated gene therapy. Previously, we constructed a novel HAC, termed 21 Δpq HAC, with a loxP site in which circular DNA can be reproducibly inserted by the Cre/loxP system. We here assessed the feasibility of lineage‐specific transgene expression by the 21Δpq HAC vector using an in vitro differentiation system with an MSC cell line, hiMSCs, which has potential for osteogenic, chondrogenic, and adipogenic differentiation. An enhanced green fluorescent protein (EGFP) gene driven by a promoter for osteogenic lineage‐specific osteopontin (OPN) gene was inserted onto the 21 Δpq HAC and then transferred into hiMSC. The expression cassette was flanked by the chicken HS4 insulators to block promoter interference from adjacent drug‐resistant genes. The EGFP gene was specifically expressed in the hiMSC that differentiated into osteocytes in coordination with the transcription of endogenous OPN gene but was not expressed after adipogenic differentiation induction or in noninduction culture. These results suggest that use of the HAC vector is suitable for regulated expression of transgenes in stem cell–mediated gene therapy.