Cell-mediated Gene Therapy Research Articles

Ex vivo cell-mediated gene therapy for metachromatic leukodystrophy using neurospheres

Metachromatic leukodystrophy (MLD) is an autosomal recessive disease caused by mutations in the gene encoding the lysosomal enzyme arylsulfatase A (ASA). In MLD, accumulation of the substrate, sulfated glycoprotein, in the central and peripheral nervous systems results in progressive motor and mental deterioration. Neural progenitor cells are thought to be useful for cell replacement therapy and for cell-mediated gene therapy in neurodegenerative diseases. In the present study, we examined the feasibility of ex vivo gene therapy for MLD using neural progenitor cells. Neural progenitor cells (neurospheres) were prepared from the striatum of E14 embryo MLD knockout mice or GFP transgenic mice and were transduced with the VSV pseudotyped HIV vector carrying the ASA gene (HIV-ASA). For in vivo study, neurospheres from GFP mice were transduced with HIV-ASA and inoculated into the brain parenchyma of adult MLD mice. HIV vector-transduced progenitor cells retained the potential for differentiation into neurons, astrocytes and oligodendrocytes in vitro. Expression of ASA in neurospheres transduced with HIV-ASA was confirmed by spectrophotometric enzyme assay and Western blotting. In vivo, GFP-positive cells were detectable 1 month after injection. These cells included GFAP- and MAP2-positive cells. Immunohistochemistry using anti-ASA antibody demonstrated localization of ASA in both GFP-positive and -negative cells. Partial clearance of accumulated sulfatide was confirmed in vivo in MLD knockout mice. The present findings suggest that ASA enzyme is released from migrated neurospheres and is able to digest sulfatide in surrounding cells. Our results suggest the potential of genetically engineered neural progenitor cells (neurospheres) for ex vivo therapy in MLD.

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 Deltapq 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 21Deltapq 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 Deltapq 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.

Murine spinal fusion induced by engineered mesenchymal stem cells that conditionally express bone morphogenetic protein—2

The authors hypothesized that spinal fusion can be achieved and monitored by using cell-mediated gene therapy. Mesenchymal stem cells (MSCs) genetically engineered to express recombinant human bone morphogenetic protein-2 (rhBMP-2) conditionally, were implanted into the paraspinal muscles of mice to establish spinal fusion. The goal was to demonstrate an MSC-based gene therapy platform in which controlled gene expression is used to obtain spinal fusion in a murine model. Mesenchymal stem cells expressing the rhBMP-2 gene were injected into the paravertebral muscle in mice. Bone formation in the paraspinal region was longitudinally followed by performing micro-computerized tomography scanning, histological studies, and an analysis of osteocalcin expression to demonstrate the presence of engrafted engineered MSCs. The minimal period of rhBMP-2 expression by the engineered MSCs required to induce fusion was determined. The results of this study demonstrate that genetically engineered MSCs induce bone formation in areas adjacent to and touching the posterior elements of the spine. This newly formed bone fuses the spine, as demonstrated by radiological and histological studies. The authors demonstrate that injected cells induce active osteogenesis at the site of implantation for up to 4 weeks postinjection. They found that a 7-day induction of rhBMP-2 expression in genetically engineered MSCs was sufficient to form new bone tissue, although the quantity of this bone increased as longer expression periods were implemented. After their injection genetically engineered MSCs can efficiently form new bone in the paraspinal muscle of the mouse to obtain spinal fusion. The extent and quantity of this newly formed bone can be monitored by controlling the duration of rhBMP-2 gene expression.

Transforming Growth Factor β1–Modified Donor Cell Transfusion Induced Allo-Heart Tolerance

The therapeutic value of the transforming growth factor beta 1 (TGF-β1) in transplantation has been reported; however, cell-mediated gene therapy using TGF-β1 is not a widespread application in organ transplantation. This study was performed to evaluate whether TGF-β1-modified donor spleen cell-specific transfusion could induce tolerogenicity and prolong allograft survival in rat heterotopic heart transplantation. Stable TGF-β1-transduced spleen cells were established. Wistar rat splenic T-cell responses to donor spleen cells that received TGF-β1-transduced were severely impaired. Survival of Sprague-Dawley cardiac allografts in Wistar rats given TGF-β1-modified donor spleen cells (5 × 10 6, 7 days before transplantation), was extended modestly but significantly. Liposome transduction of donor spleen cells to overexpress TGF-β1 is associated with marked impairment of their T-cell allostimulatory activity but only modest prolongation of allo-heart survival.

1053. Novel Exogenously Regulated Stem Cell–Mediated Gene Therapy Platform for Spinal Fusion

Cell-mediated gene therapy involving mesenchymal stem cells (MSCs) and bone morphogenetic protein (BMP) genes can provide a local supply of precursor cells and osteoinductive molecules that may promote bone formation. Spinal fusion has become a popular surgical technique for different pathological spinal conditions. We hypothesized that MSCs that overexpress the human (h)BMP-2 gene under the tetracycline (Tet)-controlled expression system (Tet-Off) can induce regulated bone formation leading to spinal fusion. We used a Tet-Off system based on genetically engineered MSCs containing an inducible hBMP-2 expression vector (ptTATop-BMP2) that had a bidirectional promoter (TATA sequence) utilizing elements of the tetracycline regulatory system. The system was regulated by the administration of doxycycline (Dox), a tetracycline analogue. In C3H/HeN mice 2 |[times]| 10^6 genetically engineered MSCs were injected into the paravertebral muscle of the lumbar spine (L2|[ndash]|6). Ten animals served as a negative control group (Dox was added to their drinking water) and 20 animals constituted the experimental group (Dox was supplied for different time periods leading to different duration of hBMP-2 expression). A longitudinal quantitative analysis of vertebrae fusion was performed using micro|[ndash]|computerized tomography (|[mu]|CT) to observe bone formation. In addition, engineered MSCs were injected in a similar manner to transgenic mice expressing the luciferase gene under human osteocalcin promoter. In these mice measuring the luciferase signal using a bioluminescence imaging system can noninvasively and quantitatively monitor osteogenic activity. From Week 1 postinjection the |[mu]|CT analysis demonstrated extensive bone formation leading to vertebrae fusion (Fig. 1A). No bone formation was observed in the control animals, in which hBMP-2 was inhibited. A minimum of 7 days of hBMP-2 expression was required to induce bone formation (Fig. 1B). Detection of luciferase gene expression in the transgenic mice 2 and 4 weeks postinjection indicated an increase in the osteogenic activity in vivo, which could be attributed to the paracrine effect of the engineered MSCs (Fig. 2). Our data demonstrate a regulated system for the induction of spinal fusion. We conclude that exogenously controlled gene therapy for spinal fusion can be achieved using the Tet-regulated hBMP-2 system.

184. Nonvirally Electrotransfected Human Mesenchymal Stem Cells: A Novel Platform for Bone Gene Therapy Based on Autocrine and Paracrine Mechanisms

Studies have shown that cell-mediated gene therapy involving virally transduced mesenchymal stem cells (MSCs) can efficiently regenerate nonunion bone defects. In these studies bone tissue regeneration was achieved by using viral gene delivery of osteogenic factors. There have been extensive reports on the limitations of virus-based approaches, most focusing on the humoral immune response generated against viral proteins. An alternative approach is the use of nonviral gene therapy, which is considered safer but lacks the high efficiency of gene transduction provided by viral vectors. DNA electrotransfer (electroporation) may be an efficient physical method of delivering genes into primary stem cells. We tested the hypothesis that in vivo bone regeneration can be achieved via autocrine and paracrine effects exerted by human (h)MSCs that are nonvirally electrotransfected with BMP genes. Primary hMSCs were electrotransfected using a Nucleofector (Amaxa Biosystems), a novel electroporation system. In the first stage of our study we utilized the gene of the enhanced green fluorescent protein (EGFP) to assess the viability of hMSCs and the efficiency of DNA transfer. In the second stage we used the osteogenic genes BMP-2 and BMP-9 to induce osteogenic differentiation via autocrine and paracrine effects. Our in vitro results demonstrated 53% cell viability, up to 70% transfection efficiency, and transgene expression that lasted at least 14 days in vitro. BMP-electrotransfected hMSCs expressing luciferase were detected in vivo by using noninvasive, quantitative bioluminescence imaging following an injection of these cells into the thigh muscles of NOD/SCID mice. Micro-computerized tomography (°CT) demonstrated new bone formation at the injection site 4 weeks postinjection (Fig. 1A & B). The electrotransfected hMSCs differentiated and integrated into the newly formed bone; this was demonstrated by masson's trichrome (Fig. 1C & D) and an anti-human mitochondria immunohistochemical analysis (Fig. 1E & F, arrowheads). Both in vitro and in vivo studies demonstrated a significantly higher osteogenic potency in BMP-9-electrotransfected hMSCs than in BMP-2-electrotransfected hMSCs. We conclude that hMSCs can be effectively transfected using this novel nonviral technique. These findings may pave the way to a safe and efficient gene therapy for human bone regeneration.

The mitotic clock in skeletal muscle regeneration, disease and cell mediated gene therapy

The regenerative capacity of skeletal muscle will depend on the number of available satellite cells and their proliferative capacity. We have measured both parameters in ageing, and have shown that although the proliferative capacity of satellite cells is decreasing during muscle growth, it then stabilizes in the adult, whereas the number of satellite cells decreases during ageing. We have also developed a model to evaluate the regenerative capacity of human satellite cells by implantation into regenerating muscles of immunodeficient mice. Using telomere measurements, we have shown that the proliferative capacity of satellite cells is dramatically decreased in muscle dystrophies, thus hampering the possibilities of autologous cell therapy. Immortalization by telomerase was unsuccessful, and we currently investigate the factors involved in cell cycle exits in human myoblasts. We have also observed that insulin-like growth factor-1 (IGF-1), a factor known to provoke hypertrophy, does not increase the proliferative potential of satellite cells, which suggests that hypertrophy is provoked by increasing the number of satellite cells engaged in differentiation, thus possibly decreasing the compartment of reserve cells. We conclude that autologous cell therapy can be applied to specific targets when there is a source of satellite cells which is not yet exhausted. This is the case of Oculo-Pharyngeal Muscular Dystrophy (OPMD), a late onset muscular dystrophy, and we participate to a clinical trial using autologous satellite cells isolated from muscles spared by the disease.

Experimental research on human insulin-like growth factor I gene transfect the cultured bone marrow mesenchymal stem cells

To investigate the effectiveness of human insulin-like growth factor I (hIGF-I) gene transferred into the cultured goat bone marrow mesenchymal stem cells with liposome, and find a new method of cell-mediated gene therapy. Bone marrow was extracted from adult goats and cultured in vitro by monolayer. Then the recombinant eukaryotic expression plasmid pIRES2-EGFP-hIGF was transfected into cells by FuGene 6. After transfection, the marker gene coding enhanced green fluorescent protein (EGFP) was observed at different time points. The hIGF concentration in the supernatant fluids was measured by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry stain of hIGF was performed to detect the target protein. Besides, reverse transcription polymerase chain reaction and flow cytometry were also adopted in order to find out the changes of cells after transfection. Bone marrow stem cells were all in the long shuttle-like shape and adhered to the disk. The expression of EGFP was first found at 4 h after transfection. The amount and intensity of EGFP increased gradually during the period of detection and got to the peak degree at 72 h, after that decreased slowly. EGFP was also seen in the second generation cells, but the intensity was relatively faint. The IGF-I concentration secreted into the supernatant was in accordance with the EGFP observed with the peak concentration at 34.75 ng/ml. The outcome of RT-PCR and immunohistochemistry was positive. The morphology of many stem cells was changed into triangular or irregular forms under the circumstance of the secreted hIGF-I. Percentage of stem cells in the S stage increased after transfection. The hIGF-I gene can be transfected efficiently and safely into BMSCs by FuGene 6, and the hIGF-I protein can be secreted into the supernatant in a relatively high level during a long period, therefore accelerate the proliferation and differentiation of the transfected cells.

Continuous Transforming Growth Factor β1Secretion by Cell-Mediated Gene Therapy Maintains Chondrocyte Redifferentiation

One of the most important factors in the production of cartilage is transforming growth factor beta1 (TGF-beta1). To obtain sustained release of TGF-beta1, a cell-mediated gene therapy technique was introduced. We infected chondrocytes with a retroviral vector carrying the TGF-beta1 gene. The single clone derivative showed sustained TGF-beta1 secretion. It also showed constitutive type II collagen expression. Whereas the TGF-beta1 protein itself is unable to induce formation of cartilage in vivo, human chondrocytes engineered to express a retroviral vector encoding TGF-beta1 showed cartilage formation in vivo when cells were injected into nude mice intradermally. These data suggest that cell-mediated gene therapy using TGF-beta1 as a transgene would be a promising treatment for osteoarthritis.

Cytokines transduced bone marrow stromal cell lines promote immunohematopoietic reconstitution in mice after allogeneic bone marrow transplantation

Impaired immune reconstitution following allogeneic T-cell depleted bone marrow transplantation (allo-TCD-BMT) is a major obstacle to its clinical application. Stromal cell line QXMSC1, established from bone marrow cells of BALB/c(H-2 d), was transfected with murine IL-3 and/ or IL-2 gene, and injected into lethally irradiated C57BL/6(H2 b) mice. We evaluated its effects on immunologic and hematopoietic reconstitution after allo-TCD-BMT. The results showed that QXMSC1-IL-3 + IL-2 could significantly increase the numbers of hematopoietic primitive progenitors (CFU-S), committed progenitors (CFU-GM, and BFU-E), and lymphocytes (CD8 + cells, CD4 + cells, and B cells). Similarly, immune functions of recipient mice were significantly enhanced in the QXMSC1-IL-3 + IL-2 group. In addition, QXMSC1-IL-3 or QXMSC1-IL-2 also exerted apparent effects on accelerating immune reconstitution, but these effects were far less than that of QXMSC1-IL-3 + IL-2. Our results demonstrated that stromal cell-mediated IL-3 and IL-2 gene therapy may be a potent approach in promoting immunologic and hematopoietic reconstitution after allo-TCD-BMT.

Ganciclovir-mediated elimination of HSV-Tk + T cells and cure of graft-vs-host disease in an allogeneic bone marrow transplantation model in the rat

Suicide gene therapy for leukemia aims to benefit from T cells in the BM graft, by reducing the probability of leukemia relapse (GVL), while severe complications of graft-vs-host disease (GVHD) may be avoided. In an allogeneic rat BMT model we defined the conditions to induce a lethal GVHD with HSV-Tk gene-transduced T cells. We studied the feasibility to rescue the animals by conditional elimination of the T cells with ganciclovir (GCV) treatment. Allogeneic T cells transduced with a retroviral vector encoding the HSV-Tk suicide gene were added in varying numbers to a BM graft. Expression of HSV-Tk strongly increases the cytolytic effect of GCV, thereby allowing elimination of overreactive T cells at will. Various experimental conditions were tested in the rat model. A relation between the number of HSV-Tk(+) T cells added to the BM graft and GVHD development was found. GCV treatment resulted in selective HSV-Tk(+) T-cell elimination in blood and tissues but not in abrogation of GVHD due to persistence of HSV-Tk(-) T cells. T cells in unmanipulated rat BM normally have a low risk to induce GVH but when they are administered in combination with high numbers of HSV-Tk(+) T cells there is an apparent increase in their GVH-inducing potential. When HSV-Tk(+) T cells are added to T cell-depleted BM a consequently developing GVH can be controlled by GCV treatment with 60-70% of the animals surviving. We show that T cell-mediated suicide gene therapy within the context of allo-BMT can be applied with success. The apparent limitation in the number of transduced as well as nontransduced T cells that can be safely added to the BM graft should be taken into consideration when designing human suicide gene therapy protocols.

827. A Novel Bone Gene Therapy Platform Based on Nonvirally Transfected Human Mesenchymal Stem Cells

Several gene therapy approaches were developed aiming to achieve bone tissue regeneration, based on viral gene delivery of osteogenic factors. The limitations of viral-based approaches have been extensively reported mostly dealing with the humoral immune response generated against the viral proteins. An alternative approach is the use of non-viral gene therapy, which is considered to be safer but lacks the high efficiency of gene transduction available with viral vectors. Electroporation might be an efficient physical nonviral method to deliver DNA molecules into stem cells. We have shown that cell-mediated gene therapy using virally trasduced mesenchymal stem cells can efficiently regenerate non-union bone defects. Therefore, we hypothesized that in vivo bone formation could be achieved using human Mesenchymal Stem Cells (hMSCs) nonvirally transfected with BMP genes. HMSCs were isolated from bone marrow samples and transfected using a Nucleofector (Amaxa Biosystems), a novel electroporation-based technique. We first utilized the GFP and Luciferase reporter genes in order to optimize the viability of hMSCs, the efficiency of DNA transfer and the duration of transgene expression following gene transfer. FACS analysis, quantitative PCR and Luciferase biochemical assay were used for the analysis. Moreover, we have manipulated hMSCs to G0/G1 cell cycle phase, prior to transfection, in order to determine whether it would increase transfection efficiency. In the second stage we utilized the osteogenic genes, BMP-2,-6 and-9, in order to induce osteogenic differentiation. Transfected hMSCs, embedded in Fibrin gel carrier (Baxter) were also injected into the thigh muscle or in the lumbar region, of NOD/SCID mice. We were able to monitor injected cell survival in vivo by transfecting the cells with a Luciferase plasmid, and noninvasively detecting them quantitatively, using a CCCD system (B). The sites of injection were scanned in vivo and ex vivo, using uCT system. Anti human mitochondria assay was used to demonstrate the integration of the engineered cells in bone formation (C). Our in vitro results demonstrated 53% cell viability and up to 70% transfection efficiency. The transfection efficiency was increased when 98% of the cells were in Go/G1 phase prior to transfection, and transgene expression lasted at least 14 days in vitro. Osteogenic differentiation was demonstrated in hMSCs transfected nonvirally with different BMPs (A). In vivo bone formation was evident in the injection sites (D, E), and the nonvirally transfected hMSCs were able to induce spinal fusion in the lumbar region. We conclude that hMSCs can be effectively transfected using this nonviral novel technique. These findings could pave the way to a safe and efficient gene therapy for human bone regeneration Figure 1.

865. An Ex Vivo Gene Therapy Approach for Metachromatic Leukodystrophy Using Neural Progenitor Cells

Metachromatic leukodystrophy(MLD) is an autosomal recessive inherited disease caused by mutations of the gene encoding the lysozomal enzyme, arylsulfatase A(ASA). Accumulation of the substrate, sulfated glycoprotein, in both the central and peripheral nervous system results in progressive motor and mental deterioration. No effective therapy has been developed yet. Neural progenitor cells are thought to be useful for cell replacement therapy and also cell mediated gene therapy of neurodegenerative diseases. In the present study, we examined the feasibility of ex vivo gene therapy for MLD using neural progenitor cells. Neural progenitor cells (neurospheres) were prepared from the striatum of E14 embryo MLD knockout mice or GFP transgenic mice and transduced with the VSV pseudotyped HIV vector carrying the ASA gene (HIV-ASA). HIV vector transduced progenitor cells retained the potential for differentiation into neurons, astrocytes, and oligodendrocytes in vitro. Expression of ASA in neurospheres transduced with HIV-ASA was confirmed by the spectrophotometric enzyme assay and Western blotting. When neurospheres from GFP mice were transduced with HIV-ASA and inoculated into the brain parenchyma of adult MLD mice, GFP positive cells were detectable one month after injection. Those cells include GFAP positive astrocytes and undifferentiated cells. Immunohistochemistry using anti-ASA antibody demonstrated that ASA was localized in both GFP positive and negative cells, suggesting that ASA was secreted from the transduced cells and taken up by the surrounding cells. These results hold promise for the use of genetically engineered neural stem cells for ex vivo therapy of MLD.

Overexpression of CXCR4 on human CD34+progenitors increases their proliferation, migration, and NOD/SCID repopulation

AbstractA major limitation to clinical stem cell–mediated gene therapy protocols is the low levels of engraftment by transduced progenitors. We report that CXCR4 overexpression on human CD34+progenitors using a lentiviral gene transfer technique helped navigate these cells to the murine bone marrow and spleen in response to stromal-derived factor 1 (SDF-1) signaling. Cells overexpressing CXCR4 exhibited significant increases in SDF-1–mediated chemotaxis and actin polymerization compared with control cells. A major advantage of CXCR4 overexpression was demonstrated by the ability of transduced CD34+cells to respond to lower, physiologic levels of SDF-1 when compared to control cells, leading to improved SDF-1–induced migration and proliferation/survival, and finally resulting in significantly higher levels of in vivo repopulation of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice including primitive CD34+/CD38-/lowcells. Importantly, no cellular transformation was observed following transduction with the CXCR4 vector. Unexpectedly, we documented lack of receptor internalization in response to high levels of SDF-1, which can also contribute to increased migration and proliferation by the transduced CD34+cells. Our results suggest CXCR4 overexpression for improved definitive human stem cell motility, retention, and multilineage repopulation, which could be beneficial for in vivo navigation and expansion of hematopoietic progenitors. (Blood. 2004;103:2942-2949)

Stem cells as vehicles for orthopedic gene therapy.

Adult stem cells reside in adult tissues and serve as the source for their specialized cells. In response to specific factors and signals, adult stem cells can differentiate and give rise to functional tissue specialized cells. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into various mesenchymal lineages such as muscle, bone, cartilage, fat, tendon and ligaments. Adult MSCs can be relatively easily isolated from different tissues such as bone marrow, fat and muscle. Adult MSCs are also easy to manipulate and expand in vitro. It is these properties of adult MSCs that have made them the focus of cell-mediated gene therapy for skeletal tissue regeneration. Adult MSCs engineered to express various factors not only deliver them in vivo, but also respond to these factors and differentiate into skeletal specialized cells. This allows them to actively participate in the tissue regeneration process. In this review, we examine the recent achievements and developments in stem-cell-based gene therapy approaches and their applications to bone, cartilage, tendon and ligament tissues that are the current focus of orthopedic medicine.

The role of cell type in bone healing mediated by ex vivo gene therapy.

The ideal cellular vehicle for use in cell-mediated gene therapy to enhance bone healing has not yet been identified. The purpose of this study was to compare the capacity of two types of cells transduced with retro-bone morphogenetic protein 4 (BMP4)-muscle-derived cells (MDCs) and unfractioned bone marrow stromal cells (BMSCs). Primary rat MDCs and unfractioned rat BMSCs were transduced with a retrovirus to express BMP4. A 7-mm, critical-sized femur defect was created in adult rats, and 5 x 10(6) transduced cells were implanted into the femoral defect. Bone healing was monitored radiographically and histologically at 4, 8, and 12 weeks post-implantation. All specimens in the MDC-BMP4 group and BMSC-BMP4 group showed a bridging callus at 8 and 12 weeks. At 12 weeks post-implantation the calluses of the MDC-BMP4 femora displayed significantly higher bone photodensity than the BMSC-BMP4 femora (P<0.05). Histomorphometry revealed no difference between the two treatment groups. However, non-union between newly formed and original bone was observed in none of the MDC femora but in six femora from the BMSC-BMP4 group. Both MDCs and unfractioned BMSCs can improve healing of a critical-sized bone defect following transduction of the cells with retroBMP4. However, MDCs appear to yield superior results when compared with BMSCs in terms of improved healing of segmental defects.

Gene therapy to improve osteogenesis in bone lesions with severe soft tissue damage.

Ex vivo gene therapy can induce bone formation when delivery cells carrying the bone morphogenetic protein (BMP) gene are used. The hypothesis for this study was that the cell-mediated gene therapy could improve the healing of bony lesions with severe soft tissue damage. An animal model with a femoral osteotomy lesion associated with soft tissue damage was developed in rats. Muscle-derived cells, genetically engineered to express BMP4, were inserted within the osteotomy gap. Cells genetically engineered to express LacZ were used for the control group. The groups were subdivided with regard to the fixation method: stable and unstable fixation. The rats were killed for histological and radiographic evaluation 3 and 6 weeks post-surgery. No callus formation was found in the control group at any time point, whereas sufficient callus formation appeared in the treatment group after 6 weeks. A bridging callus with woven bone and hypertrophic chondrocytes was achieved in the treatment group when a stable fixation was used, but failed to appear in unstable fixation. The combination of muscle-derived cells expressing BMP4 and a stable fixation were able to bridge the bone defect within 6 weeks, but with prolonged osteochondral ossification. Therefore, the ex vivo gene therapy could be an efficient biological approach to improve the treatment of bone lesions with severe soft tissue damage.

Molecular cloning of feline CD34

In humans, baboons, dogs and mice CD34 is a cell surface molecule that is expressed on primitive hematopoietic cells and in all these species CD34 positive cells can be used to effect long-term haematopoietic reconstitution. CD34 positive haematopoietic cells therefore provide a convenient and relatively small cell population to target when attempting gene therapy via the haematopoietic system. In order to develop the mucopolysaccharidosis type VI (MPS VI) cat as a model for haematopoietic cell-mediated gene therapy we have isolated the feline CD34 gene as a first step in the generation of antibodies for purification of feline CD34 positive cells. The coding sequence for feline CD34 was isolated from brain cDNA using the polymerase chain reaction (PCR) with oligonucleotides designed to conserved regions of known CD34 gene sequences as primers. Sequence analysis of PCR products revealed the complete amino acid sequence of feline CD34 and allowed analysis of sequence conservation with CD34 from other species. Northern blot analysis showed a 2.6 kb CD34 transcript was present in feline brain, spleen, heart, testis and thymus, and to a lesser extent, in liver. A full-length cDNA clone of the feline CD34 coding sequence was assembled and expressed in CHO-K1 cells. The isolation and expression of the feline CD34 cDNA should facilitate the production of antibodies suitable for the purification of CD34 positive cells.

1033. Cell-Mediated Gene Therapy for Ligament and Tendon Repair

1033. Cell-Mediated Gene Therapy for Ligament and Tendon Repair

Chondrogenesis enhanced by overexpression of sox9 gene in mouse bone marrow-derived mesenchymal stem cells

We investigated chondrogenesis of cell-mediated sox9 gene therapy as a new treatment regimen for cartilage regeneration. pIRES2-EGFP vector containing a full-length mouse sox9 cDNA was transfected into bone marrow-derived mesenchymal stem cells (MSCs) by lipofection and chondrogenic differentiation of these cells was evaluated. In vitro high density micromass culture of these sox9 transfected MSCs demonstrated that a matrix-rich micromass aggregate with EGFP expressing MSCs was positively stained by Alcian blue and type II collagen. Next, sox9 transfected MSCs were loaded into the diffusion chamber and transplanted into athymic mice to analyze in vivo chondrogenesis. A massive tissue formation in about 2 mm diameter was visible in the chamber after 4 weeks transplantation. Histological examinations demonstrated that both Alcian blue and type II collagen were positively stained in the extracellular matrix of the mass while type X collagen was not stained. These results indicated that cell-mediated sox9 gene therapy could be a novel strategy for hyaline cartilage damage.