Biomedical subjects

Antoneta Radu

Publications and source records attributed to Antoneta Radu.

5 recordsLinked to original sources

PubMed2003

Human fetal trachea-SCID mouse xenografts: efficacy of vesicular stomatitis virus-G pseudotyped lentiviral-mediated gene transfer.

BACKGROUND/PURPOSE: Postnatal gene transfer in respiratory epithelium has been inefficient, particularly in submucosal gland cells, the target cells for cystic fibrosis transmembrane regulator (CFTR) gene transfer in cystic fibrosis. The authors hypothesized that fetal tracheobronchial epithelium may be more receptive to gene transfer in that precursor cells of the respiratory epithelium may be more accessible. METHODS: Vesicular stomatitis virus-G (VSV-G) pseudotyped lentiviral vector was first tested in human fetal tracheal organ culture then validated in a human fetal tracheal xenograft model in severe combined immunodeficiency (SCID) mice. Between 7 x 10(7) and 1.6 x 10(8) transducing units of lentiviral vector encoding the transgene LacZ under the control of the human CMV promoter were administered onto the lumenal surface of the xenografts (n = 6). Biopsy specimens were taken from the xenografts at one month (n = 1), 2 months (n = 2), 3 months (n = 1), 6 months (n = 4), and 9 months (n = 1) after vector administration. Analysis of transgene expression was performed on X-gal stained sections. RESULTS: Transgene expression was observed in 20.2% to 99% of the surface epithelial cells (mean, 70.8 +/- 32.3% SD) and in 28.4% to 99% of the submucosal gland cells (mean, 68.5 +/- 26.2% SD) out to 9 months after vector administration in the tracheal xenografts, equivalent to 63 weeks postconceptual age. No staining was seen in the controls. CONCLUSIONS: Excellent gene transfer in human fetal tracheal xenografts after VSV-G pseudotyped lentiviral vector administration, which may result from more accessible target precursor cells during development, suggests the feasibility of fetal gene therapy for the treatment of congenital airway disease such as cystic fibrosis.

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PubMed2002

Efficient transduction of liver and muscle after in utero injection of lentiviral vectors with different pseudotypes.

In this study we investigate the efficacy of lentiviral vectors of different pseudotypes for gene transfer to tissues of the preimmune fetus. BALB/c fetuses at 14-15 days' gestation received lentiviral vectors carrying the transgene lacZ under the control of the human cytomegalovirus (CMV) promoter by intramuscular (i.m.) or intrahepatic (i.h.) injection. We pseudotyped the lentiviral vectors with vesicular stomatitis virus (VSV-G), with Mokola virus, or with Ebola virus envelope glycoproteins. We harvested the pups at time points between 5 days and 9 months following injection and performed a detailed histologic assessment. The efficiency and distribution of transduction after in utero administration was highly dependent upon the route of administration and the pseudotype of vector used. Biodistribution studies showed widespread distribution of vector sequences in multiple tissues, albeit at very low levels, and transduced cells were found in significant numbers only in liver, heart, and muscle. Overall, VSV-G was the most efficient in transducing hepatocytes, whereas Mokola and Ebola were more efficient in transducing myocytes. Transduction of cardiomyocytes was observed after both i.m. and i.h. injection of all three vectors. Our findings of long-term transduction of skeletal myocytes and cardiomyocytes after in utero administration suggest a novel strategy for the treatment of congenital muscular dystrophies.

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PubMed2002

Adeno-associated virus (AAV)-mediated gene transfer in respiratory epithelium and submucosal gland cells in human fetal tracheal organ culture.

BACKGROUND/PURPOSE: Since the discovery of the cystic fibrosis transmembrane regulator (CFTR) gene, cystic fibrosis has been an attractive target for gene therapy. Postnatal gene transfer in the respiratory epithelium has been difficult and particularly inefficient in the submucosal gland cells, the target cells for CFTR gene transfer. The authors hypothesized that during development, there is a favorable environment for fetal gene therapy with fewer physical barriers to efficient gene transfer and more accessible epithelial and submucosal gland precursor cells. The authors tested this hypothesis in a novel human fetal tracheal organ culture system using a serotype 2 recombinant AAV that contains an enhanced green fluorescent protein (eGFP) reporter gene (AAV-CMV-eGFP). METHODS: Human fetal tracheas at between 16 and 20 weeks' gestation age were used in this study. The distal end of each trachea was ligated and secured in an upright position with the open proximal end facing up. Only the ante-lumenal surface was exposed to culture media. 5 x 10(9) particles of AAV-CMV-eGFP were administered intratracheally through the open end. Fetal tracheas were maintained in tracheal organ culture media and harvested at either 2 weeks (n = 3) or 4 weeks (n = 7) after injection. The fetal tracheas were processed for routine H&E, standard electron microscopy (EM), and fluorescence microscopy for analysis of eGFP transgene expression. RESULTS: Histology confirmed the preservation of structural integrity out to 4 weeks of fetal tracheal organ culture. EM showed intact tight junctions of the apical respiratory epithelium. At 2 weeks after AAV-CMV-eGFP injection, there was minimal transgene expression. However, at 4 weeks there was extensive transgene expression in not only the respiratory surface epithelium but also the submucosal gland cells of the human fetal tracheal organ culture. Transgene expression was seen in nearly all cells in the submucosal glands. CONCLUSIONS: AAV-mediated gene transfer in human fetal tracheal organ culture was highly efficient with excellent transgene expression at 4 weeks in both respiratory surface epithelium and submucosal gland cells. This highly efficient gene transfer may result from fewer physical barriers and more accessible target precursor cells. These results are consistent with more efficient gene transfer in fetal tracheobronchial epithelium and show the potential for fetal gene therapy using AAV for the treatment of congenital airway disease such as cystic fibrosis.

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PubMed2002

Engraftment of bone marrow and fetal liver cells after in utero transplantation in MDX mice.

BACKGROUND/PURPOSE: In utero hematopoietic stem cell transplantation (IUHSCTx) has been experimentally or clinically effective only in circumstances in which there is a survival advantage for donor cells. A survival advantage exists for normal muscle cells in muscular dystrophy. Because hematopoietic and mesenchymal stem cells may have the capacity to differentiate into muscle cells, the authors hypothesized that in utero bone marrow (BM) or fetal liver (FL) stem cell transplantation may be used to treat muscular dystrophy. METHODS: Time-dated 14-day-gestation fetal muscular dystrophy mice (mdx) were injected intraperitoneally with 1 to 5 x 10(6) BM or FL cells per fetus from Rosa26 donor mice (transgenic for lacZ). Four weeks after birth, peripheral blood from the pups was analyzed for hematopoietic chimerism by using fluorescence-activated cell sorting (FACS) for the Ly-9.1 marker. Chimeric mice (6 BM and 2 FL recipients) were sacrificed at 12 to 14 months of age, muscles were stained with X-gal, and analyzed by 1- to 2-microm plastic sections. Polymerase chain reaction (PCR) for lacZ was performed in other organs to determine systemic engraftment. RESULTS: At the time of death, all animals that were chimeric at 4 weeks continued to show hematopoietic chimerism of 0.2% to 9% by FACS. Engrafted donor cells were found in multiple sections from hindlimb skeletal muscles, diaphragms, and hearts from both BM and FL recipients. These cells had incorporated into the host muscles, and their morphology was consistent with myogenic differentiation. PCR of BM, liver, spleen, thymus, kidney, and lung for lacZ was positive in multiple animals. CONCLUSIONS: IUHSCTx leads to widespread engraftment of donor cells in multiple muscle compartments of hematopoietic chimeras. The advantage for normal myocytes offered in the mdx model allows engraftment and myogenic differentiation of transplanted BM or FL cells by morphology at a relatively higher frequency in muscle relative to other tissues, without the need for host conditioning. Because muscular dystrophy now can be detected early in gestation, such a strategy may offer a future alternative in the clinical treatment of this disease.

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PubMed2002

Bone marrow tissue engineering.

The creation of mixed hematopoietic chimerism has become an important clinical strategy for tolerance induction for cellular and organ transplantation, and for the treatment of numerous hematopoietic diseases. Clinical success has been limited however, by host immune response and by competition from host hematopoiesis. Recent data suggests that limited donor stem cell engraftment after minimally myeloablative hematopoietic stem cell (HSC) transplantation may in part be due to MHC associated microenvironmental mismatch resulting in a competitive disadvantage for donor HSC. A strategy to overcome this barrier to stable mixed hematopoietic chimerism would involve concurrent transplantation of a donor bone marrow microenvironment. To test this possibility, we set out to develop a method to tissue engineer a bone marrow microenvironment. One to two murine femurs were mechanically crushed to a fine suspension and were combined in vitro with various delivery vehicles. These constructs were transplanted into syngeneic animals in locations that are known to support transplantation of other tissues. Although bone formation was observed with several conditions, bone marrow formation was noted only within the small bowel mesentery when type I collagen was used as the delivery vehicle. No bone marrow formed when the vehicle was changed to polyglycolic acid or type IV collagen. We have demonstrated that the small bowel mesentery can support bone marrow formation under specific in vivo conditions. Future work will focus on strategies for transplantation of an engineered donor bone marrow environment to facilitate creation of allogeneic mixed hematopoietic chimerism.

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