Viral vector-mediated gene transfer to the postnatal respiratory epithelium has, in general, been of low efficiency due to physical and immunological barriers, non-apical location of cellular receptors critical for viral uptake and limited transduction of resident stem/progenitor cells. These obstacles may be overcome using a prenatal strategy. In this study, HIV-1-based lentiviral vectors (LVs) pseudotyped with the envelope glycoproteins of Jaagsiekte sheep retrovirus (JSRV-LV), baculovirus GP64 (GP64-LV), Ebola Zaire-LV or vesicular stomatitis virus (VSVg-LV) and the adeno-associated virus-2/6.2 (AAV2/6.2) were compared for in utero transfer of a green fluorescent protein (GFP) reporter gene to ovine lung epithelium between days 65 and 78 of gestation. GFP expression was examined on day 85 or 136 of gestation (term is ∼145 days). The percentage of the respiratory epithelial cells expressing GFP in fetal sheep that received the JSRV-LV (3.18 × 10(8)-6.85 × 10(9) viral particles per fetus) was 24.6±0.9% at 3 weeks postinjection (day 85) and 29.9±4.8% at 10 weeks postinjection (day 136). Expression was limited to the surface epithelium lining fetal airways <100 μm internal diameter. Fetal airways were amenable to VSVg-LV transduction, although the percentage of epithelial expression was low (6.6±0.6%) at 1 week postinjection. GP64-LV, Ebola Zaire-LV and AAV2/6.2 failed to transduce the fetal ovine lung under these conditions. These data demonstrate that prenatal lung gene transfer with LV engineered to target apical surface receptors can provide sustained and high levels of transgene expression and support the therapeutic potential of prenatal gene transfer for the treatment of congenital lung diseases.
"Fourteen day old lambs were used in this study since cells in the respiratory tract would be expected to have a higher rate of cell division than adult sheep thereby maximizing infection efficiency. Additionally, the expression level of ovine hyaluronidase 2 (Hyal2), the receptor for ENTV-1, is high in the fetus and then markedly declines in the neonate . "
[Show abstract][Hide abstract] ABSTRACT: Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the secretory epithelial cells of the nasal mucosa of sheep and goats. It is associated with the betaretrovirus, enzootic nasal tumor virus (ENTV), but a causative relationship has yet to be demonstrated. In this study, 14-day-old lambs were experimentally infected via nebulization with cell-free tumor filtrates derived from naturally occurring cases of ENA. At 12 weeks post-infection (wpi), one of the five infected lambs developed clinical signs, including continuous nasal discharge and open mouth breathing, and was euthanized. Necropsy revealed the presence of a large bilateral tumor occupying the nasal cavity. At 45 wpi, when the study was terminated, none of the remaining infected sheep showed evidence of tumors either by computed tomography or post-mortem examination. ENTV-1 proviral DNA was detected in the nose, lung, spleen, liver and kidney of the animal with experimentally induced ENA, however there was no evidence of viral protein expression in tissues other than the nose. Density gradient analysis of virus particles purified from the experimentally induced nasal tumor revealed a peak reverse transcriptase (RT) activity at a buoyant density of 1.22 g/mL which was higher than the 1.18 g/mL density of peak RT activity of virus purified from naturally induced ENA. While the 1.22 g/mL fraction contained primarily immature unprocessed virus particles, mature virus particles with a similar morphology to naturally occurring ENA could be identified by electron microscopy. Full-length sequence analysis of the ENTV-1 genome from the experimentally induced tumor revealed very few nucleotide changes relative to the original inoculum with only one conservative amino acid change. Taken together, these results demonstrate that ENTV-1 is associated with transmissible ENA in sheep and that under experimental conditions, lethal tumors are capable of developing in as little as 12 wpi demonstrating the acutely oncogenic nature of this ovine betaretrovirus.
Veterinary Research 07/2013; 44(1):66. DOI:10.1186/1297-9716-44-66 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Because of their ability to transduce nondividing cells, human immunodeficiency virus type 1 (HIV)-based vectors have great potential for the therapeutic delivery of genes to cells. We describe here a systematic study of the packaging limit of HIV-based vectors. Restriction endonuclease-generated bacterial chromosomal DNA fragments of different lengths were cloned at three different positions within a lentiviral vector. Vesicular stomatitis virus G protein (VSV G) pseudotyped lentiviral particles were prepared and the different clones were titered on mammalian cells. We observed that the restriction endonuclease site positions at the 5' and 3' ends of the genome were superior with regard to insertional capacity of foreign DNA. In all cases, viral titers decreased semi-logarithmically with increasing vector length. There appears to be no absolute packaging limit because measurable titers were obtained even when the proviral length was in excess of 18 kb. The reduction in titer appears to occur at the level of viral encapsidation, although we cannot exclude limitations in nuclear export of proviral RNA. These results suggest that HIV-based vectors may have a secondary advantage over oncoretroviral vectors because of their greater packaging limit, although the very low titers of the larger vectors will be of limited utility.
Human Gene Therapy 11/2001; 12(15):1893-905. DOI:10.1089/104303401753153947 · 3.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The field of possible application of mesenchymal stem cells in medicine and research expanded tremendously with the advent of improved Lentiviral-vectors capable of inserting stable copies of genes of interest and expressing proteins or biologically active RNA species ad libitum, performing delicate gene editing or active gene silencing or serving as advanced drug delivery systems utilized in ex vivo cell therapy. The combination of these two fields has created a number of new areas of research in the landscape of modern medicine which are now extensively studied and discussed here. These areas include tissue engineering, tissue repair, wound healing and tissue implants, anticancer therapies, angiogenesis, myocardial infarction and repair as well as understanding and treating acute lung damage and injury. In addition, genetically modified, tagged MSCs are being intensively deployed in research and therapeutic attempts of the various ailments of the central nervous system including Parkinson’s disease, Alzheimer’s disease, various phases of acute ischemia and trauma. The emergence of new and important data for type II diabetes research is being followed with treatment suggestions and studies of senescence to find novel applications for genetically engineered MSCs. We find in general that genetically modified MSCs are at the cusp of breaking through from basic research into the next phase of clinical trials.
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