Expression of human IL-1alpha after intramarrow gene transfer into healthy non-human primate by adenoviral vector

Interleukin-1alpha (IL-1alpha) is a multifunctional cytokine that stimulates myelopoiesis in macaque. However, daily systemic injections of IL-1alpha are associated with severe side effects. We therefore investigated the feasibility of a gene therapy strategy aimed at increasing the IL-1alpha local production in bone marrow with limited release of the vector into the blood circulation. Intra-medullar administration of adenoviral vector containing human IL-1alpha (huIL-1alpha) gene resulted in enhanced neutrophil, monocyte and platelet counts during the two first weeks after injection. The DNA vector, the transgene expression and the huIL-1alpha production was detected in treated bone marrow without significant detection of huIL-1alpha in the peripheral blood. Associated with huIL-1alpha production, we observed concomitant plasma C reactive protein and IL-1Ra peaks in the acellular fraction of treated bone marrow at days 3 and 7. No abnormal clinical side effects were observed in any of the animals following the adenoviral vector injection.     

Nonviral gene transfer into primary cultures of human and porcine mesothelial cells

Due to their abundance and accessibility, mesothelial cells may be suitable tools for recombinant reagent expression by gene transfer. Genetically modified porcine mesothelial cells (PMCs) may have the potential for the treatment of vascular diseases in humans. We studied the effect of various transfection reagents on the primary culture of PMCs and human mesothelial cells (HMCs). The cells were transfected with a plasmid encoding a reporter gene (luciferase or green fluorescent protein [GFP]) under the control of the cytomegalovirus promoter. Transfection was achieved using cationic lipids (DOSPER and DOTAP) or calcium phosphate/deoxyribonucleic acid coprecipitation or Fugene 6. Results showed that Fugene 6 was the most efficient and reproducible transfection reagent with both PMCs and HMCs. With Fugene 6, luciferase activity in PMCs (1.5 x 10(8) relative light units [RLU]/10(6) cells) was at least 2.5-fold higher than with the other transfection reagents, and it was 100-fold higher than in HMCs. However, the proportion of transfected cells expressing GFP was only 1%. These preliminary findings open up new avenues for developing experimental studies on the use of genetically modified PMCs.     

A novel liver-directed gene delivery system using an autonomously replicating vector specifically expressed in AFP positive hepatoma cells

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Effective repetitive dystrophin gene transfer into skeletal muscle of adult mdx mice using a helper-dependent adenovirus vector expressing the coxsackievirus and adenovirus receptor (CAR) and dystrophin

BACKGROUND: The helper-dependent adenovirus (HDAd) vector is less immunogenic and has a larger cloning capacity of up to 37 kb enough to carry the full-length dystrophin cDNA. However, high and long-term expression of dystrophin transduced to mature muscle still remains difficult. One of the main reasons for this is that the expression of the coxsackievirus and adenovirus receptor (CAR) is very low in mature muscle. METHODS: We have constructed two different HDAd vectors. One contains the LacZ and the murine full-length dystrophin expression cassette (HDAdLacZ-dys), and the other is a new, improved vector containing the CAR and the dystrophin expression cassette (HDAdCAR-dys). RESULTS: We initially demonstrated high dystrophin expression and prevention of the dystrophic pathology in mdx muscle injected during the neonatal phase with HDAdLacZ-dys. Furthermore, we demonstrated that repeated injections of HDAdCAR-dys into mature muscle led to approximately nine times greater dystrophin-positive fibers in number than a single injection, thereby recovering the expression of dystrophin-associated proteins. This data has also shown that HDAdCAR-dys enabled administration of adenovirus (Ad) vector to the host with pre-existing immunity to the same serotype of Ad. CONCLUSIONS: Repetitive injections of the HDAd vector containing the CAR and the dystrophin expression cassette could improve the efficiency of subsequent dystrophin gene transfer to mature mdx muscle. This result suggests that our new HDAd vector will provide a novel gene therapy strategy for Duchenne muscular dystrophy, raising the prospects for gene therapy of other hereditary myopathies.     

Preparation of virosomes coated with the vesicular stomatitis virus glycoprotein as efficient gene transfer vehicles for animal cells

The vesicular stomatitis virus (VSV) glycoprotein (G) was used to prepare virosomes as a model vehicle of gene transfer to animal cells, for which viral envelope functions (receptor recognition and binding and the pH-dependent membrane-fusion) were expected to work. Plasmid DNA (pEGFP-N1; Clontech) was first encapsulated into liposomes by a method of repeated freezing and thawing of the mixture of DNA and lipids (phosphatidylcholine, phosphatidylserine and cholesterol mixed at a molar ratio of 5: 1: 4). Then, particle size of the liposomes was stepwise reduced to 200 nm or less in diameter by successive filtrations through a series of plastic filters of various pore sizes (10 micro m, 2 micro m, 0.65 micro m, and then 0.45 micro m). Assembly of the VSV G protein-coated liposomes (VSV G-virosomes) was performed by mixing the DNA-encapsulated liposome suspensions with the purified VSV G proteins at pH 5.5, followed by ultracentrifugation in a discontinuous sucrose gradient. The highest gene-transducing activity was detected in a single band formed between 20% and 45% sucrose layers. Negatively stained electron microscopic images showed that the band contained spherical particles of various sizes, ranging from 40 to 140 nm in diameter, that were covered with viral spike projections. The VSV G-virosomes displayed a roughly similar level of gene-transducing activity to that mediated by cationic liposomes (e.g., Lipofectamine), which was blocked either by pretreatment with anti-VSV G antiserum or by addition of 20 m M NH(4) Cl to transfected cultures. From these results, we assume that the virosome-mediated gene-transduction was first achieved by using the whole functions of VSV G protein, and can also be used for further studies of the protein.     

Development of a simple particle bombardment device for gene transfer into plant cells

A simple particle bombardment device was designed, constructed and shown to be efficient for the delivery of DNA into plant cells. High levels of transient -glucuronidase expression were observed in alfalfa suspension-cultured cells and embryogenic soybean suspension-cultured cells. Expression of -glucuronidase in alfalfa suspension-cultured cells was used to optimize the bombardment conditions for the device. Transient gene expression in alfalfa was found to be dependent on the state of the target tissue, the size of particles employed, the helium pressure used to accelerate the particles and the distance travel led by the tungsten particles carrying DNA.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MS Murashige & Skoog (1962) medium     

Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa.

We have attempted to transfect testicular spermatozoa with plasmid DNA by direct injection into testes to obtain transgenic animals [this technique was thus termed "testis-mediated gene transfer (TMGT)"]. When injected males were mated with superovulated females 2 and 3 days after injection, (i) high efficiencies (more than 50%) of gene transmission were achieved in the mid-gestational F0 fetuses, (ii) the copy number of plasmid DNA in the fetuses was estimated to be less than 1 copy per diploid cell, and (iii) overt gene expression was not found in these fetuses. These findings suggest the possibility that plasmid DNA introduced into a testis is rapidly transported to the epididymis and then incorporated by epididymal spermatozoa. The purpose of this study was to elucidate the mechanism of TMGT by introducing trypan blue (TB) or Hoechst 33342 directly into testis. We found that TB is transported to the ducts of the caput epididymis via rete testis within 1 min after testis injection, and TB reached the corpus and cauda epididymis within 2-4 days after injection. Staining of spermatozoa isolated from any portion of epididymis was observed 4 days after injection of a solution containing Hoechst 33342. Injection of enhanced green fluorescent protein (EGFP) expression vector/liposome complex into testis resulted in transfection of epithelial cells of epididymal ducts facing the lumen, although the transfection efficiency appeared to be low. In vivo electroporation toward the caput epididymis immediately after injection of EGFP expression vector into a testis greatly improved the uptake of foreign DNA by the epididymal epithelial cells. PCR analysis using spermatozoa isolated from corpus and cauda epididymis 4 days after injection of a DNA/liposome complex into testis revealed exogenous DNA in these spermatozoa even after treatment with DNase I. These findings indicate that exogenous DNA introduced into tesits is rapidly transported to epididymal ducts via the rete testis and efferent ducts, and then incorporated by epithelial cells of epididymis and epididymal spermatozoa.