Antisense oligonucleotides have been covalently attached to asialoglycoprotein (ASGP) via disulfide bond conjugation chemistry. These conjugates were characterized extensively by an array of chemical, chromatographic, and spectroscopic means. Multiple (approximately six) oligonucleotides can be conjugated to each ASGP molecule. The molecular conjugates were used to deliver antisense oligonucleotides complementary to the mRNA of the interleukin 6 signal transduction protein (gp130) to modulate the acute phase response of hepatoma (HepG2) cells in vitro. These conjugates were biologically active, as measured by inhibition of the cytokine-stimulated up-regulation of the acute phase protein haptoglobin. The level of inhibition was comparable to that found with previous technology featuring noncovalent complexes of ASGP-poly(L-lysine) and oligonucleotide. Because of the ability to control the stoichiometry of the conjugate and its unimolecular nature (as opposed to bimolecular for the noncovalent conjugates), this methodology holds great promise for further development and application.

Asialoglycoproteins↗

PubMedDate not supplied

Rapid quantitation of recombinant retroviruses.

A method for measuring the concentration of recombinant retrovirus encoding the Escherichia coli LacZ gene is described. The assay is based on the quantitative measurement of beta-galactosidase activity in extracts of cells infected with the LacZ-encoding retrovirus. LacZ-encoded beta-galactosidase activity in transduced cells is measured using the colorimetric substrate, o-nitrophenyl beta-D-galactopyranoside (ONPG), and the results are read using an ELISA plate-reader. Because the entire assay is performed in a 96-well plate, large numbers of samples are easily measured, and the assay has the advantages of rapidity, precision, and ease of data collection. The assay was used to determine the optimal concentration of polybrene, a polycation known to enhance the infectivity of retroviruses, and can be used to evaluate other factors that affect infection, as well as the optimal conditions for production of the recombinant retrovirus.

3T3 Cells↗

PubMedDate not supplied

Stabilization of active recombinant retroviruses in an amorphous dry state with trehalose.

The disaccharide trehalose is found to be effective for stabilization of active recombinant retroviruses in an amorphous dry state achieved through ambient-temperature vacuum dehydration of retroviral supernatants. Studies revealed that trehalose is a significantly better desiccation protectant than sucrose, glucose, and dextran: dextran has essentially no protective effect on retroviral survival after drying and rehydration. X-ray diffractometry of the retroviral supernatant dried with trehalose demonstrated its amorphous nature. The ability to dehydrate retroviral stocks at ambient temperatures into a stable glassy state will have a profound effect for researchers and commercial biotechnology companies which supply retroviral vectors for human gene therapy and basic research.

3T3 Cells↗

PubMedDate not supplied

Large-scale processing of recombinant retroviruses for gene therapy.

Gene therapy is a new therapeutic modality with the potential of treating inherited and acquired diseases. Several viral and physicochemical vehicles have been used for the transfer of genes to mammalian cells, but recombinant retroviruses are used in the majority of gene therapy clinical trials today. In this communication, we review the major concerns associated with the large-scale production and processing of retroviral particles. While some of the current processes for manufacturing recombinant proteins will be applicable to recombinant retroviruses, the instability, sensitivity to inhibitors, complexity, and size of retroviral particles require that new technologies be designed and evaluated. Here, we examine those issues critical to the design of strategies for production, concentration, and purification as well as formulation and storage of recombinant retroviruses. Processes for large-scale manufacturing of recombinant retroviruses that can produce high gene transfer efficiencies will have significant impact on the clinical implementation of gene therapy.

Cell Line↗

PubMedDate not supplied

Differential inhibition of retrovirus transduction by proteoglycans and free glycosaminoglycans.

We have previously shown that the efficiency of retrovirus-mediated gene transfer is limited in part due to the presence of chondroitin sulfate proteoglycans in virus stocks. In this study, we have used a model recombinant retrovirus encoding the Escherichia coli lacZ gene, bovine aorta chondroitin sulfate proteoglycan (CSPG), various free glycosaminoglycan chains (GAGs), and quantitative assays for retrovirus transduction to explore the mechanism by which proteoglycans and glycosaminoglycans inhibit retroviruses. We found that CSPG and GAGs block an early step in virus-cell interactions but do not act by inactivating viruses or by reducing the growth rate of the target cells. CSPG and most of the GAGs tested (chondroitin sulfate A, chondroitin sulfate B, heparin, heparan sulfate, and hyaluronic acid) inhibited transduction, but with widely varying degrees of activity. The chemical structure of GAGs was found to be an important determinant of their inhibitory activity, which suggests that GAGs do not inhibit transduction simply because they are highly negatively charged polymers. When GAGs were used in combination with a cationic polymer (Polybrene), however, their inhibitory activity was neutralized, and interestingly, at optimal doses of GAG and Polybrene, transduction efficiency was actually enhanced by as much as 72%. In contrast, the inhibitory activity of CSPG, due to the influence of its core protein, was not substantially reduced by Polybrene. The importance of these findings to our understanding of retrovirus-cell interactions and to the development of more efficient retrovirus gene transfer protocols is discussed.

3T3 Cells↗

PubMedDate not supplied

Genetic modification of cultured skin substitutes by transduction of human keratinocytes and fibroblasts with platelet-derived growth factor-A.

Gene therapy promises the potential for improved treatment of cutaneous wounds. This study evaluated whether genetically modified cultured skin substitutes can act as vehicles for gene therapy in an athymic mouse model of wound healing. Human keratinocytes and fibroblasts were genetically engineered by retroviral transduction to overexpress human platelet-derived growth factor-A chain. Three types of skin substitutes were prepared from collagen-glycosaminoglycan substrates populated with fibroblasts and keratinocytes: HF-/HK-, containing both unmodified fibroblasts and keratinocytes; HF-/HK+, containing unmodified fibroblasts and modified keratinocytes; and HF+/HK-, containing modified fibroblasts and unmodified keratinocytes. Skin substitutes were cultured for two weeks before grafting to full-thickness wounds on athymic mice. The modified skin substitutes secreted significantly elevated levels of platelet-derived growth factor throughout the culture period. Expression of retroviral platelet-derived growth factor-A mRNA was maintained after grafting to mice, and was detected in all HF-/HK+ grafts and one HF+/HK- graft at two weeks after surgery. Although no differences were seen between control and modified grafts, the results suggest that genetically modified cultured skin substitutes can be a feasible mechanism for cutaneous gene therapy. The cultured skin model used for these studies has advantages over other skin analogs containing only epidermal cells; because it contains both fibroblasts and keratinocytes, it therefore offers greater opportunities for genetic modification and potential modulation of wound healing.

Animals↗

PubMedDate not supplied

Biologic aspects of expression of stably integrated transgenes in cells of the skin in vitro and in vivo.

The observation that transgenes can be stably integrated into the genome of fibroblasts using recombinant retroviruses enhanced interest in using these cells as a vector for gene therapy. This enthusiasm has lessened during the past 8 years, not because skin has lost the features that make it attractive for gene therapy, but rather because stable transgene expression in vivo has not been achieved. All investigators who have used genetically modified fibroblasts to study in vivo aspects of gene therapy have shown a decrease in transgene expression with time. This contrasts with transgene expression in similarly transduced fibroblasts in vitro, where expression is not lost or is lost very slowly. We have initiated an approach to bring further understanding to the biology of transgene expression by fibroblasts carrying stably integrated transgenes in an in vivo setting. Experiments described permit the following conclusions. Expression by and survival of genetically modified fibroblasts a) requires a persistent matrix scaffold in in vivo settings; b) is prolonged if the matrix is allowed to mature in vitro; c) is enhanced if the matrix is partially sequestered behind a coating of normal fibroblasts; and d) can be substantively prolonged in vivo by immortalizing the cells. These observations support the notion that prolonged expression of transgenes by fibroblasts can be achieved in vivo and that gene therapy utilizing fibroblasts and other cells of the skin has clinical utility.

Clone Cells↗

PubMedDate not supplied

Evaluation of acellular human dermis as a dermal analog in a composite skin graft.

This study evaluated the structure and function of a dermal substitute composed of de-epidermalized, acellular human dermis, and tested its performance as part of a composite skin graft with cultured human keratinocytes. The acellular dermis retained much of the complex structure of native dermis, as demonstrated by immunostaining for collagen Types IV and VII. Collagen Types IV and VII were found on the papillary surface, and collagen Type IV was found at sites throughout the acellular dermis. To further demonstrate the functionality of the acellular dermis material, we seeded the papillary surface with cultured keratinocytes. In response to the architecture of the dermal papillae, the keratinocytes formed a three-dimensional epidermal structure that was several cell layers thick, and recreated the original skin rete ridges at the interface with the dermis. When these composite grafts were transplanted to athymic mice, host fibrovascular cells repopulated the acellular dermis. Some vessels grew into the acellular dermis along the original pathways of the human blood vessels, as demonstrated by co-localization of human and mouse collagen Type IV. The skin that developed from these grafts repigmented completely via passenger melanocytes from the keratinocyte cultures, was durable, and remained stable for more than 5 months.

Animals↗

PubMedDate not supplied

Differentiation and barrier formation of a cultured composite skin graft.

The differentiation and barrier formation of cultured composite skin grafts (CSGs) were assessed by histology and measurements of surface electrical capacitance (SEC) in vitro and in vivo. Keratinocytes cultured on the surface of acellular dermis were lifted to the air-liquid interface and analyzed for 30 days in vitro. Initially, SEC measurements of CSGs (n = 11) were high but quickly dropped between days 4 and 6 and remained steady for 30 days, indicating barrier formation by the epidermis. Histology of the CSGs (n = 6) demonstrated stratification of the epidermal cells and partial formation of the stratum corneum by day 3 that was complete by day 7. CSGs (n = 5) were transplanted to athymic mice, where they formed a stratified and differentiated epidermis. SEC measurements of CSGs remained low after transplant, suggesting that exposure to the air-liquid interface improved the maturation of CSGs in vitro prior to transplant.

Animals↗

PubMedDate not supplied

Intermittent aortic insufficiency. An indication of a malfunctioning aortic valve prosthesis.

Explore the source record for details and available documents.

Adult↗

PubMedDate not supplied

Bioelectric potentials associated with the growing deer antler.

Bioelectric potentials associated with the developing deer antler throughout its growth cycle can be correlated with the rate of longitudinal bone growth. Electrodes were connected to a high input impediance microvoltmeter, and potentials on the surface of antlers of mature deer were measured during the antler growth cycle. The reference electrode was placed at the base of the antler, while the recording electrode was placed at the antler tip or along the antler shaft. Antler length measurements were made during the growth cycle and longitudinal growth rates were determined. Growth rates and bioelectric potentials were compared for three specified periods during the antler growth cycle. The most negative potential was recorded from the antler tip. Furthermore, electronegativity increased as the rate of growth in length increased and decreased as the rate of growth in length decreased. The potentials reached isopolarity at the end of antler growth.

Action Potentials↗