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Biomedical subjects

M A Kay

Publications and source records attributed to M A Kay.

At least 73 records · Page 4Linked to original sources
PubMed1997

Constitutive expression of murine CTLA4Ig from a recombinant adenovirus vector results in prolonged transgene expression.

The administration of soluble muCTLA4Ig around the time of adenovirus vector mediated gene transfer into murine hepatocytes has been shown to markedly prolong transgene expression, diminish the formation of adenovirus neutralizing antibody, decrease T cell proliferative response and infiltration into the liver without causing irreversible systemic immunosuppression. In this study, an E1/E3-deleted adenovirus vector constitutively expressing murine CTLA4Ig (Ad.RSV-muCTLA4Ig) was constructed in order to determine if production of muCTLA4Ig from within transduced cells (i.e. hepatocytes) would provide a more specific/localized interference with the CD28/B7-1 and B7-2 signaling pathways, and thus result in prolonged transgene expression in vivo at nonimmunosuppressive serum concentrations. In contrast to C3H mice receiving a control adenovirus, transduction with 6 x 10(9) p.f.u. of Ad.RSV-muCTLA4Ig and a reporter adenovirus (2 x 10(9) p.f.u. of Ad.PGK-hAAT) resulted in prolonged reporter gene expression, reduced anti-adenovirus and anti-hAAT antibody production, and attenuated T cell proliferation and IFN-gamma production in response to adenoviral vector. Mice given a constant total amount of adenovirus with diminishing amounts of Ad.RSV-muCTLA4Ig and a constant amount of reporter virus (2 x 10(9) p.f.u. of Ad.PGK-hAAT) demonstrated prolonged reporter gene expression and decreased anti-adenovirus and anti-hAAT antibody production only when high serum levels of muCTLA4Ig were produced. Taken together, these findings suggest that a certain threshold of muCTLA4Ig must be achieved to alter the immune responses and prolong transgene expression from adenoviral vectors.

Abatacept↗
PubMed1997

Development of a high-performance liquid chromatographic assay for G418 sulfate (Geneticin).

We have developed a chromatographic assay with high sensitivity and specificity to quantify G418 sulfate (Geneticin), an antibiotic used routinely in molecular genetics experiments for selecting eukaryotic transformants. With this method, G418 in tissues and plasma samples can be quantitated without the confounding factors often associated with biological assays. After removal of proteins in homogenized tissue or plasma samples with methanol (2:1, vol/vol), the amino group of G418 was derivatized with 1-fluoro-2,4-dinitrobenzene (DNFB) to form the UV-visible G418-DNFB product. The DNFB-derivatized G418 was separated on a reversed-phase C18 column with an acetonitrile and water gradient as the mobile phase. Under these assay conditions, the detection limit for G418 sulfate in buffer, plasma, and tissues was recorded at 78 ng/ml and the linearity was recorded for concentrations up to 100 micrograms/ml. The data obtained from this analysis indicate that this assay can be used for the quantitative determination of G418 sulfate in plasma and tissue samples.

Animals↗
PubMed1997

The role of Kupffer cell activation and viral gene expression in early liver toxicity after infusion of recombinant adenovirus vectors.

Systemic application of first-generation adenovirus induces pathogenic effects in the liver. To begin unraveling the mechanisms underlying early liver toxicity after adenovirus infusion, particularly the role of macrophage activation and expression of viral genes in transduced target cells, first-generation adenovirus or adenovirus vectors that lacked most early and late gene expression were administered to C3H/HeJ mice after transient depletion of Kupffer cells by gadolinium chloride treatment. Activation of NF-kappaB, and the serum levels of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) were studied in correlation with liver damage, apoptosis, and hepatocellular DNA synthesis. While Kupffer cell depletion nearly eliminated adenovirus-induced TNF release, it resulted in a more robust IL-6 release. These responses were greatly reduced in animals receiving the deleted adenovirus. Although there were quantitative differences, NF-kappaB activation was observed within minutes of first-generation or deleted adenovirus vector administration regardless of the status of the Kupffer cells, suggesting that the induction is related to a direct effect of the virus particle on the hepatocyte. Early liver toxicity as determined by serum glutamic-pyruvic transaminase elevation and inflammatory cell infiltrates appeared to be dependent on adenovirus-mediated early gene expression and intact Kupffer cell function. Kupffer cell depletion had little effect on adenovirus-mediated hepatocyte apoptosis but did increase hepatocellular DNA synthesis. Finally, Kupffer cell depletion decreased the persistence of transgene (human alpha1-antitrypsin [hAAT]) expression that was associated with a more pronounced humoral immune response against hAAT. The elucidation of these events occurring after intravenous adenovirus injection will be important in developing new vectors and transfer techniques with reduced toxicity.

Adenoviridae↗
PubMed1997

Persistence of recombinant adenovirus in vivo is not dependent on vector DNA replication.

Recombinant adenovirus vectors represent an efficient means of transferring genes into many different organs. The first-generation E1-deleted vector genome remains episomal and, in the absence of host immunity, persists long-term in quiescent tissues such as the liver. The mechanism(s) which allows for persistence has not been established; however, vector DNA replication may be important because replication has been shown to occur in tissue culture systems. We have utilized a site-specific methylation strategy to monitor the replicative fate of E1-deleted adenovirus vectors in vitro and in vivo. Methylation-marked adenovirus vectors were produced by the addition of a methyl group onto the N6 position of the adenine base of XhoI sites, CTCGAG, by propagation of vectors in 293 cells expressing the XhoI isoschizomer PaeR7 methyltransferase. The methylation did not affect vector production or transgene expression but did prevent cleavage by XhoI. Loss of methylation through viral replication restores XhoI cleavage and was observed by Southern analysis in a wide variety of, but not all, cell culture systems studied, including hepatoma and mouse and macaque primary hepatocyte cultures. In contrast, following liver-directed gene transfer of methylated vector in C57BL/6 mice, adenovirus vector DNA was not cleaved by XhoI and therefore did not replicate, even after a period of 3 weeks. Although replication may occur in some tissues, these results show that stabilization of the vector within the target tissue prior to clearance by host immunity is not dependent upon replication of the vector, demonstrating that the input transduced DNA genomes were the persistent molecules. This information will be useful for the design of optimal adenovirus vectors and perhaps nonviral episomal vectors for clinical gene therapy.

Adenoviridae↗
PubMed1996

Muscle differentiation during repair of myocardial necrosis in rats via gene transfer with MyoD.

Myocardial infarcts heal by scar formation because there are no stem cells in myocardium, and because adult myocytes cannot divide and repopulate the wound. We sought to redirect the heart to form skeletal muscle instead of scar by transferring the myogenic determination gene, MyoD, into cardiac granulation (wound repair) tissue. A replication-defective adenovirus was constructed containing MyoD under transcriptional control of the Rous sarcoma virus long terminal repeat. The virus converted cultured cardiac fibroblasts to skeletal muscle, indicated by expression of myogenin and skeletal myosin heavy chains (MHCs). To determine if MyoD could induce muscle differentiation in vivo, we injected 2 x 10(9) or 10(10) pfu of either the MyoD or a control beta-galactosidase adenovirus into healing rat hearts, injured 1 wk previously by freeze-thaw. After receiving the lower viral dose, cardiac granulation tissue expressed MyoD mRNA and protein, but did not express myogenin or skeletal MHC. When the higher dose of virus was administered, double immunostaining showed that cells in reparative tissue expressed both myogenin and embryonic skeletal MHC. No muscle differentiation occurred after beta-galactosidase transfection. Thus, MyoD gene transfer can induce skeletal muscle differentiation in healing heart lesions. Modifications of this strategy might eventually provide new contractile tissue to repair myocardial infarcts.

Adenoviridae↗
PubMed1996

Adenovirus-mediated hepatic gene transfer in mice: comparison of intravascular and biliary administration.

Recombinant adenoviruses have received much attention as a potential vector for gene therapy because of their ability to transduce many cell types with high efficiencies in vivo. After intravenous infusion, the majority of the vector is found in hepatocytes, but vector DNA is found to varying degrees in other tissues. In an attempt to restrict adenovirus-mediated gene transfer to the liver, we developed a microsurgical method that allowed for vector administration directly into the biliary tract of a mouse. We demonstrate that gene transfer was 4- to 10-fold more restricted to the liver after biliary tract infusion than after intravascular infusion. Intravascular infusion of recombinant adenovirus elicits a powerful immune response that limits gene expression and the ability to readminister the vector. Biliary infusion resulted in a slightly lesser immune response as determined by the lower neutralizing antibody titers directed against the vector compared with animals treated by intravascular infusion. There was no difference in the persistence of gene expression, suggesting a similar cell-mediated immune response against the vector containing cells in animals administered vector by either method. As future-generation adenovirus vectors that are safer and less immunogenic become available, the more liver specific gene transfer via the biliary tract may offer advantages over intravenous infusion for hepatic gene therapy.

Adenoviridae↗
PubMed1996

Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes.

Hepatitis C virus (HCV), a positive-strand RNA virus, is the major infectious agent responsible for causing chronic hepatitis. Currently, there is no vaccine for HCV infection, and the only therapy for chronic hepatitis C is largely ineffective. To investigate new genetic approaches to the management of HCV infection, six hammerhead ribozymes directed against a conserved region of the plus strand and minus strand of the HCV genome were isolated from a ribozyme library, characterized, and expressed from recombinant adenovirus vectors. The expressed ribozymes individually or in combination were efficient at reducing or eliminating the respective plus- or minus-strand HCV RNAs expressed in cultured cells and from primary human hepatocytes obtained from chronic HCV-infected patients. This study demonstrates the potential utility of ribozyme therapy as a strategy for the treatment of hepatitis C virus infection.

Adenoviridae↗
PubMed1996

Recombinant adenoviruses with large deletions generated by Cre-mediated excision exhibit different biological properties compared with first-generation vectors in vitro and in vivo.

In vivo gene transfer of recombinant E1-deficient adenoviruses results in early and late viral gene expression that elicits a host immune response, limiting the duration of transgene expression and the use of adenoviruses for gene therapy. The prokaryotic Cre-lox P recombination system was adapted to generate recombinant adenoviruses with extended deletions in the viral genome (referred to here as deleted viruses) in order to minimize expression of immunogenic and/or cytotoxic viral proteins. As an example, an adenovirus with a 25-kb deletion that lacked E1, E2, E3, and late gene expression with viral titers similar to those achieved with first-generation vectors and less than 0.5% contamination with E1-deficient virus was produced. Gene transfer was similar in HeLa cells, mouse hepatoma cells, and primary mouse hepatocytes in vitro and in vivo as determined by measuring reporter gene expression and DNA transfer. However, transgene expression and deleted viral DNA concentrations were not stable and declined to undetectable levels much more rapidly than those found for first-generation vectors. Intravenous administration of deleted vectors in mice resulted in no hepatocellular injury relative to that seen with first-generation vectors. The mechanism for stability of first-generation adenovirus vectors (E1a deleted) appeared to be linked in part to their ability to replicate in transduced cells in vivo and in vitro. Furthermore, the deleted vectors were stabilized in the presence of undeleted first-generation adenovirus vectors. These results have important consequences for the development of these and other nonintegrating vectors for gene therapy.

Adenovirus E1 Proteins↗
PubMed1996

Pseudotransduction of hepatocytes by using concentrated pseudotyped vesicular stomatitis virus G glycoprotein (VSV-G)-Moloney murine leukemia virus-derived retrovirus vectors: comparison of VSV-G and amphotropic vectors for hepatic gene transfer.

Recombinant retrovirus vectors are widely used for gene transfer studies. The recent development of a pseudotyped Moloney murine leukemia virus vector that contains the G envelope protein from the vesicular stomatitis virus allows for efficient concentration of vector and offers hope for potential use of these vectors for gene expression in vivo. A standard amphotropic vector expressing a serum marker protein, human alpha 1-antitrypsin, was infused into regenerating mouse liver and was 10-fold more efficient at achieving stable gene expression than was an equivalent pseudotyped vector. Discrepant results were obtained with cultured hepatocytes infected with an Escherichia coli beta-galactosidase-producing pseudotype and amphotropic vector. High rates of beta-galactosidase-positive cells were detected with the vesicular stomatitis virus G glycoprotein vector under culture conditions known to be relatively nonpermissive for retrovirus-mediated gene transfer. Subsequent studies demonstrated that beta-galactosidase protein was concentrated and copurified during pseudotype vector preparation, resulting in high rates of protein transfer rather than stable gene transfer, a process referred to as pseudotransduction. The cotransfer of protein with concentrated pseudotyped retroviruses indicates that caution must be used when interpreting gene transduction efficiencies in gene therapy experiments.

3T3 Cells↗
PubMed1996

Adenovirus-mediated expression of ribozymes in mice.

Ribozymes are a new pharmaceutical class of reagents that offer potential in treating a number of different medical disorders, including infectious diseases and cancer. As a first step towards using ribozymes for the treatment of liver disorders such as viral hepatitis, adenovirus vectors that contain a ribozyme expression cassette under the control of different promoters directed against human growth hormone (hGH) were constructed and infused into transgenic mice that produce hGH from the gastrointestinal tract and liver. Adenovirus-mediated transfer of expressed ribozymes resulted in up to a 96% reduction of hepatic hGH mRNA over a period of several weeks in the transgenic mouse model. Furthermore, the concentration of ribozyme RNA correlated with the degree of hGH mRNA reduction. This study clearly demonstrates that ribozymes can function during the period of expression in an intact organ after somatic gene transfer.

Adenoviridae↗
PubMed1996

Method for multiple portal vein infusions in mice: quantitation of adenovirus-mediated hepatic gene transfer.

For many preclinical studies, the mouse has been an invaluable model. For hepatic studies, including gene therapy, the use of the mouse has been limited because of the inability to obtain long-term portal vein access. In this study, we have developed a surgical cannula model that allows for repeat portal vein infusion in a noninvasive manner. We have used this model to establish that the tissue distribution of recombinant adenoviral vectors is similar after portal vein or peripheral vein infusion. The majority of the vector was present in the liver, ranging from 14 to 28 copies per hepatocyte. The second most prevalent tissues were the spleen and lung with 1/10 less adenoviral DNA. The brain and ovaries had the least DNA, 1/1000 less than the liver. Additional studies were performed to study the effects of secondary adenovirus infusion through the portal vein cannula. Permanent portal vein access in a mouse model will be invaluable for a large number of medical studies, including the development of new technologies for hepatic gene transfer.

Adenoviridae↗
PubMed1996

HBV-derived promoters direct liver-specific expression of an adenovirally transduced LDL receptor gene.

In vivo approaches to liver gene therapy will require restriction of transgene expression to hepatocytes. Since targeting of viral vectors exclusively to the liver is not easy to achieve, use of liver-specific promoters for driving expression of therapeutic genes is an interesting alternative. We have shown previously that regulatory elements of the hepatotrophic hepatitis B virus (HBV) are strong and liver-specific in vitro and therefore might be useful in hepatic gene therapy. Here we describe recombinant adenoviruses in which the human LDL receptor gene is under the transcriptional control of the HBV core promoter, the core promoter linked directly to HBV enhancer I, or a HBV-CMV hybrid promoter, respectively. These viruses allowed for a moderate to strong expression of the LDL receptor gene in vitro in a hepatocyte-specific manner when compared with the CMV immediate-early promoter. In vivo experiments demonstrated that the promoter gave rise to an expression level comparable to that from the CMV promoter in mouse liver, but was very weak in lung and skeletal muscle. Thus, the HBV-CMV hybrid promoter is strong and hepatocyte specific both in vitro and in vivo even in the adenoviral context and would be a good choice for driving a therapeutic gene in liver gene therapy.

Adenoviridae↗
PubMed1995

Adenovirus-mediated urokinase gene transfer induces liver regeneration and allows for efficient retrovirus transduction of hepatocytes in vivo.

Retrovirus-mediated gene transfer into hepatocytes in vivo results in long-term gene expression. Limitations include the need to remove two-thirds of the liver and the relatively low frequency of gene transfer. To increase gene transfer without surgical hepatectomy, mouse hepatocytes were transduced in vivo with a recombinant adenovirus that transiently expressed urokinase, resulting in high rates of asynchronous liver regeneration. During the regenerative phase, in vivo retroviral-mediated gene transfer in hepatocytes resulted in 5- to 10-fold greater transduction efficiencies than that obtained by conventional partial hepatectomy. In 3-4 weeks, the architecture and microscopic structure of the recipient livers were normal. The two-viral system of achieving permanent transgene expression from hepatocytes in vivo offers an alternative approach to current ex vivo and in vivo gene-transfer models.

Adenoviridae↗