Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

First-generation recombinant adenoviruses that lack E1 sequences have shown tremendous promise in animal and human models of gene therapy. Important limitations of these vectors are that recombinant gene expression is transient and inflammation occurs at the site of gene transfer. Our hypothesis for generating vectors with increased persistence is that present recombinant adenoviruses express viral proteins that stimulate cellular immune responses leading to destruction of the infected cells and repopulation of the organ with non-transgene-containing cells. This model predicts that further crippling of the virus will improve persistence and diminish pathology. We describe in this report second-generation recombinant adenoviruses harboring a beta-galactosidase-expressing transgene in which a temperature-sensitive mutation has been introduced into the E2A gene of an E1-deleted recombinant. At nonpermissive temperature, this virus fails to express late gene products, even when E1 is expressed in trans. The biology of this recombinant was studied in vivo in the context of mouse liver, a setting that is permissive for adenovirus type 5 replication. Animals that received the second-generation virus expressed the transgene for at least 70 days, whereas expression of the first-generation virus was no longer than 14 days. In addition, the inflammatory response, as measured by infiltration of CD8+ T cells, was blunted and delayed in livers infected with second-generation virus. These studies illustrate that modifications that disrupt structural protein expression in recombinant adenoviruses may be useful in enhancing their utility for gene therapy.
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PMID:Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. 801 37

The bovine adenovirus type 3 (BAV3) genome was sequenced from the left end to the HindIII site at 11%. This region comprises the entire E1 transcription unit including the open reading frames (ORF) for proteins homologous to the E1A, E1B proteins and protein IX of human adenovirus type 5 (Ad5). A portion of the BAV3 E1A protein showed significant homology with conserved region 3 (CR3), the principal transactivation region of Ad5 E1A. The BAV3 E1A protein also contains a consensus sequence known to be important for interaction with the cellular Rb protein but lacks most of the sequence corresponding to the second exon of Ad5 E1A. Promoter sequences for BAV3 E1B were not defined though the relevant region contains a 35-base pair repeat sequence. Two ORFs define the BAV3 E1B coding unit; one with regions homologous to sequences within the Ad5 E1B 19k protein, and an overlapping ORF with significant homology to the Ad5 E1B 55k protein. The encoded BAV3 E1B proteins of 157 and 420 amino acid residues (R) have predicted unmodified molecular weights of 17,393 and 46,734 respectively. Immediately following the E1B coding region there is a transcription unit containing an SP1 binding site and TATA box followed by an ORF which encodes a protein of 125R and predicted molecular weight of 13,706 with homology to protein IX of Ad5. Five concensus poly A addition sites are located in the 350 base pairs immediately following the protein IX coding region. The homology of sequences in the Ad5 E1A CR3 region and the corresponding BAV3 protein suggested that the BAV3 protein could transactivate certain Ad5 genes normally transactivated by the Ad5 E1A product. Evidence for this hypothesis was obtained in studies in which bovine cells in culture were coinfected with BAV3 and a human adenovirus type 5 (Ad5) recombinant viral vector lacking the E1A region and having a lacZ reporter gene within the E3 region dependent on E1A for its expression. Coinfection resulted in the induction of beta-galactosidase activity and the increased expression of other Ad5 early (E2A 72k) and late (hexon) proteins.
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PMID:The E1 sequence of bovine adenovirus type 3 and complementation of human adenovirus type 5 E1A function in bovine cells. 817 72

The E2A protein is a mammalian transcription factor of the helix-loop-helix family which is implicated in cell-specific gene expression in several cell lineages. Mouse E2A contains two independent transcription activation domains, ADI and ADII; whereas ADI functions effectively in a variety of cultured cell lines, ADII shows preferential activity in pancreatic beta cells. To analyze this preferential activity in an in vivo setting, we adapted a system involving transient gene expression in microinjected zebra fish embryos. Fertilized one- to four-cell embryos were coinjected with an expression plasmid and a reporter plasmid. The expression plasmids used encode the yeast Gal4 DNA-binding domain (DBD) alone, or Gal4 DBD fused to ADI, ADII, or VP16. The reporter plasmid includes the luciferase gene linked to a promoter containing repeats of UASg, the Gal4-binding site. Embryo extracts prepared 24 h after injection showed significant luciferase activity in response to each of the three activation domains. To determine the cell types in which the activation domains were functioning, a reporter plasmid encoding beta-galactosidase and then in situ staining of whole embryos were used. Expression of ADI led to activation in all major groups of cell types of the embryo (skin, sclerotome, myotome, notochord, and nervous system). On the other hand, ADII led to negligible expression in the sclerotome, notochord, and nervous system and much more frequent expression in the myotome. Parallel experiments conducted with transfected mammalian cells have confirmed that ADII shows significant activity in myoblast cells but little or no activity in neuronal precursor cells, consistent with our observations in zebra fish. This transient-expression approach permits rapid in vivo analysis of the properties of transcription activation domains: the data show that ADII functions preferentially in cells of muscle lineage, consistent with the notion that certain activation domains contribute to selective gene activation in vivo.
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PMID:An activation domain of the helix-loop-helix transcription factor E2A shows cell type preference in vivo in microinjected zebra fish embryos. 865 47

We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity. Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression. A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice. The immune response to the virally encoded beta-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver. Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CpG dinucleotides. However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter may be critical to maintain transgene expression from these attenuated adenovirus vectors.
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PMID:Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses. 915 56

The utility of adenoviral vectors for arterial gene transfer is limited by the brevity of their expression and by inflammatory host responses. As a step toward circumventing these difficulties, we used a rabbit model of in vivo arterial gene transfer to test 3 second-generation vectors: a vector containing a temperature-sensitive mutation in the E2A region, a vector deleted of E2A, and a vector that expresses the immunomodulatory 19-kDa glycoprotein (gp19k) from adenovirus 2. Compared with similar first-generation vectors, the second-generation vectors did not significantly prolong beta-galactosidase transgene expression or decrease inflammation in the artery wall. Although cyclophosphamide ablated the immune and inflammatory responses to adenovirus infusion, it only marginally prolonged transgene expression (94% drop in expression between 3 and 14 days). In experiments performed with "null" adenoviral vectors (no transgene), loss of vector DNA from the arterial wall was also rapid (>99% decrease between 1 hour and 14 days), unrelated to dose, and only marginally blunted by cyclophosphamide. Thus, the early loss of transgene expression after adenoviral arterial gene transfer is due primarily to loss of vector DNA, is not correlated with the presence of local vascular inflammation, and cannot be prevented by use of E2A-defective viruses, expression of gp19k, or cyclophosphamide-mediated immunosuppression. Adenovirus-induced vascular inflammation can be prevented by cyclophosphamide treatment or by lowering the dose of infused virus. However, stabilization of adenovirus-mediated transgene expression in the arterial wall is a more elusive goal and will require novel approaches that prevent the early loss of vector DNA.
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PMID:Second-generation adenoviral vectors do not prevent rapid loss of transgene expression and vector DNA from the arterial wall. 1084 50