David J. Spector

Expression and Function of Recombinant Endothelial Nitric Oxide Synthase Gene in Canine Basilar Artery

Endothelial NO synthase (eNOS) is an enzyme responsible for the production of a potent vasodilator and a key regulator of vascular tone, NO. In peripheral arteries, expression of a recombinant eNOS gene increases production of NO in the blood vessel wall. This approach appears to be a promising strategy for gene therapy of cerebrovascular disease. The major objective of the present study was to determine whether a recombinant eNOS gene (AdCMVNOS) can be functionally expressed in cerebral arteries. Replication-defective recombinant adenovirus vectors encoding bovine eNOS and Escherichia coli beta-galactosidase (AdCMVLacZ) genes, driven by the cytomegalovirus promoter, were used for ex vivo gene transfer. Rings of canine basilar artery were incubated with increasing titers of the vectors in MEM. Twenty-four or forty-eight hours after gene transfer, expression and function of AdCMVNOS were evaluated by (1) immunohistochemical staining, (2) isometric tension recording, and (3) cGMP radioimmunoassay. Transfection with AdCMVNOS resulted in the expression of recombinant eNOS protein in the vascular adventitia and endothelium, associated with significantly reduced contractile responses to UTP and enhanced endothelium-dependent relaxation to calcium ionophore A23187. Basal production of cGMP was significantly increased in the transfected vessels. The reduced contractions to UTP with increased cGMP production were reversed by a NOS inhibitor, N(G)-monomethyl-L-arginine. Contractions to UTP or production of cGMP were not affected in arteries transfected with AdCMVLacZ reporter gene. The results of the present study represent the first successful transfer and functional expression of recombinant eNOS gene in cerebral arteries. Our findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall.

Regulation of the appearance of cytoplasmic RNAs from region 1 of the adenovirus 2 genome.

Abstract The time-course of appearance of cytoplasmic RNA species coded by the region 9 to 10.7 on the adenovirus 2 genome (early region 1) has been examined using specific DNA probes. Hybridization with restriction endonuclease fragments of viral DNA resolved five cytoplasmic RNAs. A 22 S RNA contains sequences to the right of map position 4.4. One 13 S RNA (13 SL) maps to the left of this site, while an RNA of similar size (13 Sf) is transcribed from two separate regions to the right of 4.4. Each 13 S RNA shares 3′ sequences with a distinct 9 S RNA. Sequences in 13Sf RNA are also present in the 22 S species. These mapping studies have allowed the identification of probes that can be used to assay each RNA after fractionation by size. During productive infection, each RNA species has a characteristic rate of appearance in the cytoplasm; 22 S and 13 SL RNA accumulate at a constant rate throughout infection. Initially 13 Sf RNA appears at a rate substantially lower than the 22 S and 13 SL, RNAs. At late times accumulation of 13 Sf RNA is stimulated more than 50-fold, until its appearance exceeds that of the other “early” RNAs. The 9 S RNAs are detected only after viral DNA replication begins. The rate of labeling of each approaches that of its colinear 13 S RNA. The results demonstrate a complex regulatory pattern for region 1 RNAs. Regulatory events must account for: (1) continued synthesis of early RNAs at late times; (2) increased accumulation of 13 Sf RNA; (3) the turn on of 9 S synthesis; and (4) balanced rates of appearance of the colinear 13 S and 9 S RNAs at late times.

Participation of two human cytomegalovirus immediate early gene regions in transcriptional activation of adenovirus promoters.

The participation of human cytomegalovirus (HCMV) immediate early genes in the activation of the expression of adenovirus genes in trans (trans-activation) was examined. The initial strategy used was to determine the ability of HCMV genes to complement mutants of adenovirus E1a, an immediate early gene which encodes a trans-activator. The HCMV immediate early gene regions IE1 and IE2 complemented E1a-deficient mutants in three separate assays. IE1 and IE2 substituted for E1a in the synthesis of infectious adenovirus, late adenovirus RNA, and adenovirus DNA. Complementation by the IE2 gene region alone, but not by IE1 alone, was observed using the most discriminating assay, that for late adenovirus RNA synthesis. A role for both HCMV gene regions in positive transcriptional control was indicated by their ability to increase expression of chloramphenicol acetyltransferase (CAT) mediated by the adenovirus E2a promoter. The IE2 region alone activated CAT synthesis but IE1 alone had no detectable activity. Moreover, the activity of both gene regions was about 10-fold higher than that of IE2 alone. These data indicate that efficient complementation of E1a-deficient mutants and trans-activation of adenovirus early promoters involved the participation of both HCMV immediate early gene regions.

Ex vivo gene transfer of endothelial nitric oxide synthase to atherosclerotic rabbit aortic rings improves relaxations to acetylcholine

Cholesterol feeding results in impaired endothelium dependent vasorelaxation. The role of nitric oxide in this process is unclear. The aim of this study was to evaluate the role of nitric oxide in cholesterol-induced vasomotor dysfunction by examining the effect of overexpression of eNOS in the hypercholesterolemic rabbit aorta on vascular reactivity. Vascular rings from the thoracic aorta of hypercholesterolemic rabbits were exposed ex vivo either to an adenoviral vector encoding endothelial nitric oxide synthase (AdeNOS) or Escherichia coli beta Galactosidase (AdbetaGal). Transgene expression was examined by histochemistry for beta galactosidase, immunohistochemistry for eNOS and cyclic GMP measurements and vasomotor studies were performed. Transgene expression was found to localize to the endothelium and adventitia. cGMP levels were significantly greater in AdeNOS compared to AdbetaGal transduced rings. Acetylcholine mediated relaxation was significantly impaired in cholesterol fed rabbits and was markedly improved by overexpression of eNOS. These results suggest that reduced NO bioavailability observed in cholesterol-induced vascular dysfunction can be partially overcome by eNOS gene transfer.

[3] Construction and isolation of recombinant adenoviruses with gene replacements

Publisher Summary This chapter discusses the construction and isolation of recombinant adenoviruses with gene replacements. Human adenovirus has become a vector of choice not only for introducing foreign genes into cell culture or animals for transient expression studies but also for the rapidly developing field of human gene therapy. The advantages of the system is that the virus is easy to manipulate and store, grows to high titers, has a broad host range, and is capable of mediating high levels of expression of the transduced gene. For gene therapy applications, additional advantages include its apparent safety and the low probability of stable integration. The most common vectors are derived from adenovirus type 5, the preferred strain for genetic analysis and manipulation. Two regions have been targeted for foreign gene replacement. The E3 region is nonessential for growth of the virus in cell culture. Therefore, E3 may be deleted and replaced with a foreign gene without compromising the infectivity of the virus, at least in vitro . Region E1 (including E1a and E1b ) is essential for virus replication. There are two standard strategies for producing adenoviruses with E1 gene replacements. In one strategy, both DNA molecules are propagated as Escherichia coli plasmids. The second strategy uses one DNA molecule that is plasmid derived and one that is derived from a viral genome. The chapter discusses the elements in a linear order for the standard arrangement for a plasmid-derived substrate for recombinant adenovirus isolation. The most important variable in recombinant virus isolation is the condition of the 293 cells, which are used to propagate adenoviruses that lack E1a and E1b genes.

Complementation of an adenovirus 5 immediate early mutant by human cytomegalovirus

The initiation of the lytic cycle of adenovirus 5 requires synthesis of a transcriptional activator encoded by the viral early genetic unit, E1a. Mutant viruses lacking E1a are defective. Human cytomegalovirus activated transcription of early adenovirus genes and complemented an E1a- adenovirus mutant in cells permissive for both viruses.

Adenovirus-Mediated Gene Transfer of Macrophage Colony Stimulating Factor to the Arterial Wall In Vivo

Macrophage colony stimulating factor (MCSF) is believed to play a key role in one of the earliest events in atherosclerosis, ie, monocyte to macrophage differentiation in the arterial intima. The aim of this study was to examine the biological effects of vascular wall expression of MCSF. A recombinant adenovirus vector encoding human MCSF (AdMCSF) was generated by standard techniques of homologous recombination in 293 cells. The rabbit carotid artery was transduced with AdMCSF. As negative controls, carotid arteries were transduced with either an adenoviral vector encoding beta-galactosidase, an adenoviral vector encoding apolipoprotein E, or diluent alone. Intima-media thickness ratio was calculated 5 and 21 days after transduction. The cell type present in intimal infiltrates was analyzed by immunohistochemistry. MCSF expression was demonstrated in the vessel wall of AdMCSF-transduced vessels by reverse transcription-polymerase chain reaction and immunofluorescence. In contrast to control vessels, adenovirus-mediated MCSF expression was associated with an intimal cellular infiltrate consisting of smooth muscle cells and small numbers of macrophages. Whereas the intima-media thickness ratio was greater in AdMCSF-transduced vessels at 5 days, this difference was no longer statistically significant at 21 days. These results suggest that MCSF may play a role in recruitment of monocytes and macrophages to the vessel wall and may contribute to smooth muscle cell proliferation and migration.

The pattern of integration of viral DNA sequences in the adenovirus 5-transformed human cell line 293

The pattern of integrated adenovirus 5 DNA in the adenovirus 5-transformed human cell line 293 was analyzed by DNA blot hybridization. In contrast to a previous report, only sequences from the left end of the genome were detected. The viral DNA was contained in a unique DNA fragment generated by cleavage of 293 DNA with either EcoRI or BamHI, two enzymes which do not cut the integrated viral DNA. Further mapping studies indicated that the integrated sequence was probably colinear with viral DNA. The joining to host cell sequences occurred between viral nucleotide base pairs 1 and 270 (a site for BalI) on one end and 4123 (SmaI site) and 5372 (BalI site) at the other end. The viral DNA was not tandemly repeated. These results suggest that integration of viral sequences into the host genome probably occurred at a single site. If any subsequent duplications of viral DNA took place, then duplication of extensive cellular sequences also must have occurred.

Identification of a human cytomegalovirus virus DNA segment that complements an adenovirus 5 immediate early mutant

Human cytomegalovirus (HCMV) complements adenovirus mutant dl312, which is completely defective for expression of the adenovirus immediate early E1a gene region, for lytic growth (Tevethia and Spector, 1984). This assay defines at least one HCMV function, activation of the transcription of adenovirus early genes in trans, that must be provided for dl312 replication in coinfected cells. We show here that trans-activation depended on the expression of one or more HCMV gene. Human embryonic lung cells were transfected with dl312 DNA-protein complex and either HCMV DNA or recombinant plasmids containing the adenovirus E1a gene region or HCMV (Towne strain) DNA fragments. Replication of dl312 occurred only in cells that received both DPC and either the E1a gene, HCMV DNA, or the XbaI-E HCMV DNA fragment (0.68 to 0.77 map units). In addition, we show that HCMV also complemented adenovirus mutant pm975 for growth in 0.2% serum. Since pm975 grows poorly in low concentrations of serum due to a defect in an E1a gene product, this assay identified a second HCMV E1a-like function.

Hybridization of herpes simplex virus dna and human ribosomal DNA and RNA

A small DNA segment from the inverted repeats at the termini of the unique long sequence region of herpes simplex virus DNA was found to hybridize with human 28 S ribosomal DNA and RNA but not 18 S ribosomal DNA and RNA. The hybridization occurred under stringent conditions and was not blocked by nucleic acids high in guanine plus cytosine content. These data strongly suggest that the hybridization represented authentic base sequence homology.