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Biomedical subjects

F L Graham

Publications and source records attributed to F L Graham.

At least 91 records · Page 5Linked to original sources
PubMed1996

Human adenovirus type 5 vectors expressing rabies glycoprotein.

The prevalence of wildlife rabies throughout the world and the continued spread of this disease in North America highlights the need for oral vaccines which may be used safely and effectively to vaccinate a number of species that are reservoirs or vectors of rabies. We have previously shown that AdRG1, a replication competent recombinant human adenovirus type 5 (Ad5) expressing a rabies glycoprotein (RG), can induce immunity to rabies in rodent, canine, and skunk model systems. To improve the Ad5 vector system as a potential oral vaccine, we have constructed additional Ad5 recombinant vectors and compared RG expression in cell culture and immunogenicity in animals. Two new replication competent vectors are compared. AdRG1.3, which carries RG with accompanying SV40 poly A addition sequences within an E3 deletion, and AdRG4, which has RG in the E3 deletion but under the control of an exogenous Ad2 major late promoter, both express higher levels of RG in permissive cell culture than did AdRG1 and both elicit high levels of serum anti-rabies antibodies by parenteral or oral routes in animals. AdRG1.3 may be a more effective vaccine vector in species which are non-permissive for the replication of human Ad5.

Adenoviruses, Human↗
PubMed1996

Pathology and immunogenicity in the cotton rat (Sigmodon hispidus) model after infection with a bovine adenovirus type 3 recombinant virus expressing the firefly luciferase gene.

The histopathology of adenovirus pneumonia in cotton rats (Sigmodon hispidus) due to bovine adenovirus type 3-luciferase recombinant virus (BAd3-Luc), which has a 0.7 kb deletion from the early region 3 (E3) replaced with the firefly luciferase gene, was compared with that produced by the parental wild-type (wt) bovine adenovirus type 3 (BAd3). After intranasal inoculation of cotton rats with 3 x 10(7) p.f.u. of BAd3-Luc, the infectious virus titres in the lungs at various times post-infection were similar to those of animals infected with the parental virus. Quantitative analysis of histopathological changes and immunohistochemical staining showed that the character and severity of the lesions were indistinguishable in the two infections. Luciferase activity was detected in the lungs of BAd3-Luc-inoculated animals until 4 days post-infection (p.i.). Antibodies to both BAd3 and luciferase were detected in sera collected from BAd3-Luc-infected animals until at least 6 weeks p.i. These results show that Bad3-Luc produces pulmonary lesions in cotton rats similar to those of wt BAd3 and suggest that BAd3-based vectors may be suitable for the development of live recombinant virus vaccines.

Adenoviridae Infections↗
PubMed1995

Intratumoral injection of an adenovirus expressing interleukin 2 induces regression and immunity in a murine breast cancer model.

Rodent tumor cells engineered to secrete cytokines such as interleukin 2 (IL-2) or IL-4 are rejected by syngeneic recipients due to an enhanced antitumor host immune response. An adenovirus vector (AdCAIL-2) containing the human IL-2 gene has been constructed and shown to direct secretion of high levels of human IL-2 in infected tumor cells. AdCAIL-2 induces regression of tumors in a transgenic mouse model of mammary adenocarcinoma following intratumoral injection. Elimination of existing tumors in this way results in immunity against a second challenge with tumor cells. These findings suggest that adenovirus vectors expressing cytokines may form the basis for highly effective immunotherapies of human cancers.

Adenocarcinoma↗
PubMed1995

Identification of adenovirus E1A regions which affect MHC class I expression and susceptibility to cytotoxic T lymphocytes.

To map and characterize functional differences between the E1A oncoproteins of Ad5 and Ad12, we previously constructed a series of hybrid Ad5/12 E1A genes and used them in combination with Ad12 E1B to transform Hooded Lister rat cells. At least two regions within the first exon of Ad12 E1A which influenced tumorigenicity were identified. In this report, again using the hybrid Ad5/12 E1A (plus Ad12 E1B) transformants, we further examined the role of these regions in tumorigenicity by analyzing their effect on cell surface major histocompatibility complex class I expression and sensitivity to class I-restricted CD8+ cytotoxic T lymphocytes (CTLs). Results of these studies suggest that expression of either of the Ad12 E1A regions implicated in tumorigenicity could down-regulate cell surface class I levels. However, neither class I down-regulation nor sensitivity to allogeneic CTLs was shown to strictly correlate with the tumorigenic capacities of the transformed rat cells. Another factor influencing the tumorigenicity of Ad5 E1 and Ad12 E1 transformants may be the ability of their E1A products to encode CTL epitopes. To this end, we provide evidence suggesting that CTL epitopes may be encoded by Ad5 E1A but not by Ad12 E1A, since expression of certain portions of the Ad5 E1A protein conferred susceptibility to syngeneic Ad5 E1-specific CTLs in vitro, while Ad12 E1A expression did not confer susceptibility to syngeneic Ad12 E1-specific CTLs.

Adenovirus E1A Proteins↗
PubMed1995

Foreign gene expression by human adenovirus type 5-based vectors studied using firefly luciferase and bacterial beta-galactosidase genes as reporters.

Adenovirus (Ad) vectors have been used extensively to obtain high-level expression of foreign genes in mammalian cells and are currently being studied for use as live viral-vectored vaccines and as gene transfer vectors for gene therapy. Many Ad recombinants have been generated that express foreign genes inserted in early region 3 (E3); however, little has been done to study the importance for gene expression of regulatory sequences flanking the gene. We have generated a series of Ad5 helper-independent vectors that contain the firefly luciferase gene or the bacterial beta-galactosidase gene (LacZ) with or without simian virus 40 (SV40) regulatory sequences, combined with E3 deletions of 1.88 or 2.69 kb. The greatest levels of luciferase expression were obtained with a vector containing the luciferase gene under the control of the SV40 promoter and polyadenylation signal inserted in a 1.88-kb E3 deletion. In contrast, LacZ expression was highest with a vector containing the LacZ gene with just the SV40 polyadenylation sequence combined with a 1.88-kb E3 deletion. It was also observed that regardless of the SV40 sequences flanking the reporter gene or the E3 deletion used, expression from the luciferase recombinants was dependent on viral DNA replication, whereas expression from the LacZ recombinants was only partially reduced when DNA replication was blocked. Analyses of RNA by dot blot hybridizations revealed that the levels of reporter gene-specific mRNA for various vectors in each series did not vary significantly. These results indicate that the kinetics and efficiency of expression of genes inserted into the E3 region, in nonconditional helper-independent vectors, may be more strongly dependent on the sequences in the foreign gene insert itself than on flanking regulatory sequences such as those used here, derived from SV40.

Adenoviruses, Human↗
PubMed1995

Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.

Adenoviral vectors are widely used as highly efficient gene transfer vehicles in a variety of biological research strategies including human gene therapy. One of the limitations of the currently available adenoviral vector system is the presence of the majority of the viral genome in the vector, resulting in leaky expression of viral genes particularly at high multiplicity of infection and limited cloning capacity of exogenous sequences. As a first step to overcome this problem, we attempted to rescue a defective human adenovirus serotype 5 DNA, which had an essential region of the viral genome (L1, L2, VAI + II, pTP) deleted and replaced with an indicator gene. In the presence of wild-type adenovirus as a helper, this DNA was packaged and propagated as transducing viral particles. After several rounds of amplification, the titer of the recombinant virus reached at least 4 x 10(6) transducing particles per ml. The recombinant virus could be partially purified from the helper virus by CsCl equilibrium density-gradient centrifugation. The structure of the recombinant virus around the marker gene remained intact after serial propagation, while the pBR sequence inserted in the E1 region was deleted from the recombinant virus. Our results suggest that it should be possible to develop a helper-dependent adenoviral vector, which does not encode any viral proteins, as an alternative to the currently available adenoviral vector systems.

Adenoviruses, Human↗
PubMed1995

Gene targeting in mouse embryonic stem cells with an adenoviral vector.

We examined the ability of an E1, E3-defective adenoviral vector to act as a substrate for homologous recombination with chromosomal DNA by including host chromosomal sequence from the mouse Fgr locus that also contained a selectable marker. After infection of mouse embryonic stem cells, stable integration was selected for neomycin resistance and the efficiency of homologous recombination was evaluated. The adenoviral vector was capable of infecting mouse embryonic stem cells efficiently. Between 30-50% of the input virus reached the nuclei after 24 hours of infection. Surprisingly, even without negative selection, 25-40% of the integration resulted from homologous recombination at m.o.i. 10 and 100, although the absolute efficiency of integration was low. Our results suggest that it is possible to modify the structure of an adenoviral vector to achieve a high gene targeting efficiency, resulting in regulated and long-term expression of an introduced gene.

Adenoviridae↗
PubMed1995

High frequency recombination between loxP sites in human chromosomes mediated by an adenovirus vector expressing Cre recombinase.

An adenovirus vector (AdCre1) expressing Cre recombinase has been used to induce recombination between loxP sites in human chromosomes. G418 resistant cells with one loxP site, generated by transfection with a plasmid containing loXp between the SV40 promoter and the G418 resistance (neo) gene, were infected with AdCre1 and transfected with a plasmid containing loxP adjacent to a promoterless hisD gene. This resulted in integration of hisD downstream of the SV40 promoter with gain of histidinol and loss of G418 resistance. Since AdCre1 is non-replicating and Cre expression transient, histidinol resistant cells containing the hisD gene flanked by loxP sites were stable. Reinfection of these cells with AdCre1 induced excision of hisD in over 90% of infected cells. This high efficiency of site-specific recombination suggests that AdCre1 may be exploited for temporal and tissue-specific regulation of gene expression and for chromosome engineering in vitro and in animals.

Adenoviridae↗
PubMed1995

Methods for construction of adenovirus vectors.

Adenoviruses are attracting increasing attention as general purpose mammalian cell expression vectors, as recombinant vaccines, and potentially as vectors for gene therapy. Not only is the adenovirus genome relatively easy to manipulate by recombinant DNA techniques, but adenovirus vectors are relatively stable, grow to high titers, and can transduce a variety of cell types in cell culture and in vivo. Vectors can be designed that are either replication competent or replication defective and, in the latter case, are highly efficient at delivering and expressing genes in mammalian cells without resulting in cell killing. Methods are described for growing, titrating, and purifying adenoviruses, for extracting viral DNA from purified virions and from infected cells, for rescuing inserts of foreign DNA into the viral genome, and for assessing expression of inserted genes in adenovirus vectors.

Adenoviridae↗
PubMed1995

The use of adenoviral vectors for gene therapy and gene transfer in vivo.

Adenoviral vectors have proven to be excellent vehicles for gene delivery in vivo to a wide range of cell types. These vectors have been used to transfer genes such as CFTR to correct the defect in cystic fibrosis and, more recently, to supply serum blood factors and genetically modify tumors to enhance therapy.

Adenoviridae↗
PubMed1995

Recombinant truncated dystrophin minigenes: construction, expression, and adenoviral delivery.

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder for which there is currently no effective treatment. Although clinical application of adenoviral vector-mediated gene transfer has not been fully developed, it shows promise for the treatment of DMD. One significant problem posed by adenoviral vector-mediated gene transfer for DMD is that currently available adenoviral vectors cannot accommodate the entire 14-kb dystrophin cDNA. To address this problem, we selectively deleted regions of the murine dystrophin cDNA to produce truncated constructs. We created three constructs, each with an in-frame deletion of a segment (3.0, 4.4, and 5.7 kb) of the spectrin-like repeat region of dystrophin. As an additional modification, we removed the majority of the 3' untranslated region of the cDNA in expression vectors encoding some of these truncated constructs. Comparative quantitative expression studies after transfection into COS and C2C12 mouse muscle cells demonstrate variations in the level of expression with different deletions in the spectrin-like repeat region. Furthermore, deletion of the 3' untranslated region was tested for one recombinant construct and resulted in a reduction in the level of expression in both cell culture systems. Toward the ultimate goal of gene transfer therapy for DMD, we created an adenoviral vector from one of our truncated constructs. Using this vector, we demonstrated truncated dystrophin expression in vitro in primary mdx (dystrophin-deficient) muscle cells and in vivo in mdx mouse muscle. In vivo, recombinant dystrophin was properly localized to the muscle membrane.

Adenoviridae↗
PubMed1995

Development of cell lines capable of complementing E1, E4, and protein IX defective adenovirus type 5 mutants.

The cloning capacity of currently available E1- and E3-deleted adenovirus (Ad) vectors does not exceed 8 kb. To increase capacity and improve vector safety further, we have explored the possibility that Early Region 4 (E4) and the gene encoding protein IX (pIX) might also be deleted. To generate cell lines expressing sufficient levels of E4 and pIX proteins in trans in addition to E1-encoded proteins to complement mutations in these genes, we transformed 293 cells with constructs containing the E4 transcription unit and pIX coding sequences under the control of inducible mouse mammary tumor virus (MMTV) and metallothionein promoters, respectively. We obtained two lines, VK2-20 and VK10-9, that express both E4 and pIX proteins as well as E1. The lines could be efficiently transfected with DNA, and allowed the rescue and propagation of an adenovirus; recombinant, Ad5dlE3,4, containing a 2.7-kb E3 deletion and a 2.8-kb E4 deletion in addition to an insertion of plasmid DNA sequences in E1A. Because the E4 sequences within VK2-20 and VK10-9 cells do not overlap with the DNA sequence of Ad5dlE3,E4, the probability of regeneration of the wild-type E4 during virus propagation should be very low. Using the cell lines described in this study, it should be possible to generate Ad vectors lacking E1, pIX, E3, and E4. This would not only increase capacity over that of currently available vectors (to approximately 11 kb) but would also result in more severely attenuated vectors than those with deletions only of E1 or of E1 and E3 and, hence, safer for use in gene therapy protocols.

Adenovirus E1 Proteins↗
PubMed1995

Development of a bovine adenovirus type 3-based expression vector.

We constructed a non-defective bovine adenovirus type 3 recombinant (BAd3-Luc) containing the firefly luciferase gene inserted in the early region 3 (E3) of the BAd3 genome. Deletion of a 696 bp XhoI-NcoI E3 segment and insertion of the luciferase gene in E3 was confirmed by Southern blot analyses. Luciferase was expressed in Madin-Darby bovine kidney cells infected with BAd3-Luc as measured by enzymic assays and Western blotting. Analyses of luciferase expression in the presence or absence of 1-beta-D-arabinofuranosylcytosine indicated that approximately 70-75% of luciferase expression was dependent on viral DNA replication, suggesting that transcription of the gene was at least partially under the control of a late promoter. Although yields of infectious virus for BAd3-Luc were approximately 10-fold lower than for wild-type virus, replication of the vector was still relatively efficient. In a Western blot experiment, anti-luciferase antibody reacted with a 62 kDa protein which is of the same molecular mass as the purified firefly luciferase polypeptide. Luciferase was also expressed in the 293 cell line infected with BAd3-Luc for at least 6 days post-infection as monitored by luciferase assays. Based on these observations we suggest that BAd-based expression vectors should have excellent potential for the development of recombinant vaccines for cattle and may also be suitable as vectors for gene transfer into human cells.

Adenoviridae↗
PubMed1995

Investigation of promoter function in human and animal cells infected with human recombinant adenovirus expressing rotavirus antigen VP7sc.

Human adenovirus (Ad) vectors are being used increasingly for a variety of applications in vaccination and gene therapy. The ability of vectors to enter cells and the efficiency of promoters expressing the therapeutic gene or vaccine antigen are critical to the outcome of such experiments. To identify promoters which might be suitable for use under a variety of conditions we have investigated the expression of a rotavirus antigen, VP7sc, employing several commonly used promoters carried in E1-substituted Ad vectors both in cell types which support virus replication and in cells which do not. Although not all gene constructions were identical, wide variations in promoter function were evident even in human 293 cells which support virus replication. The simian virus type 40 (SV40) early and beta-actin promoters expressed poorly; the SV40 late promoter was somewhat better. The human IE94 cytomegalovirus (CMV) promoter and a modified Ad major late promoter were best, functioning equally well but with different kinetics. In other human cell lines the CMV promoter was more versatile, generally providing sustained expression at a significant level, in one case for at least 6 days. In addition, as mouse, rabbit and pig models of rotavirus infection are under investigation and VP7sc is a vaccine antigen, we also investigated the ability of the recombinant adenovirus to infect cells from these and other sources. VP7sc expression was detected in several heterologous cell types, illustrating the ubiquity of the human Ad receptor and the versatility of human Ad as vectors when suitable promoters are used.

Actins↗
PubMed1995

Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: a molecular switch for control of gene expression.

We have constructed replication-defective human adenovirus (Ad) type 5 vectors containing the gene for the Cre recombinase from bacteriophage P1 under control of the human cytomegalovirus immediate-early promoter (AdCre). Expression of the protein was detected in replication-permissive (293) and in nonpermissive (MRC5) cell lines, and its biochemical activity was demonstrated in a cell-free recombination assay using a plasmid containing two loxP sites. To study Cre-mediated recombination in an intracellular system, we constructed an Ad vector (AdMA19) containing the luciferase cDNA under control of the human cytomegalovirus promoter but separated from it by an extraneous spacer sequence flanked by loxP sites which blocked luciferase expression. Upon coinfection of 293 or MRC5 cells with AdMA19 and AdCre, luciferase expression was specifically induced by Cre-mediated excision of the intervening sequence. The use of Ad vectors combined with the Cre-loxP system for regulation of gene expression and other possible applications is discussed.

Adenoviruses, Human↗
PubMed1995

Expression of a mutation causing hypertrophic cardiomyopathy disrupts sarcomere assembly in adult feline cardiac myocytes.

Mutations in the beta-myosin heavy chain (beta MyHC) induce hypertrophic cardiomyopathy (HCM), cardiac hypertrophy, and sarcomere disarray, with the latter being the characteristic hallmark. Thus, we sought to determine whether expression of mutant beta MyHC in adult feline cardiac myocytes, a species known to develop HCM with a phenotype identical to that in humans, induces sarcomere disarray. A full-length beta MyHC cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg403Gln) was induced in the beta MyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant beta MyHC cDNAs were cloned into p delta E1spIB shuttle vector, downstream from a cytomegalovirus (CMV) promoter. Replication-deficient recombinant adenoviral constructs (Ad5/CMV/beta MyHC-N and Ad5/CMV/beta MyHC-403) were generated through homologous recombination of p delta E1spIB/CMV/beta MyHC-N or Ad5/CMV/beta MyHC-403 and pBHG10 after cotransfection in 293 host cells. Infection of COS-1 cells with the beta MyHC construct resulted in the expression of a full-length myosin protein. Efficiency of infection of isolated adult cardiac myocytes was > 95%. Expression of the beta MyHC constructs into mRNA at 48 hours after infection of feline cardiac myocytes was confirmed by reverse transcription-PCR. The net total protein and beta-myosin synthesis were determined by using the amount of incorporation of [3H]phenylalanine into total protein and beta-myosin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviridae↗
PubMed1995

DNA sequence of the deletion/insertion in early region 3 of Ad5 dl309.

dl309 is an adenovirus type 5 (Ad5) mutant that has been extensively utilized for construction of Ad5 mutants in early region 1 (E1), in developing vectors for use as viral vaccines, and in development of gene transfer vectors for gene therapy. Ad5 dl309 has been useful for vector construction because of its altered XbaI restriction pattern and lends itself to a variety of strategies for rescuing inserts or mutations into E1. It contains only one XbaI site at 3.7 map units (m.u.) as compared to wt Ad5 which contains 4(3.7, 29.5, 79.5, and 84.8 m.u.). The loss of the sites at 29.5 and 79.5 m.u. is due to deletions of a few bp but the loss of the site at 84.8 m.u. was the result of a deletion from approximately 83 to 85 m.u. and substitution with a fragment of foreign DNA. Because of the widespread use of dl309 and derivatives of this mutant in the construction of Ad5-based vectors and the need to have precise genetic information on the sequences present in vectors to be used as vaccines and in gene therapy, we have sequenced the alterations in dl309 which affect the XbaI sites at 79.5 and 84.8 m.u. and have determined which E3 proteins are expressed by this virus. The deletion that removes the XbaI site at 84.8 m.u. extends from Ad5 bp 30005-30750 and is substituted with 642-bp of heterologous DNA that shows homology to salmon DNA. This alteration deletes all or part of the coding sequences for the E3 14.7K, 14.5K and 10.4K proteins and these proteins are not detected in dl309 infected cells. The loss of the XbaI site at 79.5 m.u. is the result of a 6-bp deletion which removes two internal amino acids (18 and 19) from the E3 6.7K protein. The E3 6.7K protein and other E3 proteins whose coding sequences are unaffected by the alterations in dl309 (gp19K, 12.5K and 11.6K) were expressed in dl309 infected cells.

Adenoviridae↗