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PubMed1997

Gene transfer into enteric neurons of the rat small intestine in organ culture using a replication defective recombinant herpes simplex virus type 1 (HSV1) vector, but not recombinant adenovirus vectors.

We have designed a system in which to test gene transfer into gut neurons consisting of an organ culture of neonatal rat small intestine. The tissue was exposed to herpes simplex- and adenovirus-derived vectors: (1) a temperature-sensitive herpes simplex virus-1 (HSV1) vector (tsK-beta gal) containing the lacZ gene encoding beta-galactosidase (beta-gal), under the transcriptional control of the HSV1 immediate-early 3 (IE3) promoter; (2) RAd35, an E1-/E3- replication-deficient adenovirus expressing lacZ under the control of a truncated HCMV major IE promoter; and (3) RAd122, an E1-/E3- replication-deficient adenovirus expressing the lacZ under the control of the RSV LTR. Forty-eight hours after the vector was added to the organ culture, we detected beta-gal using immunohistochemistry or X-gal histochemistry in tissue sections examined by light microscopy. We encountered a distinctive staining of cells arranged in two concentric circles corresponding in location to the myenteric and submucosal plexuses. Cells in these areas were of similar size and morphology to neonatal enteric neurons, as visualized by NADPH-diaphorase histochemistry and immunocytochemical staining with antibodies to the neuronally expressed proteins PGP 9.5, or neurofilaments. Double labelling with antibodies recognizing neurofilaments and beta-galactosidase revealed that most cells infected by tsK were neurons, while the RAd35 and 122 vectors only infected non-neuronal cells. We thus demonstrate that both HSV1- and adenovirus-derived vectors can be used to transfer genes to the gut in vitro, but they transduce different populations of target cells.

Adenoviridae↗

PubMed2003

Adeno-associated virus (AAV) vectors achieve prolonged transgene expression in mouse myocardium and arteries in vivo: a comparative study with adenovirus vectors.

Plasmid DNA and adenovirus vectors currently used in cardiovascular gene therapy trials are limited by low efficiency and short-lived transgene expression, respectively. Recombinant adeno-associated virus (AAV) has recently emerged as an attractive vector for cardiovascular gene therapy. In the present study, we have compared AAV and adenovirus vectors with respect to gene transfer efficiency and the duration of transgene expression in mouse hearts and arteries in vivo. AAV vectors (titer: 5 x 10(8) transducing units (TU)/ml) and adenovirus vectors (1.2 x 10(10) TU/ml) expressing a green fluorescent protein (EGFP) gene were injected either intramyocardially (n=32) or intrapericardially (n=3) in CD-1 mice. Hearts were harvested at varying time intervals (3 days to 1 year) after gene delivery. After intramyocardial injection of 5 microl virus stock solution, cardiomyocyte transduction rates with AAV vectors were 4-fold lower than with adenovirus vectors (1.5% (range: 0.5-2.6%) vs. 6.2% (range: 2.7-13.7%); P<0.05), but similar to titer-matched adenovirus vectors (0.7%; range: 0.2-1.2%). AAV-mediated EGFP expression lasted for at least 1 year. AAV vectors instilled into the pericardial space transduced epicardial myocytes. Arterial gene transfer was studied in mouse carotids (n=26). Both vectors selectively transduced endothelial cells after luminal instillation. Transduction rates with AAV vectors were 8-fold lower than with adenovirus vectors (2.0% (range: 0-3.2%) vs. 16.2% (range: 8.5-20.2%); P<0.05). Prolonged EGFP expression was observed after AAV but not adenovirus-mediated gene transfer. In conclusion, AAV vectors deliver and express genes for extended periods of time in the myocardium and arterial endothelium in vivo. AAV vectors may be useful for gene therapy approaches to chronic cardiovascular diseases.

Adenoviridae↗

PubMed2000

Effective repeat administration with adenovirus vectors to the muscle.

Effective repeat administration of adenovirus vectors following intranasal or intravenous delivery is hindered by a strong neutralizing antibody response to the vector. Intramuscular administration of adenovirus vectors elicited a neutralizing antibody response that peaked between 14 and 21 days after infection. However, effective repeat intramuscular administration of adenovirus vectors was not hindered by the presence of neutralizing antibodies in the serum. Surprisingly, beta-galactosidase expression in the skeletal muscle of immunized mice was equivalent to that observed in control mice. As expected, these serum neutralizing antibodies effectively blocked repeat administration of adenovirus vectors when delivered via the intravenous route. These results were observed in both C57BL/6 and Balb/c mice and thus do not appear to be strain specific. Successful repeat administration of adenovirus vectors to skeletal muscle has significant implications for the use of adenovirus vectors clinically and for increasing the safety and efficacy of adenovirus vector gene delivery.

Adenoviridae↗

PubMed2001

Tetracycline-regulatable adenovirus vectors: pharmacologic properties and clinical potential.

Stringent control of gene expression in human gene therapy strategies is important for both therapeutic and safety reasons. Replication-defective vectors derived from adenoviruses have been shown to be capable of highly efficient in vivo gene delivery to a wide variety of dividing and nondividing human cells. Here, we review the progress in the development of regulatable adenovirus vectors that allow gene expression to be tightly controlled by low concentrations of tetracyclines. As an example of the potential clinical utility of this technology, we highlight our results obtained in an immunotherapy model for prostate cancer with a tetracycline-regulatable adenovirus vector expressing the cytokine interleukin-12. Recombinant adenovirus vectors with tetracycline-regulatable gene expression provide new opportunities and improved safety for gene therapy applications in humans.

Adenoviridae↗

PubMed1996

"Sero-switch" adenovirus-mediated in vivo gene transfer: circumvention of anti-adenovirus humoral immune defenses against repeat adenovirus vector administration by changing the adenovirus serotype.

Recombinant, replication-deficient adenovirus (Ad) vectors have been successfully used to transfer and express the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA in vivo in the respiratory epithelium of experimental animals and humans with cystic fibrosis (CF). Since Ad-directed gene expression wanes over time, repeat administration is necessary to achieve an effective treatment for CF. A major hurdle to such a strategy is the possibility that anti-Ad humoral immunity may prevent gene expression in individuals with pre-existing anti-Ad immunity or following repeat administration. One strategy to circumvent such a problem would be alternating the use of Ad vectors belonging to different subgroups. Neutralizing antibodies developed with the administration of one Ad serotype do not cross-react with an Ad belonging to a second serotype in a manner that blocks infection and gene expression. To test this hypothesis, an immunizing dose of wild-type Ad5 (subgroup C), Ad4 (subgroup E), or Ad30 (subgroup D) was administered intratracheally to experimental animals, followed by an intratracheal administration of a replication-deficient subgroup C-derived vector coding for marker genes (chloramphenicol acetyl transferase or beta-galactosidase) or for the normal human CFTR cDNA. As expected, studies with vectors coding for marker genes or for CFTR cDNA demonstrated that airway administration of a vector does not yield efficient gene transfer, if there has been prior recent airway administration of the same Ad subgroup. In contrast, effective expression from the second administration can be achieved with an adenovirus vector belonging to a subgroup different from the first adenovirus administered. These data support the paradigm of alternating Ad vectors derived from different subgroups as strategy to circumvent anti-Ad humoral immunity, thus permitting the use of Ad vectors as a means to treat the respiratory manifestations of CF.

Adenoviruses, Human↗

PubMed2001

Biology of E1-deleted adenovirus vectors in nonhuman primate muscle.

Adenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors.

Adenoviridae↗

PubMed2001

A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus.

Fully deleted adenovirus vectors (FD-AdVs) would appear to be promising tools for gene therapy. Since these vectors are deleted of all adenoviral genes, they require a helper adenovirus for their propagation. The contamination of the vector preparation by the helper limits the utility of currently existing FD-AdVs in gene therapy applications. We have developed an alternative system for the propagation of FD-AdVs, in which the adenoviral genes essential for replication and packaging of the vector are delivered into producer cells by a baculovirus-adenovirus hybrid. A hybrid baculovirus Bac-B4 was constructed to carry a Cre recombinase-excisable copy of the packaging-deficient adenovirus genome. Although the total size of the DNA insert in Bac-B4 was 38 kb, the genetic structure of this recombinant baculovirus was stable. Bac-B4 gave high yields in Sf9 insect cells, with titers of 5 x 10(8)p.f.u./ml before concentration. Transfection of 293-Cre cells with lacZ-expressing FD-AdV plasmid DNA followed by infection by Bac-B4 at a MOI of 2000 p.f.u./ml resulted in rescue of the helper-free vector. Subsequent passaging of the obtained FD-AdV using Bac-B4 as a helper resulted in approximately 100-fold increases of the vector titer at each passage. This resulting vector was completely free of helper virus and was able to transduce cultured 293 cells. However, scaling-up of FD-AdV production was prevented by the eventual emergence of replication-competent adenovirus (RCA). Experiments are underway to optimize this system for the large-scale production of helper virus-free FD-AdVs and to minimize the possibility of generation of replication-competent adenovirus (RCA) during vector production. This baculovirus-based system will be a very useful alternative to current methods for the production of FD-AdVs.

Adenoviridae↗

PubMed2006

[Construction of a recombinant adenovirus vector for interferon-gamma-inducible protein 10 and the adenovirus preparation].

OBJECTIVE: To construct a recombinant adenovirus vector for expressing interferon-gamma-inducible protein 10 (IP-10) by homogenous bacterial recombination. METHODS: IP-10 gene was cloned into the shuttle plasmid pAdTrack-CMV that contained the coding sequence of enhanced green fluorescent protein (EGFP). The shuttle plasmid was then transformed into E. coli BJ5183 with pAdEasy-1 vector by chemical transformation. The recombinant adenovirus vector pAd/IP-10 was identified by enzyme digestion with Pac I and the linearized plasmid was transfected into HEK293 cells. RESULTS: The positive clones were identified with enzyme digestion and polymerase chain reaction (PCR) and were further verified by DNA sequencing. The recombinant adenovirus of high titration was obtained after transfection and packaging in HEK293 cells. CONCLUSION: A recombinant adenovirus vector for expression of IP-10 has been constructed successfully and high-titer active adenovirus is obtained for functional study of IP-10 protein.

Adenoviridae↗

PubMed2000

Production of first generation adenovirus vectors: a review.

In the past decade, adenovirus vectors have generated tremendous interest, especially in gene therapy applications. In the so-called 'first generation' adenovirus vectors, the transgenes are inserted in place of the E1 region, or less often the E3 region. Although second-generation and helper-dependent adenovirus vectors will probably prevail in the future in applications that require long-term gene expression, first generation adenovirus vectors will remain very useful in other settings, such as cancer and vaccination, or simply to transfect cell lines that are refractory to other transfection methods. Until a few years ago, the construction of first generation adenovirus vectors was a labor-intensive and time-consuming process. More than 20 methods have appeared that facilitate their construction and are reviewed below.

Adenoviridae↗

PubMed2006

High-efficiency system for the construction of adenovirus vectors and its application to the generation of representative adenovirus-based cDNA expression libraries.

We here describe a convenient system for the production of recombinant adenovirus vectors and its use for the construction of a representative adenovirus-based cDNA expression library. The system is based on direct site-specific insertion of transgene cassettes into a replicating donor virus. The transgene is inserted into a donor plasmid containing the viral 5' inverted terminal repeat, the complete viral packaging signal, and a single loxP site. The plasmid is then transfected into a Cre recombinase-expressing packaging cell line that has been infected with a donor virus containing a partially deleted packaging signal flanked by loxP sites. Cre recombinase, by two steps of action, sequentially catalyzes the generation of a nonpackageable donor virus acceptor substrate and the generation of the desired recombinant adenovirus vector. Due to its growth impairment, residual donor virus can efficiently be counterselected during amplification of the recombinant adenovirus vector. By using this adenovirus construction system, a plasmid-based human liver cDNA library was converted by a single step into an adenovirus-based cDNA expression library with about 10(6) independent adenovirus clones. The high-titer purified library was shown to contain about 44% of full-length cDNAs with an average insert size of 1.3 kb. cDNAs of a gene expressed at a high level (human alpha(1)-antitrypsin) and a gene expressed at a relatively low level (human coagulation factor IX) in human liver were isolated from the adenovirus-based library using an enzyme-linked immunosorbent assay-based screening procedure.

Adenoviridae↗

PubMed1988

Expression of HBV surface antigen or HIV envelope protein using recombinant adenovirus vectors.

Recombinant adenoviruses were constructed that contained either the HBsAg coding sequence or the HIV envelope protein coding sequence. The recombinant adenoviruses can replicate normally in cultured human cells. Cells infected with the adenovirus-HBV recombinant secreted HBsAg into the tissue culture medium. This HBsAg had immunological and physical properties similar to those of the 22-nm particles found in human serum. Expression of HIV envelope protein in cells infected with the adenovirus-HIV recombinant was demonstrated using cytoimmunofluorescence and immunoprecipitation. A hamster model was developed to evaluate the immunogenic properties of adenovirus-HBV recombinants. Hamsters inoculated intranasally with live adenovirus-HBV recombinant produced antibody against both adenovirus and hepatitis B virus surface antigen.

Adenoviridae↗

PubMed1996

Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.

Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies. However, lack of standardization of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible. This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance. The methods for evaluation of the concentration of functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells. Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability. These observations may be very useful in comparison of data from different preclinical and clinical studies and may also have important implications for how adenovirus vectors can optimally be used in human gene therapy.

Adenoviridae↗

PubMed1996

Immunological instability of persistent adenovirus vectors in the brain: peripheral exposure to vector leads to renewed inflammation, reduced gene expression, and demyelination.

Nonreplicating adenovirus vectors are being developed as vehicles for the delivery of therapeutic genes in vivo. Whereas in many organs an antiviral T cell response eliminates the vector and damages local tissue, when adenovirus vectors are injected into the brain the subsequent immune attack can be ineffective, allowing the vector to persist. In the present study, E1-deleted human adenovirus vectors were injected into the caudate nucleus of rats. Two months later, expression of protein from the vector was still evident and little inflammation was seen. A subcutaneous injection of adenovirus vector at this time, however, led within 2 weeks to severe mononuclear inflammation and microglial activation in the caudate. This caused local demyelination and a decrease in detectable protein expression from the vector. Interestingly, intense microglial activation and numerous lymphocytes and monocytes were also seen in brain areas containing neurons capable of retrogradely transporting the adenovirus vector from the caudate. Control experiments established that this inflammation in distant brain areas was not a nonspecific consequence of degeneration. These experiments demonstrate that although adenovirus vectors can persist in the brain without causing chronic inflammation, they remain the potential target of a damaging cell-mediated immune response brought about by a subsequent peripheral exposure to vector. The finding of lymphocytes in brain areas that project to the caudate further shows that viral antigens that are retrogradely transported by neurons can also be the target of a T cell attack.

Adenoviridae↗

PubMed1999

Frequency and stability of chromosomal integration of adenovirus vectors.

One of the limitations of adenovirus vectors is the lack of machinery necessary for their integration into host chromosomes, resulting in short-term gene expression in dividing cells. We analyzed frequencies of integration and persistence of gene expression from integrated adenovirus vectors. Both E1-substituted and helper-dependent adenovirus vectors achieved similar integration efficiencies of approximately 10(-3) to 10(-5) per cell, with the helper-dependent vector showing slightly higher efficiencies. In stable cell pools, gene expression of the integrated vector persisted for at least 50 cell divisions without selection. However, some stable cell clones showed changes in gene expression, which were accompanied by structural changes in the integrated vector DNA.

3T3 Cells↗

PubMed2005

Effective repetitive dystrophin gene transfer into skeletal muscle of adult mdx mice using a helper-dependent adenovirus vector expressing the coxsackievirus and adenovirus receptor (CAR) and dystrophin.

BACKGROUND: The helper-dependent adenovirus (HDAd) vector is less immunogenic and has a larger cloning capacity of up to 37 kb enough to carry the full-length dystrophin cDNA. However, high and long-term expression of dystrophin transduced to mature muscle still remains difficult. One of the main reasons for this is that the expression of the coxsackievirus and adenovirus receptor (CAR) is very low in mature muscle. METHODS: We have constructed two different HDAd vectors. One contains the LacZ and the murine full-length dystrophin expression cassette (HDAdLacZ-dys), and the other is a new, improved vector containing the CAR and the dystrophin expression cassette (HDAdCAR-dys). RESULTS: We initially demonstrated high dystrophin expression and prevention of the dystrophic pathology in mdx muscle injected during the neonatal phase with HDAdLacZ-dys. Furthermore, we demonstrated that repeated injections of HDAdCAR-dys into mature muscle led to approximately nine times greater dystrophin-positive fibers in number than a single injection, thereby recovering the expression of dystrophin-associated proteins. This data has also shown that HDAdCAR-dys enabled administration of adenovirus (Ad) vector to the host with pre-existing immunity to the same serotype of Ad. CONCLUSIONS: Repetitive injections of the HDAd vector containing the CAR and the dystrophin expression cassette could improve the efficiency of subsequent dystrophin gene transfer to mature mdx muscle. This result suggests that our new HDAd vector will provide a novel gene therapy strategy for Duchenne muscular dystrophy, raising the prospects for gene therapy of other hereditary myopathies.

Adenoviridae↗

PubMed2000

Local adenovirus-mediated CTLA4-immunoglobulin expression suppresses the immune responses to adenovirus vectors in the brain.

The effect of local administration of two adenovirus vectors, one of which expressed CTLA4-immunoglobulin (AdCTLA), which blocks the B7-CD28 co-stimulatory pathway of T cell activation in the inflammatory response to adenovirus vectors was investigated. Mice injected with AdCTLA and an E1-deleted adenovirus vector that encodes the lacZ gene (AdRL) into the brain showed inflammatory cell infiltration from the early phase until day 6 after injection that was not different from that seen in control mice injected with an E1-deleted adenovirus vector containing no transgene (Ad0) and AdRL. After day 6 the inflammation in the control mice increased, peaked by day 15 and then decreased gradually but persisted until day 60. By contrast, in mice treated with AdCTLA and AdRL the inflammation, especially T cell infiltration, was suppressed after day 15. The anti-adenovirus antibody titer increased gradually until day 60 in the Ad0-AdRL control group, and whereas the mice injected with AdCTLA and AdRL showed lower anti-adenovirus antibody titers than the control group mice after day 15. Neutralizing antibody was not detected in either group. Expression of beta-galactosidase, the gene product of AdRL, at the injection site in the striatum and corpus callosum peaked on day 6 and remained until day 60 although it was very low in both groups; beta-galactosidase expression was similar in the two groups in spite of the difference in the degree and extent of the local immune response in the brain. This study demonstrated that the injection of an adenovirus vector expressing CTLA4-immunoglobulin into the brain suppressed not only local cell infiltration in the brain but also reduced the humoral immune response to adenovirus vectors.

Abatacept↗

PubMed2004

Unexpected pulmonary uptake of adenovirus vectors in animals with chronic liver disease.

When adenovirus vectors are injected intravenously, most of the virions are quickly taken up by the reticuloendothelial system, primarily by the liver macrophages known as Kupffer cells. However, little is known about the behavior of adenovirus vectors when there is pre-existing liver disease. To study this, we examined the biodistribution of intravenously injected vector in a rat model of cirrhosis induced by bile duct ligation. Using quantitative PCR and fluorescently tagged adenovirus vectors, we observed a significant reduction in vector uptake by the cirrhotic liver and increased accumulation in the lungs. Immunocytochemistry and electron microscopy demonstrated that this was due to changes in the reticuloendothelial system, with the vector being taken up by large numbers of pulmonary intravascular macrophages in the lungs of cirrhotic rats. Interestingly, expression of vector-encoded luciferase was significantly reduced in the livers of cirrhotic rats, but was not increased in the lungs. These data demonstrate that the biodistribution of adenovirus vectors in rats is altered by cirrhosis, which suggests the possibility that these vectors might behave unexpectedly in patients with pre-existing liver conditions, particularly since pulmonary reticuloendothelial changes are known to occur in human disease.

Adenoviridae↗

PubMed1996

Adenovirus vector infection of chronic lymphocytic leukemia B cells.

Adenovirus vectors have several features that make them attractive for potential use in gene therapy, including a broad tissue tropism and an ability to infect quiescent or postmitotic cells. In light of this, we examined whether recombinant adenovirus vectors could transfer genes into neoplastic cells of patients with chronic lymphocytic leukemia (CLL), a leukemia of "resting" B cells. Using high-titer recombinant adenovirus vectors, we found we could transfer genes encoding beta-galactosidase or murine CD80 (B7-1) into the CLL B cells of all patients tested (n = 10). The efficiency of gene transduction into CLL B cells was approximately 100 to 1,000-fold lower than into HeLa cells at any given multiplicity of infection (MOI). At a MOI of 500, 10% to 70% of the CLL B cells from different patients were made to express the transgene, as assessed by multiparameter flow cytometric analysis. Sustained levels of expression with little loss in the percentage of infected cells were maintained for up to 9 days, at which point the analysis was stopped. We found that CLL B cells have markedly lower expression levels of integrins that facilitate internalization of adenovirus particles into target cells, perhaps accounting, in part, for the reduced efficiency of adenovirus-mediated gene transfer compared with that in HeLa cells. Although HeLa cells express high levels of alpha(v)beta5, and detectable amounts of alpha(v)beta3, we find CLL cells from all patients tested express only low amounts of alpha(v)beta3, and no detectable alpha(v)beta5. Activation of CLL cells via CD40 cross-linking enhances expression of alpha(v)beta3, and induces expression of alpha(v)beta5. This phenotypic change is associated with a fivefold increase in the efficiency of adenovirus-mediated gene transfer into such activated CLL B cells. This study demonstrates that adenovirus vectors can transduce genes into CLL B cells and that the efficiency of gene transduction is enhanced by activation via CD40 cross-linking. This is the first demonstration that high proportions of CLL B cells can be made to express a selected transgene, suggesting that such gene transfer methods may become useful for the study of the pathogenesis and/or treatment of this disease.

Adenoviridae↗