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PubMed1999

Stable alphavirus packaging cell lines for Sindbis virus and Semliki Forest virus-derived vectors.

Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alphavirus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly inducible and was detected only after synthesis of an authentic subgenomic mRNA by the vector-encoded replicase proteins. Efficient production of biologically active vector particles occurred after introduction of Sindbis virus vectors into the PCLs. In one configuration, the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in vector packaging levels of 10(7) infectious units/ml, but reducing the generation of contaminating replication-competent virus below the limit of detection. Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replication-competent virus, suggesting utility for production of large-scale vector preparations. Furthermore, both Sindbis virus-based and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirus-derived vectors.

Alphavirus↗
PubMed2004

Conditionally replication-competent adenoviral vectors with enhanced infectivity for use in gene therapy of melanoma.

To generate vector Ad.Tyr-E1A, which is cytolytic for tyrosinase-positive melanoma cells, we replaced the adenoviral E1A promoter with a human tyrosinase enhancer/promoter. To overcome the low transduction efficiency in populations of melanoma cells that exhibit a low level of the coxsackievirus-adenovirus receptor (CAR), we inserted an RGD-4C peptide into the HI loop of the fiber knob domain of the Ad.Tyr-E1A vector. The resulting vector was named Ad.Tyr-E1A(RGD). As a result of these changes, the transduction efficiency of the RGD-modified vector was increased both in vitro and in vivo. Western blot analysis proved that infection of cells with the Ad.Tyr-E1A(RGD) vector led to expression of the E1A gene selectively in tyrosinase-positive melanoma cell lines, but not in tyrosinase-negative cell lines. The Ad.Tyr-E1A(RGD) vector was as potent in its cytotoxic effect as a tumor nonselective vector (Ad.CMV-E1A) in tyrosinase-positive melanoma cell lines. The Ad.Tyr-E1A(RGD) vector produced a higher vector particle yield in tumor cells than did the Ad.Tyr-E1A vector. Intratumoral injection of the Ad.Tyr-E1A(RGD) vector into xenotransplanted human melanoma tumors led to tumor regression in vivo. The combination of tumor-specific replication and enhanced infectivity generates a more potent CRAD vector for gene therapy of melanoma.

Adenoviridae↗
PubMed1999

Persistence of an [E1-, polymerase-] adenovirus vector despite transduction of a neoantigen into immune-competent mice.

The ability of a uniquely modified [E1-, polymerase-] adenovirus (Ad) vector to persist after the transduction of the bacterial beta-galactosidase gene into the livers of nontolerant, immune-competent adult mice was compared with an identical gene transfer attempt with an [E1-] Ad vector. After transduction, the E1-deleted vector was rapidly eliminated, but the modified vector persisted for at least 2 months (experiment duration). Modified vector persistence was also accompanied by prolonged transgene expression and decreased hepatotoxicity profiles. This result was in contrast to several reports suggesting that the transgene expressed by an Ad vector is the primary determinant of Ad vector elimination in vivo. Our results implied that the rapid immune clearance of Ad-transduced cells in vivo is codependent on the presence of two stimuli, or "hits." Hit 1 is due to Ad vector-derived gene expression while hit 2 is due to transgene immunogenicity. Attenuation of the first hit by the use of a significantly modified vector (such as the [E1-, polymerase-] Ad vector) allowed for extended persistence, despite the continued presence of the transgene-derived stimulus (hit 2). We discuss how the two-hit hypothesis is in fact congruent with a number of other reports that have analyzed Ad vector persistence in immune-competent animals. On the basis of our results, [E1-, polymerase-] Ad vectors should have broad benefits for use in human gene therapy situations in which the encoded transgene may be perceived as a neoantigen by the intact human immune system.

Adenoviridae↗
PubMed1999

DNA from both high-capacity and first-generation adenoviral vectors remains intact in skeletal muscle.

Previous studies of the use of adenoviral vectors in animal models of gene therapy have focused on the immune response against transduced cells as the major limiting factor to long-term transgene expression. In this study we eliminated the variable of immunity induced by expression of the transgene in order to investigate vector DNA stability of both first-generation and high-capacity adenoviral vectors after gene transfer to skeletal muscle. Transgene expression from a high-capacity adenoviral vector remained at a high level for at least 20 weeks and was accompanied by persistence of intact vector genomes. In contrast, transgene expression from a first-generation adenoviral vector markedly diminished by 6 weeks after gene transfer and was accompanied by mild and variable inflammatory cell infiltrates. Surprisingly, despite this loss of transgene expression, the first-generation adenoviral vector genomes persisted like the high-capacity adenoviral vector genomes. Therefore, in the absence of immunity to transgene proteins, loss of expression from the first-generation vector was due to inhibition of transgene expression rather than to the elimination of vector-containing cells. DNA stability and persistent expression of the high-capacity adenoviral vector supports the potential of this vector for clinical applications of muscle gene transfer.

Adenoviridae↗
PubMed1997

Circumvention of anti-adenovirus neutralizing immunity by administration of an adenoviral vector of an alternate serotype.

Effective gene transfer and expression following repetitive administration of adenoviral (Ad) vectors in experimental animals is limited by anti-Ad neutralizing antibodies. Knowing that anti-Ad humoral immunity is serotype-specific, we hypothesized that anti-Ad neutralizing immunity could be circumvented using Ad vectors of different serotypes (Ad2, Ad5) within the same subgroup (C) to transfer and express beta-glucuronidase (beta glu) in the lung. Sprague-Dawley rats received an intratracheal administration of either Ad2 beta glu or Ad5 beta glu, and, 14 days later, repeat administration of either the same vector or a vector of a different serotype. Analysis of serum and bronchoalveolar lavage fluid following initial vector administration demonstrated systemic and local serotype-specific neutralizing antibodies. For both the Ad2 and Ad5 vectors, beta glu expression 24 hr following the second administration of the same serotype was < 30% of that of naive animals. In contrast, beta glu expression 24 hr following second administration of a different serotype Ad vector was similar to expression at 24 hr of naive animals receiving a single administration (Ad5 beta glu followed by Ad2 beta glu, as well as Ad2 beta glu followed by Ad5 beta glu; p > 0.2 both comparisons). Although the alternative serotype bypassed anti-Ad neutralizing immunity, persistence of expression was reduced compared to that following administration to naive animals. Compatible with this observation, systemic administration of the same vectors to C57B1/6 mice demonstrated induction of cytotoxic T lymphocytes directed against the beta glu transgene, as well as products of the Ad genome. Interestingly, intratracheal administration of vectors with different serotypes and different transgenes to rats resulted in longer expression (but still not normalized) compared to that achieved with vectors of different serotypes but the same transgene. These observations demonstrate that alternate use of Ad vectors from different serotypes within the same subgroup can circumvent anti-Ad humoral immunity to permit effective gene transfer after repeat administration, although the chronicity of expression is limited, likely by cellular immune process directed against both the transgene and viral gene products expressed by the vector.

Adenoviridae↗
PubMed1997

Role of alveolar macrophages in rapid elimination of adenovirus vectors administered to the epithelial surface of the respiratory tract.

To evaluate the hypothesis that innate immune mechanisms play a major role in eliminating adenovirus (Ad) vectors from the lung, the fate of adenoviral genome of an Ad vector was quantified in the first 24 h after intratracheal administration of an Ad vector coding for beta-galactosidase (beta gal) to mice. Southern analysis with an Ad specific probe showed that 70% of the Ad genome was lost within 24 h, in both immunocompetent and immunodeficient animals. When alveolar macrophages were eliminated by administration of liposomes containing dichloromethylene-biphosphanate, subsequent administration of Ad vector was associated with a 100%+/-8% increase in lung Ad DNA and 96%+/-9% rise in beta gal expression at 24 h compared to control animals. In vitro infection of mouse, rat, and human alveolar macrophages with an Ad vector resulted in 65% loss of vector genome within 24 h, whereas the vector genome was stable in lung epithelial cell lines. PCR in situ hybridization demonstrated that the Ad vector genome persisted A549 lung epithelial cell in vitro but not in alveolar macrophages. Finally, alveolar macrophages recovered from the mouse lung 30 min following intratracheal administration of an Ad vector showed large amounts of vector genome, whereas much less was evident in alveolar macrophages recovered after 24 h. These observations demonstrate that alveolar macrophages play an important role in elimination of Ad vectors from the lung and suggest that strategies to transiently suppress this major innate immune defense system might be rewarding in enhancing the efficiency Ad vectors for lung gene therapy.

Adenoviridae↗
PubMed1997

Comparison of retroviral and adeno-associated viral vectors designed to express human clotting factor IX.

Several different designs for retroviral and adeno-associated virus (AAV) vectors were developed to express human clotting factor IX. Seven separate retroviral vectors were constructed, including chimeric long terminal repeat (LTR)-based designs, vectors containing splice donor/acceptor sites with internal ribosome entry sites (IRES), and vectors with an internal cytomegalovirus (CMV)- or hepatitis B virus (HBV)-derived promoter. Five AAV vectors were produced using the same cassette design where a viral promoter was used to transcribe a bicistronic mRNA containing factor IX and an IRES/neo gene. In the human hepatocyte cell line HepG2, the constructs were tested for factor IX production by ELISA, Northern blot, and Western blot, and for biological activity by normalization of the prolonged activated partial thromboplastin time (APTT) of factor IX-deficient plasma. All of the constructs produced biologically active factor IX in the range of 0.23-152 ng/24 hr per 10(6) cells (the HBV-promoted factor IX AAV vector was the least effective, and the CMV-promoted retroviral vector was the most active). Primary fibroblasts of both human and rabbit origin were also evaluated for factor IX production following transduction with viral vectors. Fibroblasts produced substantially more factor IX than the HepG2 cell line, with the best AAV vector synthesizing > 250 ng/24 hr per 10(6) cells and the best retroviral vector making > 900 ng/24 hr per 10(6) cells. Generally, we observed lower transduction efficiency and poorer expression with the AAV vectors versus retroviral vectors in these cell types.

Animals↗
PubMed1997

Adenoviral vector-mediated gene expression in the nervous system of immunocompetent Wistar and T cell-deficient nude rats: preferential survival of transduced astroglial cells in nude rats.

In the present paper, we examined the effect of the adenoviral vector dosage, the role of T cells, and the influence of the presence of replication-competent adenovirus (RCA) in adenoviral vector stocks, on the efficacy of adenoviral vector-directed transgene expression in the facial nucleus of immunocompetent Wistar and athymic nude rats. A small number of motor neurons and glial cells was transduced at low dosages of viral vector (1 x 10(6) pfu) and in the absence of RCA, and transgene-expressing cells persisted throughout the 3-week period of observation. Intraparenchymal infusion of 2 x 10(7) pfu of a recombinant adenoviral vector free of RCA was required for optimal transduction of facial motor neurons. In Wistar rats, a biphasic immune response occurred at higher dosages of the vector (5 x 10(6) and 2 x 10(7) pfu) that was characterized by early infiltration of macrophages and the occurrence of T cells during the second week after injection of the vector. The immune response was associated with the loss of transduced neural cells. In nude rats, administration of an adenoviral vector free of RCA resulted in a macrophage response comparable to that in the Wistar rat and long-term survival of transduced astroglial cells. However, transduced motor neurons degenerated according to a similar time course as observed in Wistar rats. Small amounts of RCA (2 x 10(5) pfu) injected with 2 x 10(7) pfu recombinant viral vector particles resulted in an accelerated T cell response and a rapid elimination of transduced cells within 1 week in Wistar rats, whereas in nude rats transgene expression continued during this period. Taken together, these observations suggest that at the high viral vector loads necessary to achieve optimal transduction of the facial nucleus, T cells play a role in the degeneration of adenoviral vector-transduced astroglial cells. The adverse effects on neurons appear to be due to the observed inflammatory response or to direct adenoviral vector toxicity.

Adenoviridae↗
PubMed2004

Coexpression of tyrosine hydroxylase, GTP cyclohydrolase I, aromatic amino acid decarboxylase, and vesicular monoamine transporter 2 from a helper virus-free herpes simplex virus type 1 vector supports high-level, long-term biochemical and behavioral correction of a rat model of Parkinson's disease.

Parkinson's disease is due to the selective loss of nigrostriatal dopaminergic neurons. Consequently, many therapeutic strategies have focused on restoring striatal dopamine levels, including direct gene transfer to striatal cells, using viral vectors that express specific dopamine biosynthetic enzymes. The central hypothesis of this study is that coexpression of four dopamine biosynthetic and transporter genes in striatal neurons can support the efficient production and regulated, vesicular release of dopamine: tyrosine hydroxylase (TH) converts tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), GTP cyclohydrolase I (GTP CH I) is the rate-limiting enzyme in the biosynthesis of the cofactor for TH, aromatic amino acid decarboxylase (AADC) converts L-DOPA to dopamine, and a vesicular monoamine transporter (VMAT-2) transports dopamine into synaptic vesicles, thereby supporting regulated, vesicular release of dopamine and relieving feedback inhibition of TH by dopamine. Helper virus-free herpes simplex virus type 1 vectors that coexpress the three dopamine biosynthetic enzymes (TH, GTP CH I, and AADC; 3-gene-vector) or these three dopamine biosynthetic enzymes and the vesicular monoamine transporter (TH, GTP CH I, AADC, and VMAT-2; 4-gene-vector) were compared. Both vectors supported production of dopamine in cultured fibroblasts. These vectors were microinjected into the striatum of 6-hydroxydopamine-lesioned rats. These vectors carry a modified neurofilament gene promoter, and gamma-aminobutyric acid (GABA)-ergic neuron-specific gene expression was maintained for 14 months after gene transfer. The 4-gene-vector supported higher levels of correction of apomorphine-induced rotational behavior than did the 3-gene-vector, and this correction was maintained for 6 months. Proximal to the injection sites, the 4-gene-vector, but not the 3-gene-vector, supported extracellular levels of dopamine and dihydroxyphenylacetic acid (DOPAC) that were similar to those observed in normal rats, and only the 4-gene-vector supported significant K(+)-dependent release of dopamine.

Animals↗
PubMed1992

Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy.

Recombinant retroviral vectors producing multicistronic mRNAs were constructed. Picornavirus putative internal ribosome entry sites (IRES) were used to confer cap-independent translation of an internal cistron. Internal cistrons were engineered by ligation of various lengths of the IRES of encephalomyocarditis (EMC) virus or polio virus to the E. coli chloramphenicol acetyltransferase (CAT) gene. The IRES/CAT fusions were introduced into retroviral vectors 3' to the translation stop codon of the neomycin phosphotransferase (NEO) gene, and the molecular constructs transfected into retroviral vector packaging lines. Retroviral vector producer cells efficiently express the internal CAT gene product only when the full length IRES is used. Both the EMC/CAT and polio/CAT retroviral vectors produced high titer vector supernatant capable of productive transduction of target cells. To test the generality of this gene transfer system, a retroviral vector containing an IRES fusion to the human adenosine deaminase (ADA) gene was constructed. Producer cell supernatant was used to transduce NIH/3T3 cells, and transduced cells were shown to express NEO, and ADA. Novel three-gene-containing retroviral vectors were constructed by introducing the EMC/ADA fusion into either an existing internal-promoter-containing vector, or a polio/CAT bicistronic vector. Producer cell clones of the three-gene vectors synthesize all three gene products, were of high titer, and could productively transduce NIH/3T3 cells. By utilizing cap-independent translation units, IRES vectors can produce polycistronic mRNAs which enhance the ability of retroviral-mediated gene transfer to engineer cells to produce multiple foreign proteins.

Adenosine Deaminase↗
PubMed1998

Cloning and stable maintenance of DNA fragments over 300 kb in Escherichia coli with conventional plasmid-based vectors.

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) systems were previously developed for cloning of very large eukaryotic DNA fragments in bacteria. We report the feasibility of cloning very large fragments of eukaryotic DNA in bacteria using conventional plasmid-based vectors. One conventional plasmid vector (pGEM11), one conventional binary plasmid vector (pSLJ1711) and one conventional binary cosmid vector (pCLD04541) were investigated using the widely used BAC (pBeloBAC11 and pECBAC1) and BIBAC (BIBAC2) vectors as controls. The plasmid vector pGEM11 yielded clones ranging in insert sizes from 40 to 100 kb, whereas the two binary vectors pCLD04541 and pSLJ1711 yielded clones ranging in insert sizes from 40 to 310 kb. Analysis of the pCLD04541 and pSLJ1711 clones indicated that they had insert sizes and stabilities similar to the BACs and BIBACs. Our findings indicate that conventional plasmid-based vectors are capable of cloning and stably maintaining DNA fragments as large as BACs and PACs in bacteria. These results suggest that many existing plasmid-based vectors, including plant and animal transformation and expression binary vectors, could be directly used for cloning of very large eukaryotic DNA fragments. The pCLD04541 and pSLJ1711 clones were shown to be present at at least 4-5 copies/cell. The high stability of these clones indicates that stability of clones does not seem contingent on single-copy status. The insert sizes and the copy numbers of the pCLD04541 and pSLJ1711 clones indicate that Escherichia coli can stably maintain at least 1200 kb of foreign DNA per cell. These results provide a new conceptual and theoretical basis for development of improved and new vectors for large DNA fragment cloning and transformation. According to this discovery, we have established a system for large DNA fragment cloning in bacteria using the two binary vectors, with which several very large-insert DNA libraries have been developed.

Animals↗
PubMed1996

Gene therapy of metastatic pancreas cancer with intraperitoneal injections of concentrated retroviral herpes simplex thymidine kinase vector supernatant and ganciclovir.

OBJECTIVE: The objective of this study was to determine the efficacy of intraperitoneal (IP) injections of a new concentrated herpes simplex thymidine kinase (HS-tk) retroviral vector and ganciclovir (GCV) for peritoneal metastases from pancreas cancer. SUMMARY BACKGROUND DATA: Metastatic pancreas cancer is fatal. Gene therapy may provide a novel approach for this disease. Gene therapy with adeno- or retroviral-mediated transfer of the HS-tk gene into tumor cells renders the cells susceptible to GCV. Intratumoral or intracavity injections of retroviral vectors have been ineffective in previous studies. METHODS: Pancreatic cancer B x PC3 cells (3 x 10(7)) were injected into the tail of pancreas in nude mice. Mice received IP injections of a concentrated HS-tk vector (5 x 10(7)) cfu/mliters) or a control vector (G1Na) without the tk gene for 10 days and GCV (100 mg/kg) for 14 days. To determine whether the vector would survive in the milieu of the peritoneal cavity, the authors examined the effects of ascitic fluid on the vector. Pancreas cancer cells were transduced in vitro with HS-tk vector in presence of media or ascitic fluid and treated with GCV. RESULTS: Highly significant reductions in the mass of metastatic peritoneal tumor deposits were found in HS-tk-treated group (124 +/- 27 mg; n = 11) compared with G1Na vector controls (910 +/- 168 mg; n = 8; p < 0.0001). Results of polymerase chain reaction analysis demonstrated integration of the vector in the tumors, and on immunohistochemistry, expression of the TK protein was seen in the number of surviving colonies (representing nontransduced cells) were similar in both groups, suggesting that the vector effectively transduced tumor cells bathed in the ascitic fluid. CONCLUSIONS: Results demonstrate that IP administration of concentrated retroviral HS-tk vectors is effective treatment for pancreas cancer metastatic to the peritoneal cavity; furthermore, the vector is active in the presence of ascitic fluid. Intraperitoneal retroviral HS-tk may provide a novel approach to treatment of metastatic pancreas cancer.

Animals↗
PubMed2006

Generation of an adenoviral vaccine vector based on simian adenovirus 21.

Adenoviral vectors can be used to generate potent humoral and cellular immune responses to transgene products. Use of adenoviral vectors based on non-human isolates may allow for their utilization in populations harbouring neutralizing antibodies to common human serotypes. A vector chimera was constructed using simian adenovirus 22 (a serotype belonging to the species Human adenovirus E) and simian adenovirus 21 (a serotype belonging to the species Human adenovirus B) expressing the Ebola (Zaire) virus glycoprotein (Ad C5/C1-ZGP). This chimeric adenovirus vector was used as a model to test its efficacy as a genetic vaccine and comparisons were made to a vector based on the commonly used human adenovirus C serotype 5 (Adhu5-ZGP). Ebola glycoprotein-specific T- and B-cell responses were measured in B10BR mice vaccinated with either Adhu5-ZGP or Ad C5/C1-ZGP vectors. Both vectors resulted in Ebola glycoprotein-specific gamma interferon-expressing T cells, although the Ad C5/C1-ZGP vector appeared to induce lower frequencies with kinetics slower than those elicited by the Adhu5-ZGP vector. The total immunoglobulin G response to Ebola glycoprotein was similar in sera from mice vaccinated with either vector. Two rhesus macaques vaccinated with the Ad C5/C1-ZGP vector were found to mount T-cell and antibody responses to the Ebola glycoprotein. It was found that a single administration of the chimeric Ad C5/C1-ZGP vector protected mice against a lethal challenge with a mouse-adapted strain of the Ebola (Zaire) virus.

Adenoviruses, Simian↗
PubMed1996

Sindbis virus DNA-based expression vectors: utility for in vitro and in vivo gene transfer.

Several DNA-based Sindbis virus vectors were constructed to investigate the feasibility and potential applications for initiating the virus life cycle in cells transfected directly with plasmid DNA. These vectors, when transfected into mammalian cells, have been used to produce virus, to express heterologous genes, and to produce infectious vector particles. This approach involved the conversion of a self-replicating vector RNA (replicon) into a layered DNA-based expression system. The first layer includes a eukaryotic RNA polymerase II expression cassette that initiates nuclear transcription of an RNA which corresponds to the Sindbis virus vector replicon. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the vector, proceeds according to the Sindbis virus replication cycle and results in expression of the heterologous gene. The Sindbis virus DNA vectors expressed reporter genes in transfected cells at levels that were comparable to those of in vitro-transcribed RNA replicons and were approximately 10-fold higher than the levels produced by conventional RNA polymerase II-dependent plasmids in which the promoter and reporter gene were linked directly. Reporter gene expression was also observed in rodent muscle following injection with Sindbis virus DNA vectors. In a second application, packaged vector particles were produced in cells cotransfected with complementing replicon and defective helper DNAs. The Sindbis virus-derived DNA vectors described here increase the utility of alphavirus-based vector systems in general and also provide a vector with broad potential applications for genetic immunization.

Animals↗
PubMed1999

Herpes simplex virus type 1 vector-mediated expression of nerve growth factor protects dorsal root ganglion neurons from peroxide toxicity.

Nerve growth factor beta subunit (beta-NGF) transgene delivery and expression by herpes simplex virus type 1 (HSV-1) vectors was examined in a cell culture model of neuroprotection from hydrogen peroxide toxicity. Replication-competent (tk- K mutant background) and replication-defective (ICP4(-);tk- S mutant background) vectors were engineered to contain the murine beta-NGF cDNA under transcriptional control of either the human cytomegalovirus immediate-early gene promoter (HCMV IEp) (e.g., KHN and SHN) or the latency-active promoter 2 (LAP2) (e.g., KLN and SLN) within the viral thymidine kinase (tk) locus. Infection of rat B103 and mouse N2A neuronal cell lines, 9L rat glioma cells, and Vero cells with the KHN or SHN vectors resulted in the production of beta-NGF-specific transcripts and beta-NGF protein reaching a maximum at 3 days postinfection (p.i.). NGF protein was released into the culture media in amounts ranging from 10.83 to 352.86 ng/ml, with the highest levels being achieved in B103 cells, and was capable of inducing neurite sprouting of PC-12 cells. The same vectors produced high levels of NGF in primary dorsal root ganglion (DRG) cultures at 3 days. In contrast to HCMV IEp-mediated expression, the LAP2-NGF vectors showed robust expression in primary DRG neurons at 14 days. The neuroprotective effect of vector produced NGF was assessed by its ability to inhibit hydrogen peroxide-induced neuron toxicity in primary DRG cultures. Consistent with the kinetics of vector-mediated NGF expression, HCMV-NGF vectors were effective in abrogating the toxic effects of peroxide at 3 but not 14 days p.i. whereas LAP2-NGF vector transduction inhibited apoptosis in DRG neurons at 14 days p.i. but was ineffective at 3 days p.i. Similar kinetics of NGF expression were observed with the KHN and KLN vectors in latently infected mouse trigeminal ganglia, where high levels of beta-NGF protein expression were detected at 4 wks p.i. only from the LAP2; HCMV-NGF-driven expression peaked at 3 days but could not be detected during HSV latency at 4 weeks. Together, these results indicate that (i) NGF vector-infected cells produce and secrete mature, biologically active beta-NGF; (ii) vector-synthesized NGF was capable of blocking peroxide-induced apoptosis in primary DRG cultures; and (iii) the HCMV-IEp functioned to produce high levels of NGF for several days; but (iv) only the native LAP2 was capable of long-term expression of a therapeutic gene product in latently infected neurons in vivo.

Animals↗
PubMed1999

Effects of stuffer DNA on transgene expression from helper-dependent adenovirus vectors.

We have analyzed transgene (lacZ) expression from a first-generation adenovirus (Ad) vector in comparison to helper-dependent (hd) Ads deleted for various portions of the viral coding sequences and generated by using the Cre/loxP helper-dependent system (R. J. Parks et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996). An hd vector deleted for approximately 70% of the Ad genome (AdRP1001) provided levels and durations of transgene expression similar to those of a control first generation Ad vector containing an identical expression cassette. Deletion of all Ad sequences from the hdAd and replacement with a approximately 22-kb fragment of lambda DNA resulted in a decrease in the level and duration of lacZ expression which could not be reversed by the inclusion of a matrix attachment region. However, substitution of the lambda stuffer in the fully deleted hdAd with sequences from the human hypoxanthine-guanine phosphoribosyltransferase gene resulted in significantly improved transgene expression. In vitro assays for cytotoxic T lymphocytes (CTL) directed against putative peptides encoded by the vector backbone showed that, although CTL were generated against the vector containing the lambda DNA, no such CTL were generated against the vector containing the hypoxanthine-guanine phosphoribosyltransferase (HPRT) sequences. Surprisingly, the rate of loss of the HPRT- and lambda-containing vectors from mouse liver was similar, despite the differences in expression kinetics, indicating that the lambda stuffer-directed CTL were inefficient at eliminating the transduced cells. Thus, the nature of the DNA backbone of hdAds can have important effects on the functioning of the vector. Since most fully deleted vectors require "stuffer" DNA as part of the vector backbone to maintain optimum vector size, these observations must be taken into account in the design of hdAd vectors.

Adenoviridae↗
PubMed1999

Variability of human systemic humoral immune responses to adenovirus gene transfer vectors administered to different organs.

Administration of adenovirus (Ad) vectors to immunologically naive experimental animals almost invariably results in the induction of systemic anti-Ad neutralizing antibodies. To determine if the human systemic humoral host responses to Ad vectors follow a similar pattern, we evaluated the systemic (serum) anti-Ad serotype 5 (Ad5) neutralizing antibodies in humans after administration of first generation (E1(-) E3(-)) Ad5-based gene transfer vectors to different hosts. AdGVCFTR.10 (carrying the normal human cystic fibrosis [CF] transmembrane regulator cDNA) was sprayed (8 x 10(7) to 2 x 10(10) particle units [PU]) repetitively (every 3 months or every 2 weeks) to the airway epithelium of 15 individuals with CF. AdGVCD.10 (carrying the Escherichia coli cytosine deaminase gene) was administered (8 x 10(8) to 8 x 10(9) PU; once a week, twice) directly to liver metastasis of five individuals with colon cancer and by the intradermal route (8 x 10(7) to 8 x 10(9) PU, single administration) to six healthy individuals. AdGVVEGF121.10 (carrying the human vascular endothelial growth factor 121 cDNA) was administered (4 x 10(8) to 4 x 10(9.5) PU, single administration) directly to the myocardium of 11 individuals with ischemic heart disease. Ad vector administration to the airways of individuals with CF evoked no or minimal serum neutralizing antibodies, even with repetitive administration. In contrast, intratumor administration of an Ad vector to individuals with metastatic colon cancer resulted in a robust antibody response, with anti-Ad neutralizing antibody titers of 10(2) to >10(4). Healthy individuals responded to single intradermal Ad vector variably, from induction of no neutralizing anti-Ad antibodies to titers of 5 x 10(3). Likewise, individuals with ischemic heart disease had a variable response to single intramyocardial vector administration, ranging from minimal neutralizing antibody levels to titers of 10(4). Evaluation of the data from all trials showed no correlation between the peak serum neutralizing anti-Ad response and the dose of Ad vector administered (P > 0.1, all comparisons). In contrast, there was a striking correlation between the peak anti-Ad5 neutralizing antibody levels evoked by vector administration and the level of preexisting anti-Ad5 antibodies (P = 0.0001). Thus, unlike the case for experimental animals, administration of Ad vectors to humans does not invariably evoke a systemic anti-Ad neutralizing antibody response. In humans, the extent of the response is dictated by preexisting antibody titers and modified by route of administration but is not dose dependent. Since the extent of anti-Ad neutralizing antibodies will likely modify the efficacy of administration of Ad vectors, these observations are of fundamental importance in designing human gene therapy trials and in interpreting the efficacy of Ad vector-mediated gene transfer.

Adenoviruses, Human↗
PubMed1999

Ovine adenovirus vectors overcome preexisting humoral immunity against human adenoviruses in vivo.

Recombinant human adenoviruses (hAd) have become widely used as tools to achieve efficient gene transfer. However, successful application of hAd-derived vectors in clinical trials is limited due to immunological and potential safety problems inherent in their human origin. In this study, we describe a recombinant ovine adenovirus (OAV) as an alternative vector for gene transfer in vivo. In contrast to an hAd vector, the OAV vector was not neutralized by human sera. An OAV vector which contained the cDNA of the human alpha1-antitrypsin (hAAT) gene linked to the Rous sarcoma virus promoter was generated and administered systemically to mice. The level and duration of hAAT gene expression was similar to that achieved with an hAd counterpart in both immunocompetent and immunodeficient mice. However, the tissue distribution of the OAV vector differed from that observed for hAd vectors in that the liver was not the dominant target. Significantly, we demonstrated efficient gene transfer with the OAV vector into mice immunized with hAd vectors and vice versa. We also confirm that the immune response to a transgene product can prevent its functional expression following sequential application of a vector. Our results suggest a possible solution to endemic humoral immunity against currently used hAd vectors and should therefore have an impact on the design of improved gene therapy protocols utilizing adenovirus vectors.

Adenoviruses, Human↗