Ad Gene Research Articles

Adenovirus hexon T-cell epitope is recognized by most adults and is restricted by HLA DP4, the most common class II allele.

The immunogenicity of adenovirus (Ad) vectors is enhanced by virus-specific memory immune responses present in most individuals as a result of past exposure to these ubiquitous pathogens. We previously identified the first human T-cell epitope from the major capsid protein hexon, H910-924, and found that it is highly conserved among different Ad serotypes. Memory/effector T-cell responses to H910-924 were detected in 14 of 18 (78%) healthy adults by an interferon-gamma ELISPOT assay. Hexon peptide-specific CD4 T-cell lines were generated from three HLA-typed donors and analyzed using a panel of HLA homozygous B-cell lines and monoclonal antibodies to HLA class II loci. These studies reveal that the hexon epitope is restricted by HLA DP4, a class II allele present in 75% of the population. Analysis of overlapping peptides and peptides with single residue mutations identified a HLA DP4-binding motif. Additionally, antibodies to the hexon peptide were detected in all donor sera by dot blot assay and ELISA. Therefore, most individuals exhibit both memory B- and T-cell responses to this highly conserved epitope on hexon, an obligate component of all Ad vectors, including 'gutted' vectors. These data suggest that current strategies for the use of Ad gene therapy vectors will not evade memory immune responses to Ad.

O3-02-07 Linkage analysis for AD using amyloid beta 42 levels shows evidence for a novel AD gene on chromosome 19

O3-02-07 Linkage analysis for AD using amyloid beta 42 levels shows evidence for a novel AD gene on chromosome 19

O3-06-07 Multi-compartmental pharmacodynamic assessment of the functional gamma-secretase inhibitor LY450139 in PDAPP transgenic mice and non-transgenic mice

β-Amyloid peptide (Aβ) immunization is regarded as the most promising therapy to Alzheimer’ s disease. The full length Aβ as antigen might induce meningoencepholontis adverse effect since the middle and C-terminal fragments of Aβ contain T cell epitopes. While N-terminal fragment of Aβ, containing B cell epitope, has weak or no immunogenicity. To improve the immunogenicity, we used HBV core antigen as carrier to make fusion protein containing 2 Aβ1–15. The fusion protein was expressed in Escherichia coli harboring the recombinant plasmid pET/c-2Aβ15-c. Transmission electron microscope (TEM) showed that fusion protein could form virus-like particles (VLPs). After 7-weeks immunization with Aβ-HBc VLPs through subcutaneous injection, the titer of anti-Aβ antibody in sera of BALB/c mice reached up to 105, higher than Aβ peptide immunization. Aβ-HBc VLPs immunization did not elicit Aβ-specific T cell proliferation. The main isotypes of antibody in mice immunized with Aβ-HBc VLPs were IgG1 and IgG2b, while isotype in mice immunized with Aβ1–42 was IgG2a. When the antisera from mice immunized with Aβ-HBc VLPs were co-incubated for 1 week at 37 °C with Aβ, fibers of aggregated Aβ was reduced or diminished. The antibodies also prevented PC12 cells from injury by toxicity of Aβ.In conclusion, recombinant c-2Aβ15-c gene can be expressed in E. coli. The expressed protein could form VLPs and has strong immunogenicity. The antisera prevented Aβ fiber formation and protected the PC12 cells against toxicity of Aβ. This study lays the foundation for the experimental study of AD gene engineering vaccine.

117. Antitumor Immunity Induced by Adenovirus Vector-Mediated Transduction of OX40 Ligand

As the field of Adenovirus (Ad) gene therapy continues to develop, alternate serotypes have been explored as a means to circumvent the disadvantages of Ad serotype 5 (Ad5)-based vectors. One such group of alternate serotypes that has been intensively investigated is the Ad subgroup B. Members of the Ad subgroup B have been found to efficiently transduce cells of the hematopoeitic system as well as more efficiently transduce malignant cells in a variety of neoplastic contexts compared to Ad5-based vectors. Whereas the majority of human Ad utilize the Coxsackie and Adenovirus Receptor (CAR) to infect cells, none of the Ad subgroup B utilize CAR. It has previously been demonstrated that Ad subgroup B, which are further divided into two groups: B1 and B2, utilize at least two distinct receptors. In this regard, CD46 has been implicated as the B2-specific receptor. Recently, Ad serotype 3 (Ad3), a member of Ad subgroup B1, was found able to utilize CD80 and CD86 as a primary cellular attachment receptors on human dendritic cells and the cervical carcinoma cell line HeLa. CD80 and CD86 are cell surface markers expressed on human dendritic cells and mature B-lymphocytes, and play a critical role in stimulating T-lymphocyte responses by their interaction with the ligands CD28 and CTLA-4. Both CD80 and CD86 are upregulated during the process of dendritic cell maturation and are often over-expressed in a variety of lymphocytic neoplasms and other neoplastic contexts. Whereas, CD80 and CD86 serve as the primary receptor for Ad3 on human dendritic cells, they play a more secondary role in HeLa infection. There exists a close homology between the knob domains of the Ad fiber, the receptor-interacting protein of the virion, of several members of subgroup B Ad. On this basis, we hypothesized that other members of the Ad subgroup B may utilize CD80 and CD86 as primary cellular attachment receptors. Recombinant knob domain of the Ad fiber from both B1 and B2 Ad could efficiently block CD80 and CD86-dependent infection of Ad3 vectors. All subgroup B members showed specific binding to cells expressing CD80 and CD86. Members of both B1 and B2 demonstrated CD80 and CD86-specific infection of CHO cells heterogenously expressing CD80 and CD86. Therefore, it appears that all of the members of Ad subgroup B can utilize CD80 and CD86 for infection of cells. Though these may not represent the only primary cellular receptors utilized by these viruses on all cells in the case of Ad3 they appear to play a critical role in the infection of human dendritic cells. This finding may help facilitate the construction of Ad subgroup B based vectors in the application of vaccine development and for vectors targeted towards CD80 and CD86 expressing neoplasms.

381. Self-Cleaving Multimeric Hammerhead Ribozymes Abolish Mutant Collagen Transcripts In Cellulo and Exhibit Activity In Vivo

Current progress in adenoviral (Ad) gene therapy for Duchenne muscular dystrophy (DMD), a degenerative, inherited neuromuscular disease characterized by a lack of a functional dystrophin protein, has revealed many physical and biochemical obstacles affecting the transduction of muscle. To avoid barriers such as the basal lamina surrounding each muscle fiber and a mature immune system, fetal gene therapy could be an attractive method to deliver therapeutic genes to afflicted individuals. The ability to use a small vector load to transduce target organs in utero, combined with the possibility of transducing progenitor or stem cells, provides a key advantage over post-natal treatment. High-capacity Ad (HC-Ad) vectors devoid of all viral genes have shown more persistent expression after transduction in utero, as compared to first-generation Ad vectors, and have the added ability to carry the full-length 14 kb murine or human dystrophin cDNA. In a previous transduction study in C57BL/6 mice in utero, transgene expression was stable for at least 5 months by intramuscular injection. Furthermore, a lack of neutralizing antibody response to the Ad vector in injected animals was observed, which may allow for repeat administration of vector later in life. For this study we wanted to determine if a single intramuscular in utero injection of an HC-Ad vector carrying the full-length murine dystrophin cDNA could ameliorate the dystrophic phenotype, restore the dystrophin-glycoprotein complex, and provide functional correction of transduced muscles in the mdx mouse, an animal model for DMD. Using a surgical approach on pregnant mdx mice carrying embryonic gestation day 16 fetuses (E-16), one hind limb of fetal mice was injected with an HC-Ad vector carrying dystrophin. The injected pups were delivered naturally and analyzed at 9 weeks of age. In vitro physiological studies showed modest functional correction as assessed by specific tetanic force and muscle weight in an in utero transduced tibialis anterior muscle demonstrating recombinant dystrophin expression. Furthermore, the dystrophin-glycoprotein complex was restored in those fibers expressing dystrophin. These studies provide the first evidence that dystrophin gene delivery in utero has the potential for therapeutic effect in DMD.

543. Ad DNA Polymerase and Preterminal Protein (pTP) Genes Each Mediate Inhibition of HIV-1 Replication in Human Alveolar Macrophages

Macrophages serve as a reservoir for HIV-1 even in the presence of highly active antiretroviral therapy. Adenovirus (Ad) gene transfer vectors (E1−E3−) inhibit HIV-1 replication in human alveolar macrophages (AM) at a step in the HIV-1 life cycle at, or prior to, HIV-1 long terminal repeat (LTR) transcription. The inhibition of HIV-1 replication by Ad is independent of the transgene, the Ad E4 region and the Ad capsid (Am J Respir Cell Mol Biol. 2002; 27: 214–219). We previously observed that both fully deleted Ad vectors or 1st generation vectors (E1−E3−) with an additional deletion of the Ad E2b region lose the ability to inhibit HIV-1 replication in AM, implicating E2b as the inhibitor of HIV-1 replication. The Ad E2b region includes the Ad DNA polymerase and the preterminal protein (pTP) genes that are needed for efficient initiation of Ad DNA replication and transcription of late proteins in wild type Ad. To determine their relative contributions, we used Ad vectors with an additional deletion in the Ad pol, but containing the pTP gene. One vector expressed the β-galactosidase gene (Adpol−lacZ); another expressed the human acid-alpha-glucosidase gene (Adpol−hGAA). Both Adpol−lacZ and AdE2b−lacZ mediated expression of the β-galactosidase gene in THP-1 cells at a dose of 2.5×104 particle units (pu)/cell. To quantify the relative contribution of pTP, AM (2×105/well), were infected with AdNull, AdE2b−lacZ, Adpol−lacZ, Adpol−hGAA, or empty Ad capsids 6×103 to 105 pu/cell or no Ad for 1 hr. After Ad infection, the AM were infected with HIV-1 strain JRFL, 103 TCID50/well and p24 in the media was quantified by ELISA. For the Ad dose of 2.5×104 pu/cell on day 25 post-infection, p24 levels were: AdNull 23±18, AdE2b-lacZ 5410±431, Adpol−lacZ 23±20, Adpol−hGAA 10±0, empty Ad capsids 9853±1170, no Ad 9942±217 pg/ml (p<0.05, ANOVA). This represents a greater than 2 log inhibition by AdNull, Adpol−lacZ and Adpol−hGAA, but little inhibition by empty Ad capsids or AdE2b−lacZ. To quantify the relative contribution of Ad pol, we used an Ad vector with an additional deletion in the Ad pTP, but containing the Ad DNA polymerase gene and expressing the green fluorescent protein gene (AdpTP−GFP). AZT 10 μg/ml was used as an additional control. For the Ad dose of 2.5×104 pu/cell on day 24 post-infection, p24 levels were: AdNull 931×338, AdpTP−GFP 940×88, AZT 932×68 no Ad 6368×1220 pg/ml (p<0.05, ANOVA). Taken together with our previous observations on the lack of inhibitory effect of E2b− and fully deleted Ad vectors, these data imply that the presence of either the Ad pTP or Ad DNA polymerase genes alone in the context of a 1st generation Ad vector are sufficient to inhibit HIV-1 replication in human AM.

E1A and E1B proteins inhibit inflammation induced by adenovirus.

Replication-defective human adenovirus (Ad) group C transducing vectors, most of which have the E1A, E1B, and E3 genes deleted, are highly inflammatory despite the fact that the parental viruses typically cause subclinical or mild infections. To investigate this paradox, the roles that the E1A, E1B, and E3 genes play in inflammation were tested by using replication-incompetent viruses carrying a deletion of the preterminal protein gene. The viruses were injected into BALB/c mouse ears, and edema was monitored as a sensitive surrogate marker of inflammation. A virus deleted for the E1A 289R (transcription activating) protein was noninflammatory, and inhibited edema induced by empty virus particles. The E1A 243R and E1B 55-kDa (p53 binding) proteins play the most important roles in inhibition of inflammation by the noninflammatory virus. The E1B 19-kDa antiapoptotic protein inhibited edema when both the E1A 243R and E1B 55-kDa proteins were expressed but strongly induced edema when only one was expressed. E3 proteins had their greatest effect on the inhibition of edema induced by the E1A 289R protein. The results support a model in which inflammation is countered through a mechanism that involves complex genetic interactions between Ad early region proteins and offer promise for the design and construction of noninflammatory Ad gene therapy vectors that are relatively easy to grow and purify.

Modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses.

Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing beta-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the beta-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing beta-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to beta-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-beta-galactosidase antibody levels following vector administration. However, cellular responses to beta-galactosidase were significantly enhanced, with the frequency of CD4(+) as well as the CD8(+) beta-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P < 0.01). Importantly, this enhanced cellular immune response of the AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing beta-galactosidase: BALB/c mice implanted with the CT26 syngeneic beta-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These data demonstrate that addition of an RGD motif to the Ad fiber knob increases the infectibility of DC and leads to enhanced cellular immune responses to the Ad-transferred transgene, suggesting that the RGD capsid modification may be useful in developing Ad-based vaccines.

Identification of multiple genetic loci that regulate adenovirus gene therapy

A key aspect of the immune response to adenovirus (Ad) gene therapy is the generation of a cytotoxic T-cell (CTL) response. To better understand the genetic network underlying these events, 20 strains of C57BL/6 x DBA/2 (BXD) recombinant inbred (RI) mice were administered with AdLacZ and analyzed at days 7, 21, 30, and 50 for liver beta-galactosidase (LacZ) expression and CTL response. Sera levels of interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were analyzed at different times after AdLacZ. There was a distinct strain-dependent expression of LacZ, which was strongly correlated with the CTL response. Among the five BXD RI strains that exhibited significantly prolonged LacZ expression, four also exhibited a marked defect in the production of Ad-specific CTL. There was a strong correlation between the sera levels of IFN-gamma, TNF-alpha, and IL-6, but cytokine responses were not significantly correlated with LacZ expression or the CTL response. Quantitative trait loci regulating LacZ on day 30 were found on chromosome (Chr) 19 (33 cM) and Chr 15 (42.8 cM). Cytotoxicity mapped to Chr 7 (41.0 and 57.4-65.2 cM), Chr 15 (61.7 cM), and Chr X (27.8 cM). IFN-gamma production mapped to Chr 18 (22, 27, and 32 cM) and Chr 11 (64.0 cM). TNF-alpha and IL-6 production mapped to Chr 6 (91.5 cM) Chr 9 (42.0 cM) and Chr 8 (52 and 73.0 cM). These results indicate that different strains of mice exhibit different pathways for effective clearance of AdLacZ depending on genetic polymorphisms and interactions at multiple genetic loci.

Adenovirus protein IX sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation.

The product of adenovirus type 5 (Ad5) gene IX, protein IX (pIX), is a multifunctional protein that stabilizes the viral capsid and has transcriptional activity. We show that pIX also contributes to the Ad5-induced reorganization of the host-cell nuclear ultrastructure: pIX induces the formation of specific and dynamic nuclear inclusions, and the host promyelocytic leukaemia (PML) protein, which is the main structural organizer of PML bodies, is stably relocated and confined within the pIX-induced inclusions late in infection. Our results suggest that Ad5 has evolved a unique strategy that leads to the sustained neutralization of PML bodies throughout infection, thereby ensuring optimal viral proliferation.

Engineering Regulatory Elements for Conditionally-Replicative Adenoviruses

Virus-mediated oncolysis is a rapidly growing field with the potential to dramatically alter the future of cancer therapy. Replication-selective viruses are superior to non-replicating vectors in several aspects, such as the amplification of the initial low-dose viral inoculum up to 10(3)-10(3)-fold, lateralization into neighboring cells, introduction of novel cell killing mechanisms, and a potential for a safe profile. However, due to their capacity to replicate, the importance of tumor selectivity is further underscored. Of the replication-selective viruses, adenoviruses (Ad) possess several attributes that appear essential for targeting and eliminating tumor cells. These include susceptibility to genomic modifications, convergence with cellular pathways implicated in carcinogenesis, and a high oncolytic capacity. A primary tumor targeting strategy of oncolytic Ad is based on re-engineering the viral genome viruses to construct conditionally replicative adenoviruses (CRAds). In this regard, modification of CRAd genome is traditionally designated as type I or type II. Type I CRAds are based on mutation or deletion of early Ad genes. Type II CRAds are based on the placement of essential early Ad genes under tissue/tumor-specific regulatory elements in a heterologous context. Thus, both strategies confer varying degrees of tumor-specific replication. Recent data, however, indicate that type III CRAds, embodying the paradigms of both type I and II, offer better replication selectivity for tumor cells while maintaining efficient oncolysis. These characteristics of CRAds yield therapeutic indices unprecedented heretofore in cancer therapy. However, other biological aspects of CRAds should also be addressed before these agents prove as first-line antitumor agents. When these issues are resolved, novel tumor cell killing potential of CRAds may truly be realized and dramatically alter future cancer therapy.

Modulation of renal glomerular disease using remote delivery of adenoviral-encoded solubletype II TGF-beta receptor fusion molecule.

Systemic adenoviral (Ad) gene therapy for renal disorders is largely hampered by the unique architecture of the kidney. Consequently, currently available Ad vectors are of only limited therapeutic utility in the context of glomerular and fibroproliferative renal diseases. The Ad vectors studied in the context of blocking renal fibrosis were AdTbeta-ExR and AdCATbeta-TR. AdTbeta-ExR encodes a chimeric soluble molecule comprising the entire ectodomain of the human type II TGF-beta receptor, genetically fused to the Fc fragment of the human IgG1 (sTbetaRII), while AdCATbeta-TR encodes only the dominant-negative truncated ectodomain of the human type II TGF-beta receptor. The biologic activity of the type II TGF-beta receptor was evaluated in vitro by its ability to inhibit cellular proliferation and in vivo in a unilateral ureter obstruction fibrosis model. Renal targeting with sTbetaRII was evaluated immunohistochemically after intramuscular (IM) delivery of AdTbeta-ExR. The renal antifibrotic effect of the Ad vectors was evaluated in a lupus murine model with both light and electron microscopy and urinalysis. sTbetaRII was detected in the glomeruli after remote IM injection of AdTbeta-ExR, but not the control AdCATbeta-TR, indicating renal deposition of the heterologous soluble fusion protein after its expression in the muscle and secretion into the circulation. AdTbeta-ExR, but not AdCATbeta-TR, could transiently inhibit mesangial expansion, glomerular hypercellularity, proteinuria and cortical interstitial fibrosis in a murine lupus model. However, the autoimmune renal disease eventually surpassed the antifibrotic effect. These results indicate the superiority of a soluble type II TGF-beta receptor over a dominant-negative, non-soluble type II TGF-beta receptor in the context of blocking renal fibrosis in murine models.

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3.

Matrix metalloproteinases (MMPs) are believed to be pivotal enzymes in the invasion of articular cartilage by synovial tissue in rheumatoid arthritis (RA). Here, we investigated the effects of gene transfer of tissue inhibitors of metalloproteinases (TIMPs) on the invasiveness of RA synovial fibroblasts (RASF) in vitro and in vivo. Adenoviral vectors (Ad) were used for gene transfer. The effects of AdTIMP-1 and AdTIMP-3 gene transfer on matrix invasion were investigated in vitro in a transwell system. Cartilage invasion in vivo was studied in the SCID mouse co-implantation model for 60 days. In addition, the effects of AdTIMP-1 and AdTIMP-3 on cell proliferation were investigated. A significant reduction in invasiveness was demonstrated in vitro as well as in vivo in both the AdTIMP-1- and AdTIMP-3-transduced RASF compared with untransduced SF or SF that were transduced with control vectors. in vitro, the number of invading cells was reduced to 25% (P<0.001) in the AdTIMP-1-transduced cells and to 13% (P<0.0001) in the AdTIMP-3-transduced cells (% of untransduced cells). Cell proliferation was significantly inhibited by AdTIMP-3 and, less, by AdTIMP-1. In conclusion, overexpression of TIMP-1 and TIMP-3 by Ad gene transfer results in a marked reduction of the invasiveness of RASF in vitro and in the SCID mouse model. Apart from the inhibition of MMPs, a reduction in proliferation rate may contribute to this effect. These results suggest that overexpression of TIMPs, particularly TIMP-3 at the invasive front of pannus tissue, may provide a novel therapeutic strategy for inhibiting joint destruction in RA.

Packaging cell lines for generating replication-defective and gutted adenoviral vectors.

Gene transfer using adenovirus (Ad) can be a highly efficient method for transducing cells and tissues. Wild-type Ad has a 36-kb double-stranded linear genome that encodes numerous overlapping open reading frames (). Because wild-type Ad is infectious to humans, vectors used in the laboratory generally contain a deletion of one or more genes to limit their growth and replication on nonpermissive cells. One drawback to Ad vectors is that infection of a target cell results in transfer not only of the desired transgene of interest, but also of the Ad genes. Expression of these other genes can be toxic, or in vivo, can elicit a potent host-immune response against transduced cells. Consequently, methods have been developed by many labs to engineer Ad vectors that display greatly reduced expression of viral genes except in specifically generated packaging cell lines designed for propagation of the vectors. In general, two types of alterations are made to the Ad genome. In the first, individual genes, or combinations thereof, are deleted from the Ad genome and placed into a packaging cell line under control of a constitutive or inducible promoter. In the second method, all viral genes are removed from the Ad vector, and vector growth is regulated by coinfection with a helper virus. The fully deleted, or gutted, Ad vectors must then be purified away from helper virus prior to use. Both of these strategies can also be combined, whereby one uses a multiply deleted Ad vector in a specialized complementing cell line as a helper virus to grow gutted vectors.

Gene transfer of extracellular SOD to the penis reduces O2-* and improves erectile function in aged rats.

Increased superoxide anion (O(2)(-).) may contribute to vascular dysfunction in aging. In aged cavernosal tissue, lucigenin-enhanced chemiluminescence demonstrated a threefold increase in superoxide formation, and the oxidative fluorescent probe hydroethidine indicated higher superoxide levels throughout the aged penis. This increase in superoxide was associated with impaired cavernosal nerve-mediated and agonist-induced erectile responses, increased nitrotyrosine staining, and lower cGMP levels, but no compensatory change in cavernosal extracellular (EC)-superoxide dismutase (EC-SOD) mRNA or protein. In vivo adenoviral (Ad) gene transfer of EC-SOD to the penis resulted in higher expression of EC-SOD mRNA, protein, SOD activity, cGMP levels, and lower nitrotyrosine staining. Transfection with AdCMVEC-SOD resulted in a significant increase in erectile response to cavernosal nerve stimulation, ACh, and zaprinast to a magnitude similar to young rats. These data provide evidence in support of the hypothesis that erectile dysfunction associated with aging is related in part to an increase in cavernosal O(2)(-). formation. Gene-transfer of EC-SOD reduces superoxide formation and restores age-associated erectile function and may represent a novel therapeutic target for the treatment of erectile dysfunction.

Differential display analysis of presenilin 1-deficient mouse brains

Missense mutations in presenilin 1 (PS1) gene are the most common cause of early onset familial Alzheimer’s disease (FAD). AD pathogenic PS1 mutations result in elevated γ-secretase cleavage of APP and diminished S3-site cleavage of Notch. We have previously described a PS1-hypomorphic mouse line that could survive postnatally with markedly reduced γ-secretase cleavage of APP and S3-site cleavage of Notch, resulting in a Notch developmental phenotype similar to PS1-null mice. This model was exploited to identify genes whose expression is altered due to the loss of PS1. A global gene expression study by differential display was performed on whole brains of PS1-hypomorphic mice and their wild type siblings. In total, more than 16 000 bands corresponding to cDNAs were compared between the mutant and wild-type brains. This analysis identified 19 cDNAs showing significantly altered expression resulting from PS1 deficiency. Four of the identified cDNAs corresponded to genes that could be associated with AD or presenilin function. Hypoxia inducible factor 1a (Hif1a), NPRAP (δ-catenin) and cell division cycle 10 (CDC10) showed significantly reduced expression in the PS1-hypomorphic compared to wild-type brains, whereas expression of nucleoside diphosphate kinase sub-unit A (NDPK-A) was markedly elevated in the respective brains. Clarification of the possible role of these genes in AD and the basis for their differential expression induced by PS1-deficiency may provide insight into the disease, presenilin function and consequences of its loss, as well as possible deleterious effects of AD therapeutics aimed at inhibiting PS1.

Inhibition of HIV-1 replication in alveolar macrophages by adenovirus gene transfer vectors.

To assess the hypothesis that infection of alveolar macrophages (AM) with adenovirus (Ad) gene transfer vectors might prevent subsequent human immunodeficiency virus (HIV)-1 replication in AM, AM isolated from normal volunteers were infected with increasing doses of first generation (E1(-)) Ad vectors, followed 72 h later by infection with HIV-1(JRFL), an R5/M-tropic strain that preferentially uses the CCR5 coreceptor. As a measure of HIV-1 replication, p24 Ag was quantified by enzyme-linked imunosorbent assay in supernatants on Days 4 to 14 after HIV-1infection. Pretreatment of the AM with an Ad vector resulted in a dose- and time-dependent suppression of subsequent HIV-1 replication. The Ad vector inhibition of HIV-1 replication was independent of the transgene in the Ad vector expression cassette and E4 genes in the Ad backbone. Moreover, it did not appear to be secondary to a soluble factor released by the AM, nor was it overridden by the concomitant transfer of the CCR5 or CXCR4 receptors to the AM before HIV-1 infection. These observations have implications regarding pulmonary host responses associated with HIV-1 infection, as well as possibly uncovering new therapeutic strategies against HIV-1 infection.

An adenoviral expression system for AAV rep78 using homologous recombination.

The construction and amplification of adenoviral (Ad) vectors expressing biologically active transgenes that are cytotoxic or inhibit Ad replication can be extremely difficult, if not impossible. In this study, we harnessed the ability of Ad genomes to undergo efficient homologous recombination to reconstitute the adeno-associated virus (AAV) rep78 gene, a cytotoxic gene that strongly inhibits Ad replication, which was divided between two parental, first-generation Ad vectors. A functional open reading frame was generated by recombination only upon co-infection of both parental vectors and after the onset of viral DNA replication. We were able to amplify both parental rep78 vectors to normal titers without any signs of inhibition or toxicity and could use them to generate progeny vectors containing a functional rep78 gene without any Ad genes. Using this vector recombination system in AAV rescue assays demonstrated that no Ad protein was essential for Rep78 mediated rescue of AAV ITR flanked DNA from plasmid or Ad backbones; the amount of rescue product generated was substantially greater in the presence of Ad infection; neither cellular nor viral DNA replication was necessary for rescue to occur; and progeny vector genomes were efficiently co-replicated along with conventional, first-generation Ad vectors.

Hepatic DR5 induces apoptosis and limits adenovirus gene therapy product expression in the liver.

A major limitation of adenovirus (Ad) gene therapy product expression in the liver is subsequent elimination of the hepatocytes expressing the gene therapy product. This elimination is caused by both necrosis and apoptosis related to the innate and cell-mediated immune response to the Ad. Apoptosis of hepatocytes can be induced by the innate immune response by signaling through death domain receptors on hepatocytes including the tumor necrosis factor alpha (TNF-alpha) receptor (TNFR), Fas, and death domain receptors DR4 and DR5. We have previously shown that blocking signaling through TNFR enhances and prolongs gene therapy product expression in the liver. In the present study, we constructed an Ad that produces a soluble DR5-Fc (AdsDR5), which is capable of neutralizing TNF-related apoptosis-inducing ligand (TRAIL). AdsDR5 prevents TRAIL-mediated apoptosis of CD3-activated T cells and decreases hepatocyte apoptosis after AdCMVLacZ administration and enhances the level and duration of lacZ transgene expression in the liver. In addition to blocking TRAIL and directly inhibiting apoptosis, AdsDR5 decreases production of gamma interferon (IFN-gamma) and TNF-alpha and decreases NK cell activation, all of which limit Ad-mediated transgene expression in the liver. These results indicate that (i) AdsDR5 produces a DR5-Fc capable of neutralizing TRAIL, (ii) AdsDR5 can reduce activation of NK cells and reduce induction of IFN-gamma and TNF-alpha after Ad administration, and (iii) administration of AdsDR5 can enhance Ad gene therapy in the liver.

Efficient integration of recombinant adeno-associated virus DNA vectors requires a p5-rep sequence in cis.

The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.