Ad5 Fiber Research Articles

145. Evaluation of Biodistribution and Safety of Adenovirus Vectors Containing B-Group Fibers after Intravenous Injection into Baboons

Top of pageAbstract Vectors containing group B adenovirus (Ad) fibers have been shown to efficiently transduce gene therapy targets that are refractory to infection with standard Ad serotype 5 (Ad5) vectors, including malignant tumor cells, hematopoietic stem cells, and dendritic cells. Preliminary studies in mice indicate that B-group fiber containing Ads do not efficiently transduce most organs and cause less acute toxicity than Ad5 vectors after intravenous injection. However, biodistribution and safety studies in mice are of limited value because the mouse analogue of the B-group Ad receptor, CD46, is expressed only in the testis, while in humans, CD46 is expressed on all nucleated cells. Unlike mice, baboons have CD46 expression patterns and levels that closely mimic those in humans. We conducted a biodistribution and toxicity study of group B Ad vectors in baboons. Animals received intravenous injection of PBS, a standard Ad5 vector, or chimeric vectors containing B-group Ad11 or Ad35 fibers (Ad5/35 or Ad5/11) at dose of 2|[times]|1012 vp/kg and biodistribution and safety was analyzed over 3 days. Analysis of Ad5/35 and Ad5/11 vector genome distribution by quantitative PCR revealed uptake into most tissues at levels 1-3 orders of magnitude below that of Ad5. Significant Ad5/35 and Ad5/11-mediated transgene (b-galactosidase) expression was only seen in the marginal zone of splenic follicles. Compared to the animal that received the Ad5 vector, all animals injected with B-group fiber containing Ad vectors had lower elevations in serum pro-inflammatory cytokine levels. Gross and histopathology were normal in animals that received B-group Ad fiber containing Ads, in contrast to the Ad5 infused animal which showed widespread endothelial damage and inflammation (which is in agreement with studies performed by others). In a further study, a chimeric Ad5/35 vector carrying pro-apoptotic TRAIL and Ad E1A genes under tumor-specific regulation was well tolerated in a 30 day toxicity study. No major clinical, serologic or pathologic abnormalities were noticed in this animal. Overall, this study suggests that Ad vectors possessing B-group Ad fibers have a better safety profile after intravenous injection than conventional Ad5-based vectors. This makes them potential candidates for gene therapy, for example in treatment of metastatic cancer or cardiovascular diseases.

1091. Analysis of T-Cell Responses to Helper-Dependent Serotype 5-Based Capsid-Modified Adenovirus (HD.5/35) or First-Generation Serotype 11- Based (Ad11) Vectors

Top of pageAbstract Cellular immunity to adenovirus serotype 5 (Ad5) is an obstacle for Ad5-based gene therapy. We asked if this could be overcome by either a helper-dependent capsid-modified Ad5 vector possessing the Ad35 fiber (HD.5/35), or a first-generation vector completely derived from Ad serotype 11 (Ad11). Ad35 and Ad11 fiber containing Ad vectors efficiently transduce dendritic cells (DC) in vitro and are being evaluated for immunotherapy strategies. We transduced human peripheral blood mononuclear cells (PBMC) from 11 donors (including cancer patients) with GFP expressing Ad11 or HD.5/35 vectors, or first generation Ad5, Ad5/35 or Ad5/11 vectors as controls. Regardless of the vector used, all donors showed IFN-|[gamma]| secreting cells, detected by ELISpot assays (200|[ndash]|400 spots in 1|[times]|10e6 PBMC). The Ad-specific T-cells against the HD vector indicates a response to Ad capsid epitopes from the incoming particles. It was speculated that this could interfere with responses to a vaccination or therapy antigen expressed from Ad vectors. We evaluated this in vivo and in vitro. For in vivo studies, we injected C57Bl/6 mice transgenic for CD46 (the receptor for Ad5/35) with Ad vectors expressing the model antigen hepatitis B virus e antigen (Ad5.HBeAg, Ad5/35.HBeAg and HD.5/35.HBeAg). Fourteen days later, their splenocytes were mixed with splenocytes from na|[iuml]|ve mice in vitro pulsed with HBeAg recombinant protein, or transduced with the same or control Ad vectors. A strong anti-Ad T cell response was detected by an IFN-|[gamma]| ELISpot assay irrespective of the vector used. The frequency of HBeAg specific T cells was similar with all the vectors tested, and at least two orders of magnitude lower than that to the Ad vectors. For the in vitro human studies, we used the HD.5/35.Her2.Fc vector. It expresses a target antigen in breast cancer, Her-2/neu. We stimulated twice 5|[times]|10e7 PBMC from a normal donor with 5|[times]|10e6 transduced and matured DC. The T cell line was then stimulated with DC pulsed with a library of 168 15mer-overlapping peptides covering the extracellular domain (ECD) of Her2/neu, or transduced with a control vector (HD.5/35.GFP), or pulsed with an Ad5 hexon-derived peptide or other control peptides in an IFN-|[gamma]| ELISpot plate. A strong T cell response against Ad transduced DC and against the hexon epitope was observed. No ECD specific T cell was detected. In conclusion, i) we found a strong T cell response to Ad11 vectors, which was surprising considering previous studies from our lab and others that showed a low serum-prevalence of neutralizing anti-Ad11 antibodies in humans; this would indicate either pre-existing T-cell immunity to Ad11 or, most likely, T-cell cross-reactivity between Ad serotypes; ii) the use of HD vectors did not prevent induction of anti Ad T cells in na|[iuml]|ve animals, and the incoming Ad particles activated memory T cell responses in in vitro human studies; iii) we could not detect strong antigen specific immune responses in our experiments. Then, these studies support the idea that cellular immunity, directed to the Ad capsid epitopes, is an important problem to be solved for antigen specific vaccination/immunotherapy purposes.

528. Infectivity Enhancement of a Genetic Fiber Mosaic Adenovirus Vector in Ovarian Cancer Cells

Despite aggressive surgical debulking and chemotherapy, the 5-year survival rate for advanced ovarian cancer is very low. Thus, gene therapy is a promising treatment alternative; however, clinical cancer gene therapy trials with recombinant human adenovirus (Ad) serotype 5 have seen limited success. The efficiency of Ad gene transfer to ovarian cancer cells has been limited by low expression of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR). Thus, development of novel Ad vectors providing CAR-independent tropism may lead directly to therapeutic gain. We have previously shown that reovirus |[sigma]|1, a fiber-like attachment protein, incorporated into Ad5 virions, contributed to achieve infectivity enhancement in ovarian cancer cells via CAR-independent manner. Also we have reported that ovarian cancer cells possess a primary receptor for Ad3, which is independent from CAR. We hypothesized that an Ad5 vector containing both reovirus and Ad3 fibers would result in expanded tropism based on cellular expression of CAR independent receptors, thereby providing increased gene delivery to ovarian cancer cells. We have constructed a mosaic fiber Ad5 vector, designated Ad5/3-|[sigma]|1, encoding a luciferase reporter gene and two fibers. The first fiber is the chimeric fiber possessing the reovirus |[sigma]|1 and the second fiber is the chimeric fiber possessing the Ad3 fiber knob. Virus particles were purified and characterized by standard manners. Ad5/3-|[sigma]|1 gene delivery, as measured by luciferase transgene activity, was analyzed in a panel of ovarian cancer cell lines and primary ovarian cancer tissues. Characterization of Ad5/3-|[sigma]|1 confirmed the incorporation of both fibers in the same virus particle. Functionally, Ad5/3-|[sigma]|1 was able to utilize primary receptors for both reovirus and Ad3 for attachment to cells. Ad5/3-|[sigma]|1 provided 28 to 62-fold greater gene transfer versus the isogenic control Ad5 vector in a panel of ovarian cancer cell lines, known for relatively low CAR expression. Additionally, Ad5/3-|[sigma]|1 yielded 4.5-fold higher gene transfer to primary ovarian cancer tissue slices. We developed an infectivity enhanced Ad5 vector to address the key issue of limited Ad5 vector efficiency. The expanded tropism of the Ad5/3-|[sigma]|1 represents a crucial attribute for an Ad-based gene therapy vector for ovarian cancer, and may represent an agent with future clinical utility.

30. AdenoLibrary for Construction and Selection of Targeted Adenoviruses

Top of pageAbstract Vector systems derived from the human adenovirus serotype 5 (Ad5) have shown promise as gene delivery vehicles for a variety of clinical applications. However, the paucity of the Ad5 primary receptor (coxsackie and adenovirus receptor, CAR) on target cells of interest has rendered many tissues refractory to Ad-mediated transduction. Additionally, this endogenous tropism may potentially lead to undesired transduction of tissues expressing CAR. In view of this, strategies have been proposed to modify Ad5 to achieve targeted gene delivery, by ablating native tropism while redirecting Ad to target cell markers of interest. Such strategies involve modification of the trimeric capsid fiber ligands tend to lose specificity and/or affect virion integrity. As an alternative, several so-called knob-less fiber platforms have been utilized. These protein, whose C-terminal knob domain has been shown to dictate the native tropism associated with Ad5. While approaches employing genetic fusion of simple targeting ligands to the fiber have shown promise in retargeting, in many cases, the incorporated modifications are based on the concept of replacing the native fiber with an alternative protein capable of providing the trimerization functions that the knob usually confers. Such a strategy would accrue the additional benefit of an expanded repertoire of incorporable ligands, based on removal of the structural constraints imposed by the fiber knob. Previously, our group has utilized the bacteriophage T4 fibritin protein for fiber replacement, with subsequent display of complex targeting ligands providing for expanded tropism. However, vectors employing these knob-less fiber platforms often exhibit poor capsid incorporation of the alternate fibers in comparison to that of wild-type Ad5 fiber, which consequently leads to limited gene transfer efficacy and difficulty in vector propagation. In view of these obstacles, we have hypothesized that direct analysis of modified adenoviruses during their rescue/propagation is the only straightforward approach that guarantees the selection of functional, targeted vectors with optimized structural integrity and targeting efficiency. Herein, we describe a new schema for Ad vector construction, which allows for the selection of vectors with the optimal modified capsid configuration at the level of viral rescue. Based on our first generation fiber-fibritin molecule, several new fibers employing variant amounts of fibritin and Ad5 fiber shaft were analyzed via this library selection schema. Our selected variant, composed of the entire Ad5 fiber shaft connected to 12th coiled-coil segment of fibritin, is capable of efficient fiber incorporation, ligand display, and CAR-independent transduction. Furthermore, we have concluded that various targeting ligands require individual optimization, which may be accomplished by utilizing this adenoviral library approach.

29. Redirection of Adenoviral Tropism through Capsomers Other Than Fiber Is Generally Inefficient and Dependent on Ligand-Receptor Biology

We have generated a panel of metabolically biotinylated adenoviral vectors based on the genetic insertion of a biotin acceptor peptide (BAP) into the fiber, protein IX, and hexon capsomers of the Ad5 virion. Here we demonstrate that avidin-based redirection of Ad transduction through biotinylated ligand:receptor interactions is greatly dependent on the nature of the BAP-modified capsomer. While transduction of Ad-Fiber-BAP can generally be targeted through a wide range of ligand:receptor pairs, Ad-IX-BAP and Ad-Hexon-BAP are surprisingly inefficient as targeting platforms, despite having a higher display of biotins than Ad-Fiber-BAP. We reason this difference is due to the nature of the interactions between viral capsomers and cell surface receptors. Ad5 fiber, the natural cell-binding capsomer, has evolved to bind very tightly to cell surface CAR. Upon endosomal uptake fibers then dissociate from the capsid, releasing the virion from the receptor and enabling trafficking to continue. We believe that targeting Ad through IX or hexon capsomers results in the entrapment of virions on cellular receptors thereby leading to very inefficient transduction. Indeed, preliminary evidence suggests that protein IX stays tightly associated with the virion and does not dissociate at early stages of vector trafficking like fiber. We reason that targeted transduction through IX or hexon may be feasible with ligands that naturally release from their receptors upon endocytosis, resulting in release of the incoming virions. These initial results highlight the importance of knowledge of capsomer, ligand, and receptor biology when engineering new tropism into viral capsids and may be of great importance for vectors like AAV, which do not shed capsomers as they traffic. Current progress on the analysis of the differences in trafficking between the different BAP-modified vectors will be presented.

886. An Adenovirus Vector with a Chimeric Fiber Derived from Ovine Adenovirus for Novel Tropism Discovery

Gene therapy vectors based on human adenovirus (Ad) serotypes 2 and 5 continue to show increasing promise as gene therapy delivery vehicles, particularly in the context of cancer gene therapy. However, many clinically important tissues are refractory to Ad5 infection, including numerous cancer tissue types, due to reduced levels of the coxsackie and adenovirus receptor (CAR). Thus, development of novel Ad vectors demonstrating CAR-independent tropism may lead directly to therapeutic gain. Ovine adenovirus type 7, isolate 287 (OAdV7-287), has recently been described as the prototype of the new genus Atadenovirus, which contains several animal Ads. OAd287 encodes a fiber molecule containing 25 repeating units, and has a unique C-terminal knob domain of 121 amino acids. OAdV7-287 abortively infects several human, rodent and rabbit cell lines in vitro, and efficiently transduces human cells in vitro. Unlike Ad5, however, systemically administered OAd-based vectors do not predominately localize to the liver in rodents, but are distributed more evenly among the major organs. Based on these observations, we hypothesized that replacement of the Ad5 fiber with the non-human OAdV7-287 fiber would result in an Ad5 vector with a novel, CAR-independent, tropism in vitro and liver |[ldquo]|detargeting|[rdquo]| in vivo. We constructed an E1-deleted, luciferase transgene Ad5 vector encoding a chimeric fiber from ovine adenovirus 7-287 that includes the Ad5 tail region (Ad5/OAdV7), utilizing fiber-switching technology established in our laboratory. Viral particles were harvested from 911 cells, purified by standard cesium chloride gradient centrifugation and yielded titers in excess of 2.7|[times]|10e12 vp/mL. Characterization of Ad5/OAdV7 vector infectivity in CAR-deficient CHO Chinese hamster ovary cells, low-CAR MCF7 human breast cancer cells, T24 human bladder cancer cells and PC-3 human prostate cancer cells showed 10-to-30 fold greater luciferase reporter gene activities versus the isogenic AdLuc1 control vector. Furthermore, competitive inhibitory knob blocking assays in high-CAR cells using Ad5 recombinant knob protein demonstrated these increases to be independent of CAR receptor binding. Herein, we have demonstrated the construction, rescue, purification, and initial infectivity characterization of a novel CAR-independent, infectivity enhanced chimeric fiber Ad vector. These data strongly suggest that our genetic xeno-fiber paradigm alters Ad vector tropism, a key gene therapy vector design principle.

138. Blood Factors Affect Adenovirus Infectivity and Bio-Distribution In Vivo

An incomplete understanding of adenovirus (Ad) interactions with host factors has significantly hampered application of Ad-based vectors for human gene therapy. This is particularly true following intravascular delivery, when the bulk of the vector dose is rapidly cleared from the blood and transduction of liver tissue and innate immune responses contribute to the overall poor performance of Ad as a therapeutic vector. Accumulating data suggest that liver-mediated Ad clearance from the blood and Ad infection of liver cells do not occur through the classical model of Ad infection, which relies on the presence of the coxsakie- and adenovirus receptor, CAR, and alpha-v integrins. Both, CAR and/or integrin binding-ablated vectors exert dramatic innate immune responses and are rapidly cleared by the liver upon systemic application. Recently, we discovered that blood coagulation factor IX directly binds to the Ad fiber knob domain and mediates hepatocyte and Kupffer cell infection in vivo through interaction with novel hepatocellular receptors. Our data, however, indicate that in addition to FIX, other blood factors are likely to contribute to Ad liver cell infection. To study in a systematic way host factors interacting with Ad upon intravascular application, we incubated freshly purified mouse plasma with recombinant Ad5 or Ad35 fiber knob domains immobilized on agarose beads, and Ad fiber knob-bound proteins were recovered and analyzed by tandem mass spectrometry. These analyses revealed that multiple plasma proteins efficiently bind to the Ad fiber knob domain. We confirmed that most of them (including alpha-2 macroglobulin, fibrinogen, complement C3, and complement C4-binding protein, C4BP) are capable of direct binding to Ad fibers in a slot-blot assay. To investigate whether any of the identified Ad-binding plasma proteins can mediate liver cell infection, we infected human hepatocarcinoma cells with an Ad5F|[ast]| vector, possessing a single point mutation abrogating its binding to CAR. While supplementation of Ad5F|[ast]| virus containing media with alpha-2 macroglobulin, fibrinogen or complement C3 resulted in very low background levels of infection, supplementation of media with C4BP completely restored Ad5F|[ast]| infectivity toward these cells. Additionally, we confirmed that C4BP-mediated infection was dose dependent, mediated by the fiber knob domain, and could be efficiently competed with ligands that bind heparan sulfate proteoglycans and LDL-related receptor protein, LRP. C4BP is one of the most abundant plasma proteins, present at an average concentration of 250 |[mu]|g/ml. Clearly, upon intravascular administration, Ads will be exposed to large amounts of C4BP capable of targeting them to liver cells. While further studies should show whether Ad binding to other blood factors is of any physiological significance, our data indicate that there is a complex network of interactions between Ad and plasma proteins, that contribute to the observed pharmacokinetics, bio-distribution and toxicity after intravascular application. Our study helps better understand Ad-host interactions in vivo, when vector applied systemically. It also provides the basis for developing rational approaches toward construction of safe and efficient Ad vectors for human gene therapy applications.

529. Oncolytic Virotherapy Employing flt-1 Driven mda-7/IL-24 Gene Delivery

Top of pageAbstract Vascular-targeted anticancer therapy using conditionally replicating adenoviruses (CRAd) is an attractive strategy for cancer treatment. For specific retargeting of CRAd, selective transcription of viral genes utilizing tumor-specific promoters has been developed. CRAd targeting using insertion of the tumor-specific binding RGD ligand in the Ad fiber has shown increased infectivity. A cancer growth-suppressing gene, mda-7/IL-24, has demonstrated profound and selective anticancer activity. The central hypothesis of this study was that selective transcriptional and transductional targeting of CRAd encoding the mda-7/IL-24 gene using flt-1 promoter for specific E1a expression and RDG-4C modification of the Ad fiber-knob region can increase efficacy via direct killing of tumor endothelial cells and flt-1-positive cancer cells. Specific mda-7/IL-24 overexpression should enhance the anti-tumor efficacy of CRAd oncolytic virotherapy alone. The flt-1 promoter-driven firefly luciferase expression was determined using replication-defective Adflt-Luc and AdCMV-Luc vectors on a panel of human glioma, breast, ovarian and prostate cancer cells and human or mouse endothelial cells. The cytotoxicity of cancer and endothelial cells to CRAd infection was analyzed using the crystal violet inclusion assay. The CRAd progeny production was determined using real time PCR and a standard 50% tissue culture infectious dose assay. Tumor xenograft models were used to determinate the therapeutic efficacy of oncolytic virotherapy to treat human glioma, breast, ovarian and prostate tumors. Specificity of flt-1 promoter for human and mouse endothelial cells and U251MG glioma, PA-1 ovarian, LCC6 breast, and DU145 prostate cancer cells was demonstrated. We have developed a CRAd using the flt-1 promoter element for specific E1a gene expression, encoding the mda-7/IL-24 gene (CRAdRGDflt-mda-7) and a CDCGRDCFC-containing peptide in the HI loop of the fiber knob domain. The cancer cells demonstrated different levels of cytotoxicity after CRAdRGDflt-mda-7 infection. There was a significant decrease in viability of U251MG, PA-1, LCC6, and DU145 cells infected with CRAdRGDflt-mda-7. In vivo therapy studies demonstrated that intratumoral injection of CRAdRGDflt-mda-7 produced significant inhibition of subcutaneous LCC6 breast, OVCAR-3 ovarian, and DU145 prostate cancer xenografts and delay of D54MG human glioma tumor growth in athymic nude mice. These results suggest that selective targeting of oncolytic virotherapy with enhanced potency via mda-7/IL-24 pro-apoptotic/anti-angiogenic gene expression would be a superior strategy for treatment of human cancer.

28. Adenovirus Targeting Via Leucine Zipper Peptide-Mediated Ligand Attachment

Gene therapy vectors based on human adenovirus (Ad) serotypes 2 and 5 continue to show increasing promise as gene therapy delivery vehicles. Initial gene therapy efforts, however, revealed that the clinical benefit of these gene delivery modalities was dependent on localization of the therapeutic agent. Therefore, a fundamental requirement for somatic cell gene therapy is targetable gene delivery vectors. Ad vectors incorporating genetic modifications have provided gains in vector efficiency; however, specific vector targeting to specific cell-surface molecules is still required. For cell-specific targeting, the use of antibodies or single chain antibody fragments (scFv) remains a very attractive concept made possible by the definition of disease-specific membrane antigens and a wide selection of cognate antibody molecules. However, a barrier to the development of Ad vectors capsid-incorporated scFvs occurs when the antibody requires posttranslational modifications not available to Ad capsid proteins due to their cytoplasmic routing during virion assembly. To address this problem, we have developed a targeting strategy that uses a secreted scFv that undergoes the requisite posttranslational modification (disulfide bond formation) in the endoplasmic reticulum and is trafficked for secretion. Formation of the vector/ligand targeting complex is achieved via genetic tagging of the Ad capsid and scFv with synthetic leucine zipper-like dimerization domains (Zippers) that have been optimized for structural and biological compatibility with the Ad capsid and the secreted ligand. Importantly, we have designed our Zipper domains in pairs, such that only compatible heterodimers can associate, thereby eliminating the possibility of aggregate formation. We have incorporated Zippers into the C-terminus of Ad5 fiber and a knob-deleted chimeric fiber molecule, fiber-fibritin/11F (11F). The ligand is comprised of a scFv fused with the kappa-chain IgG signal peptide and a Zipper peptide. Our results show that Zipper domain-containing fiber or 11F molecules trimerize and associate with the virion penton base protein and that these vectors can be amplified. Further, we demonstrate that peptide Zippers can be genetically fused to scFv without decreasing ligand recognition of antigen. Most important, we show that Zipper-tagged virions and ligands can self-associate and form targeting complexes capable of binding to target receptors. This approach to targeting ligand design solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.

975. Immunization with an Adenovirus Vector Expressing a P. aeruginosa OprF Epitope in the Capsid Enables Boosting of Anti-Epitope Immune Responses and Protects Against Pulmonary Infection with P. aeruginosa

Top of pageAbstract Pseudomonas aeruginosa causes chronic infections of the respiratory tract, particularly in cystic fibrosis (CF). The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and our prior studies have shown that an adenovirus (Ad) vector expressing a secreted form of OprF (AdOprF), evokes protective immunity against P. aeruginosa. The present study is focused on development of a 2nd generation clinical vaccine candidate (AdOprF.RGD.Epi8) by two innovations to AdOprF to enhance the immunogenicity: (1) genetic modification of the Ad hexon to incorporate an epitope (Epi8) from OprF; and (2) genetic modification of the Ad fiber to incorporate an RGD sequence to enhance gene delivery to antigen presenting cells. We first evaluated the immunogenic properties of an Ad vector in which the Ad hexon protein was modified to include the 14-mer OprF epitope Epi8 in the hexon (AdZ.Epi8), with focus on the hypothesis that immune responses against epitopes that are part of the Ad capsid could be augmented with repeat administration. To evaluate the protective effect of immunization with AdZ.Epi8 against pulmonary infection with P. aeruginosa, C57Bl/6 mice, immunized with either AdZ.Epi8 or AdZ were challenged with a lethal dose of agar-encapsulated P. aeruginosa (laboratory strain PAO1 or two isolates of P. aeruginosa derived from patients with CF). Unimmunized mice or mice infected with AdZ died within the first 3 days. In contrast, more than 60% of the mice immunized with AdZ.Epi8 survived more than 2 wk (p 0.1, all comparisons). Similar results were observed in the CD4 Epi8-specific IL-4 as well as CD4 and CD8 Epi8-specific |[gamma]|-interferon cellular responses, suggesting that the addition of an OprF epitope to the Ad capsid enables boosting of the anti-OprF response. Finally, we evaluated the immunogenic properties of the combined AdOprF.RGD.Epi8 vector. Mice received AdOprF.RGD.Epi8, AdOprF, AdNull, (2|[times]|1011pu) or PBS and the anti-OprF antibody titers were determined at 2 and 4 wk. No anti-OprF antibodies were detected in the AdNull or PBS group. The mean titer for AdOprF was 5710 and 8602 at 2 and 4 wk, respectively, vs 11841 and 10632 for the AdOprFRGD.Epi8 injected mice, suggesting a faster and more robust induction of the anti-OprF humoral response with AdOprFRGD.Epi8. We conclude that the capsid modifications in AdOprF.RGD.Epi8 vector make it a more effective candidate vector for vaccination against P. aeruginosa.

533. Enhanced Transduction of Dendritic Cells by FCgRI-Targeted Adenovirus Vectorsγ

The production of an efficacious HIV-1 vaccine capable of inducing the humoral and cellular arms of the immune system continues to remain a major challenge in AIDS research. Dendritic cells (DC) are an attractive target for vaccine delivery because vaccine antigens presented to DC can potentially activate effector cells involved in immune defense. Adenovirus (Ad) has been widely used as a vector to express foreign proteins in a variety of cell types and tissues. However, this broad tropism represents a drawback when selective gene delivery is desired. In particular, inefficient gene delivery to antigen presenting cells (APC) is attributed to the absence of the Coxsackie and Adenovirus receptor (CAR). Based on the hypothesis that Ad vectors with improved infectivity of APC may be able to promote an enhanced immune response against a vectored antigen, our objective is to selectively deliver Ad vectors to APC by directing virus to the high affinity Fc-gamma Receptor I (FcgRI), constitutively expressed on DC. To this end, we constructed a bi-specific targeting molecule, H22sCAR, consisting of the extracellular domain of CAR (sCAR), a bacteriophage T4 fibritin trimerization domain, and a single-chain antibody molecule (H22) specific for the human FcgRI receptor. H22sCAR was produced in a baculovirus expression system, and was demonstrated to bind both the Ad fiber knob protein and cellular FcgRI. Ad vectors pre-complexed with H22sCAR enhanced the delivery of GFP and luciferase reporter genes to FcgRI-expressing cell lines, and to monocyte-derived DC, when compared with Ad alone or Ad targeted with an irrelevant control protein (Her2sCAR). These data support the hypothesis that FcgRI-targeted Ad vectors may enhance the delivery of target antigen to APC, and lend support to future studies on the use of FcgRI-targeted adenovirus vectors for HIV-1 vaccine development.

1029. Integrin and Serotype 3 Receptor-Targeted Virotherapy Using a Telomerase-Selective Oncolytic Adenovirus

Conditionally replicative adenovirus (CRAd) is a cervical cancer gene therapy strategy which seeks to achieve selective viral replication in tumor cells. Oncolysis of infected cells creates a |[ldquo]|bystander effect|[rdquo]|, leading to infection of neighboring cancer cells with progeny virus. However, cervical cancer cells are relatively resistant to infection by adenovirus (Ad), thus limiting the capability of CRAd agents to achieve effective lateralization within the tumor. The objective of this proposal was to establish an efficient and selective infectivity-enhanced CRAd-based therapeutic modality for cervical cancer. We have developed methods to dramatically improve the ability of Ad to infect cervical cancer cells by replacing the knob region of Ad serotype 5 fiber with Ad serotype 3 knob (Ad5/3 chimera) and by putting an Arg-Gly-Asp (RGD)-4C motif in the HI loop of the Ad fiber knob region (RGD modification). Human telomerase reverse transcriptase (hTERT) promoter, which is active in many cancers but mostly inactive in normal organs including the liver, maintains this useful profile in our CRAd construct for cervical cancer. We employed recombinant Ad vectors containing luciferase reporter genes under the control of cytomegalovirus (CMV) promoter with or without fiber mutation (Ad5/3LucRGD and Ad5Luc) to evaluate transductional activity in cervical cancer cell lines in vitro. Then, we evaluated the oncolytic effect of hTERT-CRAd (Ad5/3hTERT E1-RGD and AdhTERT E1) in cervical cancer cell lines by both an MTS assay and crystal violet assay. hTERT-CRAd showed selective oncolysis in hTERT-positive cervical cancer cells but not in the hTERT negative, normal cells. Ad5/3Luc-RGD Ad showed increased reporter gene expression in cervical cancer cell line, HeLa (12.7 times more compare to Ad5Luc in a luciferase assay). Moreover, oncolysis with hTERT-CRAd in cervical cancer cells was much enhanced in Ad5/3RGD retaining the specificity both in a MTS assay and crystal violet assay. These results suggest that infectivity enhancement with mutant fiber is especially effective for enhancing oncolysis. This infectivity-enhanced hTERT-CRAd may thus be useful in the gene therapy of cervical cancer.

541. Oncolytic Effects of Conditionally Replicating Adenoviruses (CRAds) with Mutations in E1A and E1B Regions

Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for the treatment of cancer. For the aim of improving adenoviral vectors for cancer gene therapy, we have constructed genetically attenuated replicating adenoviruses (Ads) with different combinations of E1A & E1B mutant genes and investigated the possibility of enhanced oncolytic and replication effects of these engineered replication-competent Ads. We show here that cytolytic potency of each gene attenuated replicating adenoviruses differed significantly depending on the presence or deletion of E1B55 kDa and E1B19 kDa function. More specifically, among the constructed (Ad-DE1B19, Ad-DE1B55, Ad-DE1B19/55, and Ad-wt), E1B19 kDa-inactivated adenovirus (Ad-DE1B19) was the most potent against all tumor cells tested, inducing the largest-sized plaques and marked CPE. As an additional effort to increase cancer cell-selectivity of replicating adenovirus, we have generated eleven E1A-mutant Ads (Ad-mt|[num]|1 |[sim]||[num]|11) with deletion or substitution in retinoblastoma (Rb) binding sites of E1A. Of these viruses, Ad-mt|[num]|7 demonstrated significantly improved CPE and viral replication in a cancer cell-specific manner. To further increase the cancer cell-specific cell killing effect of Ad-mt|[num]|7 adenovirus, both E1B19 kDa and E1B55 kDa gene was deleted, resulting in an Ad-|[Delta]|B7 Ad. As assessed using CPE assay, MTT assay, and Ad fiber immunoblot analysis, Ad-|[Delta]|B7 exerted markedly enhanced cancer cell-specific killing effect as well as viral replication in comparison to either Ad-mt|[num]|7 or Ad-|[Delta]|E1B19/55. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-DB7. In summary, we have developed replication competent adenoviruses with significantly improved therapeutic profiles for cancer treatment.

CryoEM Structure at 9 Å Resolution of an Adenovirus Vector Targeted to Hematopoietic Cells

We report a sub-nanometer resolution cryo-electron microscopy (cryoEM) structural analysis of an adenoviral vector, Ad35F, comprised of an adenovirus type 5 (Ad5) capsid pseudo-typed with an Ad35 fiber. This vector transduces human hematopoietic cells via association of its fiber protein with CD46, a member of the complement regulatory protein family. Major advances in data acquisition and image processing allowed a significant improvement in resolution compared to earlier structures. Analysis of the cryoEM density was enhanced by docking the crystal structures of both the hexon and penton base capsid proteins. CryoEM density was observed for hexon residues missing from the crystal structure that include hypervariable regions and the epitope of a neutralizing monoclonal antibody. Within the penton base, density was observed for the integrin-binding RGD loop missing from the crystal structure and for the flexible beta ribbon of the variable loop on the side of the penton base. The Ad35 fiber is flexible, consistent with the sequence insert in the third beta-spiral repeat. On the inner capsid surface density is revealed at the base of the hexons and below the penton base. A revised model is presented for protein IX within the virion. Well-defined density was assigned to a conserved domain in the N terminus of protein IX required for incorporation into the virion. For the C-terminal domain of protein IX two alternate conformations are proposed, either binding on the capsid surface or extending away from the capsid. This model is consistent with the tolerance of the C terminus for inserted ligands and its potential use in vector retargeting. This structural study increases our knowledge of Ad capsid assembly, antibody neutralization mechanisms, and may aid further improvements in gene delivery to important human cell types.

The Role of the Extracellular Domain in the Biology of the Coxsackievirus and Adenovirus Receptor

The Coxsackievirus B and Adenovirus Receptor (CAR) plays a dual role as a homotypic junctional adhesion protein and as a viral receptor. CAR is a transmembrane protein and a member of the Immunoglobulin (Ig) superfamily with two extracellular Ig-like domains. The most distal Ig-like domain (D1) mediates the homophilic interaction and is also responsible for the high-affinity binding of the adenovirus (Ad) fiber protein. Currently, no activity has been ascribed to the proximal Ig-like domain (D2). To further understand the function of the extracellular domain in the biological activities of CAR, we created extracellular deletion mutants and evaluated cellular localization, adhesion, and viral infection. Deletion of any segment of the extracellular domain results in loss of adhesion and mislocalization as explained by a model, termed "diffusion trapping," that suggests adhesion is the driving force in junctional localization. Loss of junctional localization and adhesion was particularly apparent in polarized human airway epithelia, where mutant CAR expression was basolateral but not limited to the lateral junctions between cells. Surprisingly, the D2 domain was required for adenovirus fiber-knob binding and infection. In summary, the entire extracellular domain of CAR is of vital importance to the biology of this highly conserved and important protein.

Complex mosaicism is a novel approach to infectivity enhancement of adenovirus type 5-based vectors

The use of adenovirus type 5 (Ad5) for cancer therapy is limited by deficiency of its primary cell attachment receptor, coxsackie and adenovirus receptor (CAR), on cancer cells. Ad5 retargeting to alternate receptors through fiber genetic modification can be used to circumvent CAR dependence of its tropism, and thereby achieve infectivity enhancement. Here we propose and test a novel "complex mosaicism" approach for fiber modification, which combines serotype chimerism with peptide ligand(s) incorporation in a single-fiber molecule. We incorporated integrin-binding peptide RGD-4C in the HI-loop, at the carboxy (C)-terminus, or both locales of the Ad3 knob, in the context of Ad5/3 chimera fiber in order to retarget simultaneously the Ad vector to integrins and Ad3 receptors. The infectivity enhancement of the fiber modifications was assessed in various cancer cell lines as cancer-targeting models. Replication-defective complex mosaic Ad-luc vectors bearing chimeric fiber (F.5/3), with or without C-terminal RGD-modification of Ad3 knob, demonstrated up to 55-fold gene transfer increase in bladder cancer cell lines. Although this augmentation was primarily due to Ad3 receptor targeting, some contribution of RGD-mediated integrin-targeting was also observed, suggesting that complex mosaic modification can function in a dual-receptor targeting via a single Ad3 fiber knob.

Deletion of Penton RGD Motifs Affects the Efficiency of both the Internalization and the Endosome Escape of Viral Particles Containing Adenovirus Serotype 5 or 35 Fiber Knobs

Adenovirus (Ad) vectors are widely used for gene delivery in vitro and in vivo. A solid understanding of the biology of this virus is imperative for the development of novel, effective, and safe vectors. For the group C adenovirus serotypes 2 and 5 that use CAR as a primary attachment receptor, it is known that the penton base RGD motifs interact with cellular integrins and that this interaction promotes virus internalization. However, the RGD motif's impact on the efficiency of postinternalization steps, such as the escape of the virus particle from the endosome, is less defined. Furthermore, the role of penton-integrin interactions remains unknown for new vectors possessing group B Ad fiber knobs that use CD46 as a primary virus attachment receptor. In this study, we used vectors with the RGD motif deleted that contained Ad5 and B-group Ad35 fiber knobs and long fiber shafts and studied the role of RGD-integrin interactions in virus internalization and endosome escape. The deletion of the RGD motif in the penton base did not affect virus attachment, regardless of the type of cellular receptor used for attachment. RGD motif deletion, however, significantly reduced the rate of virus internalization for both the Ad5 and Ad35 fiber knob-containing vectors. This study also demonstrates the role of penton RGD motifs in facilitating the endosome escape step of virus infection and indicates that penton-integrin interactions are involved in internalization of capsid-chimeric CD46-interacting Ads with long fiber shafts.

Development of Adenoviral Vectors for Detection of Proliferation and Differentiation Stage Dependent Telomerase Reverse Transcriptase Expression in Single Living Hematopoietic Cells.

Elevated telomerase activity is an important molecular signature for proliferating hematopoietic progenitors, lymphocytes and a variety of leukemic cells. The human telomerase activity is predominantly determined by the transcriptional control of the catalytic subunit telomerase reverse transcriptase (hTERT). For prospective identification of single living hematopoietic cells with telomerase activity, we have developed telomerase-reporting adenoviral vectors encoding destabilized enhanced green fluorescence protein with a half-life of 2 hours (d2EGFP) driven by the hTERT promoter. To shield the hTERT promoter from adenoviral backbone cis-acting DNA sequences, vectors encoding the chicken β-globin locus HS4 insulator sequence or bovine growth hormone transcription stop signal were also engineered. Moreover, these vectors were retargeted with the Ad35 fiber receptor specificity (Ad5F35 vectors) and utilize ubiquitously expressed CD46 as a cellular receptor. The Ad5F35 vectors allow efficient gene delivery into hematopoietic cells (J Gene Med 6:631, 2004). The telomerase activity dependent d2EGFP expression was demonstrated in telomerase positive HeLa, A549 or negative fibroblasts and WI-38 cells by infection with the telomerase-reporting vectors at 2000 physical particles per cells. Successful gene delivery was achieved as about 90% of the cells were EGFP+ following infection with control Ad5F35-CMV-EGFP vector. Between 40 to 95% of the individual HeLa and A549 cells were d2EGFP+, and the percentages of the d2EGFP+ cells positively correlated with the extent of the telomerase activity as assessed in Trap assay. In contrast, a barely detectable fraction of the WI-38 cells and lower than 6% of the fibroblasts were expressing lower levels of d2EGFP. The vector carrying the HS4 insulator sequence generated the lowest background d2EGFP expression in fibroblasts and WI-38 cells (called cTERTdGFP vector). Therefore the cTERTdGFP vector was used for all the subsequent studies. Importantly, no measurable d2EGFP expression was observed in normal primary myeloid cells. The cTERTdGFP vector mediated d2EGFP expression in hematopoietic cells is dynamically regulated during cell proliferation and differentiation. Promyelocytic leukemic HL-60 cells were infected with the cTERTdGFP or the control Ad5F35-PGK-EGFP vector. Nearly 100% of the HL-60 cells were EGFP+ following the control vector infection, however, around 50% of the HL-60 cells were d2EGFP+ following cTERTdGFP vector infection. The d2EGFP expression was reduced 2-fold with lower d2EGFP intensity in retinoic acid induced differentiating HL-60 cells compared with non-treated HL-60 cells. HL-60 cells expanded from sorted d2EGFP+ or d2EGFP- cells generated identical frequencies of d2EGFP+ and d2EGFP- cells following re-infection with the cTERTdGFP vector. Cell cycle analysis assessing cellular DNA content in combination with Ki-67 antigen staining or BrdU pulsing showed that the sorted d2EGFP+ HL-60 cells contained significantly fewer cells in the G1, more cells in the S/G2/M phase of cell cycle compared with the d2EGFP- HL-60 cells. In conclusion, our studies can provide a powerful tool for isolation of living normal hematopoietic or leukemic cells with telomerase activity from heterogeneous cell populations.

Comparison of the Efficiency of Transduction of Leukemic Cells by Fiber-Modified Adenoviruses

Efficient gene transfer with adenoviral type 5 (Ad5) vectors depends on the initial attachment of their fiber, which binds the coxsackie-adenovirus receptor (CAR), and their subsequent internalization, mediated by the interaction of viral penton base with target cell alphav integrins. We previously demonstrated that human leukemic cells lack these receptors and are therefore resistant to Ad5 transduction, limiting efforts to genetically modify these cells. Human leukemic blasts are, however, susceptible to transduction with an adenovector made CAR independent by substitution of a chimeric Ad5/35 fiber [Yotnda et al. (2001). Gene Ther. 8, 930-937]. Other receptors can also be targeted with recombinant ligand moieties incorporated into adenovirus fiber. We have determined which of these fiber-modified adenovectors is most effective at modifying human primary leukemia cells, and lines. We used a replication-incompetent Ad5-beta-gal vector, in which the Ad5 fiber was replaced with fiber from various adenovirus serotypes (Ad35 and Ad11), or modified either with variable length polylysine (K4, K7, K21) or RGD-4C peptide. All the modified fiber vectors transduced primary leukemia cells and cell lines more efficiently than Ad5. Polylysine-substituted Ad5F/K21 and peptide-modified Ad5F/RGD vectors were most effective overall (up to 100% efficiency), whereas Ad5F/RGD was the most effective at transducing B cell acute lymphoblastic leukemia cells (90% efficiency). Ad5F/K21 and Ad5F/RGD should be of value for the genetic modification of human primary leukemia cells.

Targeted Adenovirus Vectors

Recombinant adenovirus (Ad) vectors continue to be the preferred vectors for gene therapy and the study of gene function because they are relatively easy to construct, can be produced at high titer, and have high transduction efficiency. However, in some applications gene transfer with Ad vectors is less efficient because the target cells lack expression of the primary receptor, coxsackievirus and adenovirus receptor (CAR). Another problem is the wide biodistribution of vector in tissue following in vivo gene transfer because of the relatively broad tissue expression of CAR. To overcome these limitations, various approaches have been developed to modify Ad tropism. In one approach, the capsid proteins of Ad are modified, such as with the addition of foreign ligands or the substitution of the fiber with other types of Ad fiber, in combination with the ablation of native tropism. In other approaches, Ad vectors are conjugated with adaptor molecules, such as antibody and fusion protein containing an anti-Ad single-chain antibody (scFv) or the extracellular domain of CAR with the targeting ligands, or chemically modified with polymers containing the targeting ligands. In this paper, we review advances in the development of targeted Ad vectors.