Chimeric Fiber Research Articles

Efficient gene transfer into hematopoietic cells by a retargeting adenoviral vector system with a chimeric fiber of adenovirus serotype 5 and 11p

Adenoviral vectors (Ad) were widely used in gene therapy and study of gene function, but the commonly used serotype 5 adenovirus-based vectors (Ad5) could poorly transduce hematopoietic cells because of low expression of viral receptors on these cells. To overcome this limitation, we developed a retargeting adenovector with a chimeric fiber of Ad5 and Ad11p (Ad5F11p) and evaluated its gene transfer ability in hematopoietic cells. An Ad11p fiber pseudotyped Ad5 vector was generated by modifying the fiber gene of pAdEasy-1 backbone plasmid. Ad5F11p-GFP encoding enhanced green fluorescence protein (GFP) gene was transferred into human leukemic cell lines, primary leukemic cells, and CD34(+) hematopoietic cells. The gene transduction efficiency was determined by fluorescence-activated cell sorting assay. More than 90% of U937 or K562 cells could be infected by Ad5F11p-GFP at a moderate multiplicity of infection (MOI). Ad5F11p-GFP is also significantly more effective than control Ad5-GFP in infection of primary myeloid leukemic cells. At 200 MOI, GFP-positive percentages of Ad5F11p-GFP transduced myeloid leukemic cells range from 10.58% to 92.63% with a median of 28.65%. Ad5F11p-GFP could transduce about 50% human hematopoietic stem/progenitor (CD34(+)) cells, while Ad5-GFP could transduce <15% at 200 MOI. CD46 was reported to be the receptor of Ad11p. Our data suggest that CD46 participates in the process of Ad5F11p-GFP infection but is not the unique molecule determining its gene transfer efficiency of host cells. We established a retargeting adenovector system, which could infect hematopoietic cells effectively and would benefit research work on Ad tropism.

Generation and selection of targeted adenoviruses embodying optimized vector properties

The utility of adenovirus serotype 5 (Ad5)-based vectors for gene therapy applications would be improved by cell-specific targeting. However, strategies to redirect Ad5 vectors to alternate cellular receptors via replacement of the capsid fiber protein have often resulted in structurally unstable vectors. In view of this, we hypothesized that the selection of modified adenoviruses during their rescue and propagation would be a straightforward approach that guarantees the generation of functional, targeted vectors. Based on our first generation fiber-fibritin molecule, several new chimeric fibers containing variable amounts of fibritin and the Ad5 fiber shaft were analyzed via a new scheme for Ad vector selection. Our selected chimera, composed of the entire Ad5 fiber shaft fused to the 12th coiled-coil segment of fibritin, is capable of efficient capsid incorporation and ligand display. Moreover, transduction by the resultant vector is independent of the expression of the native Ad5 receptor. The incorporation of the Fc-binding domain of Staphylococcus aureus protein A at the carboxy terminus of this chimeric fiber facilitates targeting of the vector to a variety of cellular receptors by means of coupling with monoclonal antibodies. In addition, we have concluded that Ad5 vectors incorporating individual targeting ligands require individual optimization of the fiber-fibritin chimera, which may be accomplished by selecting the optimal fiber-fibritin variant at the stage of rescue of the virus in cells of interest, as described herein.

Influence of Fiber Detargeting on Adenovirus-Mediated Innate and Adaptive Immune Activation

The major adenovirus (Ad) capsid proteins hexon, penton, and fiber influence the efficiency and tropism of gene transduction by Ad vectors. Fiber is the high-affinity receptor binding protein that serves to mediate cell attachment in vitro when using coxsackie-adenovirus receptor (CAR)-containing cell lines. This contrasts with transduction efficiency in macrophages or dendritic cells that lack high concentrations of CAR. To determine how fiber influences gene transduction and immune activation in a murine model, we have characterized Ad type 5 (Ad5) vectors with two classes of chimeric fiber, CAR binding and non-CAR binding. In a systemic infection, Ad5 fiber contributes to DNA localization and vector transduction in hepatic tissue. However, the majority of vector localization is due to Ad5 fiber-specific functions distinct from CAR binding. CAR-directed transduction occurs but at a modest level. In contrast to CAR binding vectors, the F7 and F7F41S non-CAR-binding vectors demonstrate a 2-log decrease in hepatic transduction, with a 10-fold decrease in the amount of vector DNA localizing to the hepatic tissue. To characterize the innate response to early infection using fiber chimeric vectors, intrahepatic cytokine and chemokine mRNAs were quantified 5 hours postinfection. Tumor necrosis factor alpha mRNA levels resulting from Ad5 fiber infections were elevated compared to viruses expressing serotype 7 or 41 fiber. Levels of chemokine mRNA (gamma interferon-inducible protein 10, T-cell activation gene 3, and macrophage inflammatory protein 1beta) were 10- to 20-fold higher with CAR binding vectors (Ad5 and F41T) than with non-CAR-binding vectors (F7 and F7F41S). In spite of quantitative differences in vector localization and innate activation, fiber pseudotyping did not significantly change the outcome of anti-Ad adaptive immunity. All vectors were cleared with the same kinetics as wild-type Ad5 vectors, and each induced neutralizing antibody. Although non-CAR-binding vectors were impaired in transduction by nearly 2 orders of magnitude, the level of antitransgene immunity was the same for each of the vectors. Using primary bone marrow-derived macrophages and dendritic cells, we demonstrate that transduction, induction of cytokine/chemokine, and phenotypic maturation of these antigen-presenting cells are independent of fiber content. Our data support a model where fiber-mediated hepatic localization enhances innate responses to virus infection but minimally impacts on adaptive immunity.

An adenovirus vector with a chimeric fiber incorporating stabilized single chain antibody achieves targeted gene delivery

Adenovirus (Ad) vectors are of utility for many therapeutic applications. Strategies have been developed to alter adenoviral tropism to achieve a cell-specific gene delivery capacity employing fiber modifications allowing genetic incorporation of targeting motifs. In this regard, single chain antibodies (scFv) represent potentially useful agents to achieve targeted gene transfer. However, the distinct biosynthetic pathways that scFv and Ad capsid proteins are normally routed through have thus far been problematic with respect to scFv incorporation into the Ad capsid. Utilization of stable scFv, which also maintain correct folding and thus functionality under intracellular reducing conditions, could overcome this restriction. We genetically incorporated a stable scFv into a de-knobbed, fibritin-foldon trimerized Ad fiber and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of cells. We have shown that the scFv employed in this study retains functionality and that stabilizing the targeting molecule, per se, is critical to allow retention of antigen recognition in the adenovirus capsid-incorporated context.

A human adenoviral vector with a chimeric fiber from canine adenovirus type 1 results in novel expanded tropism for cancer gene therapy

The development of novel therapeutic strategies is imperative for the treatment of advanced cancers like ovarian cancer and glioma, which are resistant to most traditional treatment modalities. In this regard, adenoviral (Ad) cancer gene therapy is a promising approach. However, the gene delivery efficiency of human serotype 5 recombinant adenoviruses (Ad5) in cancer gene therapy clinical trials to date has been limited, mainly due to the paucity of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human cancer cells. To circumvent CAR deficiency, Ad5 vectors have been retargeted by creating chimeric fibers possessing the knob domains of alternate human Ad serotypes. Recently, more radical modifications based on 'xenotype' knob switching with non-human adenovirus have been exploited. Herein, we present the characterization of a novel vector derived from a recombinant Ad5 vector containing the canine adenovirus serotype 1 (CAV-1) knob (Ad5Luc1-CK1), the tropism of which has not been previously described. We compared the function of this vector with our other chimeric viruses displaying the CAV-2 knob (Ad5Luc1-CK2) and Ad3 knob (Ad5/3Luc1). Our data demonstrate that the CAV-1 knob can alter Ad5 tropism through the use of a CAR-independent entry pathway distinct from that of both Ad5Luc1-CK2 and Ad5/3-Luc1. In fact, the gene transfer efficiency of this novel vector in ovarian cancer cell lines, and more importantly in patient ovarian cancer primary tissue slice samples, was superior relative to all other vectors applied in this study. Thus, CAV-1 knob xenotype gene transfer represents a viable means to achieve enhanced transduction of low-CAR tumors.

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.

328. An Orthotopic Murine Model of Advanced Breast Cancer for Imaging and Comparison of Targeting Moieties

Top of pageAbstract Background: Serotype 5 adenovirus (Ad5) displays high gene transfer efficacy to cells with abundant coxsackie-adenovirus receptor (CAR) expression. However, CAR is variably and often poorly expressed in many tumor types. This has not been comprehensively studied in the context of breast cancer, the most common cancer in women. Fortunately, adenoviruses can be modified to attach to other cellular receptors in order to circumvent the dependence on CAR and to achieve enhanced gene transfer to tumors. Ad5.pK7 (GL) (contains GFP- and luciferase genes) is modified with a polylysine motif allowing CAR independent Ad binding to cell-surface receptors with polyanionic motifs such as heparan sulphate. Ad5lucRGD contains an RGD-4C motif allowing binding to cellular integrins, Ad5.RGD.pK7 (GL) contains both of these modifications and Ad5/3luc1 has a chimeric fiber with the serotype 3 knob and binds to a yet unknown receptor distinct from CAR. Our hypothesis was that we could enhance the infectivity of breast cancer cell lines, clinical samples and orthotopic xenografts by using these targeting moieties. Methods: Gene transfer was studied with firefly luciferase expressing viruses. We also studied cell killing by the respective oncolytic, conditionally replicating adenoviruses (CRAds), featuring a 24 bp deletion in the E1A region, which leads to selective replication in Rb-p16 pathway defective cells. An orthotopic murine model was created by injections of GFP positive MDA-MB-435 cells into mammary fat pads of mice. Results: Some of the modified viruses displayed improved gene transfer to cancer cells in vitro and showed enhanced oncolytic activity over a virus with a wild type capsid. Capsid modified viruses also showed enhanced gene transfer to tumors in a murine model of advanced breast cancer. Conclusions: Mature in vitro and in vivo data will be presented and discussed. In vivo data will include imaging in an orthotopic murine model of advanced breast cancer.

1018. Infectivity Enhancement for Ovarian Cancer and Glioma Via a Novel Human Adenoviral Vector with a Chimeric Fiber Derived from Canine Adenovirus Type 1

The exploitation of novel therapy strategies merits a high priority in the treatment of advanced cancers like ovarian cancer and glioma, which are resistant to most traditional treatment modalities. In this regard, adenoviral (Ad) cancer gene therapy is a promising approach. However, the gene delivery efficiency of human serotype 5 recombinant adenoviruses (Ad5) in cancer gene therapy clinical trials to date has been limited, mainly due to the paucity of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human cancer cells. To circumvent CAR deficiency, Ad5 vectors have been retargeted by creating chimeric fibers possessing the knob domains of alternate human Ad serotypes. Recently, more radical modifications based on |[ldquo]|xenotype|[rdquo]| knob switching with nonhuman adenovirus have been exploited. Herein, we present the characterization of a novel vector derived from a recombinant Ad5 vector containing the canine adenovirus serotype 1 (CAV-1) knob (Ad5Luc1-CK1), the tropism of which has not been previously described. We compared the function of this vector with our other chimeric viruses displaying the CAV-2 knob (Ad5Luc1-CK2) and Ad3 knob (Ad5/3Luc1). Our data demonstrate that the CAV-1 knob can alter Ad5 tropism through the use of a CAR-independent entry pathway distinct from that of both Ad5Luc1-CK2 and Ad5/3-Luc1. In fact, the gene transfer efficiency of this novel vector in ovarian cancer cell lines, and more importantly in patient ovarian cancer primary tissue slice samples, was superior relative to all other vectors applied in this study. Thus, CAV-1 knob xenotype gene transfer represents a viable means to achieve enhanced transduction of low-CAR tumors.

1026. In Vivo Imaging for Evaluation of Adenoviral Capsid Modifications for Systemic Delivery in an Orthotopic Model of Locally Advanced Lung Cancer

Background: Low or variable expression of the coxsackie and adenovirus receptor (CAR) has limited gene transfer efficacy to many types of cancer cells. To circumvent the problem, adenoviruses (Ads) have been modified by improving their ability to attach to heterologous receptors more regularly present on tumor cells. In the context of lung cancer, the most common cause of cancer associated mortality worldwide, targeting approaches have not been comprehensively evaluated. Our hypothesis was that we could use previously described targeting moieties for increasing gene transfer to NSCLC cells and clinical samples in vitro and in vivo. Ad5lucRGD is modified with an RGD-4C motif allowing binding to integrins. Ad5/3luc1 has a chimeric fiber protein featuring the knob domain derived from Ad3 and it uses the serotype 3 receptor for entry. One can also add a polylysine tail to the C-terminal end of the fiber. Polylysins bind to heparan sulfate proteoglygans (HSPGs). For improving tumor penetration and subsequent oncolysis, conditionally replicating adenoviruses (CRAds) have been constructed. In this study, we used CRAds with same targeting moieties as in the non-replicating Ads.

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.

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.

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.

Promoter-controlled infectivity-enhanced conditionally replicative adenoviral vectors for the treatment of gastric cancer

Gastric cancer is the fourth most common malignancy worldwide. Adenoviral vectors (Ads) have been applied for gene therapy of various cancers because of their high transduction efficiency. However, the infectivity of gastrointestinal cancer cells is poor due to the limited expression of the Coxsackie-adenovirus receptor (CAR). In addition, few tumor-specific promoters (TSPs) have been characterized for this type of cancer. To overcome these problems, we proposed TSP-driven conditionally replicating adenoviruses (CRAds) with fiber modification for virotherapy of gastric cancer. We assessed the expression profile of eight TSPs in gastric cancer cell lines and evaluated promising candidates in the context of CRAd cytocidal effect. Next, infectivity enhancement by fiber modifications was analyzed in the gastric cancer cell lines. Finally, we combined the TSP-driven CRAds of choice with the fiber modifications to augment the killing effect. Out of the eight TSPs, the midkine (MK) and cyclooxygenase-2 (Cox-2M and Cox-2L) promoters showed high transcriptional activity in gastric cancer cells. When these promoters were used in a CRAd context, Cox-2 CRAds elicited the strongest cytocidal effect. The greatest infectivity enhancement was observed with adenoviral vectors displaying 5/3 chimeric fibers. Likewise, Cox-2 CRAds with 5/3 chimeric fibers showed the strongest cytocidal effect in gastric cancer cell lines. Therefore, Cox-2 CRAds with 5/3 chimeric fiber modification showed good selectivity and infectivity in gastric cancer cells to yield enhanced oncolysis. Cox-2 CRAds with 5/3 chimeric fiber modification are promising for virotherapy of gastric cancer.

An adenovirus vector with a chimeric fiber derived from canine adenovirus type 2 displays novel tropism

Many clinically relevant tissues are refractory to Ad5 transduction because of negligible levels of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR). Thus, development of Ad vectors that display CAR-independent tropism could lead directly to therapeutic gain. The Toronto strain of canine adenovirus type 2 (CAV2) exhibits native tropism that is augmented by, but not fully dependent upon, CAR for cellular transduction. We hypothesized that an Ad5 vector containing the nonhuman CAV2 knob would provide expanded tropism and constructed Ad5Luc1-CK, an E1-deleted Ad5 vector encoding the fiber knob domain from CAV2. Ad5Luc1-CK gene delivery to CAR-deficient cells was augmented up to 30-fold versus the Ad5 control vector, and correlated with increased cell surface binding. Further, we confirmed the importance of cellular integrins to Ad5Luc1-CK transduction. Herein, we present the rationale, design, purification, and characterization of a novel tropism modified, infectivity-enhanced Ad vector.

Efficient delivery and stable gene expression in a hematopoietic cell line using a chimeric serotype 35 fiber pseudotyped helper-dependent adenoviral vector

Certain human cell populations have remained difficult to infect with human adenovirus (Ad) serotype 5 because of their lack of coxsackievirus B-adenovirus receptor (CAR). Native adenovirus fiber compositions, although diverse, cannot infect all tissue types. Recently, a chimeric Ad5/35 fiber was created, which displays an altered tropism from Ad5. We incorporated this chimeric fiber into a helper-dependent (HD) adenovirus vector system and compared HD to E1-deleted (E1Δ) vectors by transgene expression, cell transduction efficiency, and cytotoxicity. K562 cells were infected ∼50 times more efficiently with the chimeric Ad5/35 fiber compared with the Ad5 fiber. Short-term transgene expression was sustained longer from HD Ad5/35 than E1Δ Ad5/35 vector after in vitro infection of actively dividing K562 cells. Rapid loss of transgene expression from E1Δ Ad5/35 infection was not due to the loss of vector genomes, as determined by quantitative real-time PCR (QRT-PCR), or cytotoxicity, but rather through a putative silencing mechanism.

971. New Generation Conditionally Replicative Adenovirus for Hormone-Refractory Prostate Cancer

Top of pageAbstract Although anti-androgen therapy is initially effective for advanced prostate cancer, all patients eventually progress to hormone-refractory (HR) prostate cancer (PC) due to biological selection. In this stage, a median survival is only 6-12 months. Therefore, new therapeutic approaches for hormone-refractory prostate carcinoma (HRPC) are needed. Conditionally replicative adenoviruses (CRAds) are attractive anti-cancer agents. These agents are designed to replicate specifically in tumor cells followed by spread of the viral progeny to neighboring cancer cells. We used the cyclooxygenase-2 (COX-2) promoter for transcriptional targeting of tumor, which is active in many cancers but minimally active in normal tissues, including the liver. The COX-2 promoter (-1432/+59: COX-2L) was tested with luciferase (Luc) expression vectors in PC-3, Du-145, and LnCap prostate cancer cell lines. Very strong COX-2 promoter activity comparable to the CMV promoter was observed in PC-3 and Du-145 HRPC cell lines. We next examined infectivity enhancement in the PC cells by using fiber modification of Ad5 vectors, including incorporation of an RGD4C motif into the HI loop (RGD) and replacement of the Ad5 knob region with the Ad3 knob (Ad5/3 chimera),. Three CMV promoter driven Luciferase expression vectors with the wild type fiber (Ad5Luc1), RGD insertion (AdRGDLuc1), and Ad5/3 chimera fiber (Ad5/3Luc1) were used. The RGD-modified vector did not show enhanced infectivity in either of these PC cell lines, while the Ad5/3 chimera demonstrated significantly higher levels of infection in PC-3 and Du-145 HRPC cells. To analyze the effect of fiber modification on the cytocidal effect of CRAds we constructed COX-2 CRAds with wild type, RGD-modified, and Ad5/3 chimeric fibers, respectively. All COX-2 CRAds showed replication and subsequent oncolytic killing in both PC-3 and Du-145 cell lines while the COX-2 negative LnCap cell line was not affected. Ad5/Ad3 chimera CRAds demonstrated the strongest cytocidal effect among COX-2 CRAds in PC3 and Du-145 cell lines. Analysis of in vivo anti-tumor efficacy was performed using Du-145 prostate carcinoma subcutaneous xenografts. COX-2 CRAd agents with unmodified, RGD-modified, or Ad5/3 chimera fibers as well as wild type Ad5 and a non-replicative E1 deleted vector were used for intratumoral administration (5×109 vp/tumor). Our data indicate that the anti-tumor effect of COX-2 CRAd with Ad5/3 chimera fiber was better than unmodified or RGD-modified CRAds. The present results demonstrate that selective replication of CRAd under the control of the COX-2 promoter and infectivity enhancement with Ad5/3 chimeric fiber may be combined to generate promising viral therapeutic agents for the treatment of hormone refractory prostate carcinomas.

112. Gene Transfer into a Hematopoietic Cell Line Using a Helper-Dependent Adenoviral Vector with the Chimeric Ad5/35 Fiber

Certain human cell populations have remained difficult to infect with vectors based on human adenovirus (Ad) serotype 5 due to their lack of CAR receptor. Recently, a chimeric Ad5/35 fiber, comprising of the Ad5 tail domain and the Ad35 knob and shaft domains, was created. This chimeric fiber utilizes CD46 as a receptor and displays an altered tropism from Ad5. We incorporated this chimeric fiber into a helper-dependent adenovirus vector (HD AdV) system and compared HD to E1-deleted (ED) vectors by transgene expression, cell transduction efficiency, and cytotoxicity.

662. Adenovirus Targeting Via Leucine Zipper Peptide Ligand Attachment

Gene therapy vectors based on human adenovirus (Ad) serotypes 2 and 5 continue to show increasing promise as gene therapy delivery vehicles. Early gene therapy efforts, however, revealed that the clinical benefit of these gene delivery modalities was irrevocably linked to specific localization of the therapeutic agent. Hence, a fundamental requirement for somatic cell gene therapy is targetable gene delivery vectors. To this end, Ads 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 discovery of disease-specific membrane antigens and a wide selection of cognate antibody molecules. However, a barrier to the development of Ad vectors with genetically incorporated scFvs occurs when the antibody requires posttranslational modifications not available to Ad capsid proteins due to their cytoplasmic routing during virion assembly. To overcome 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 occurs following cell lysis and is achieved via genetic tagging of the Ad capsid and scFv with synthetic leucine zipper-like dimerization domains that have been optimized for structural and biological compatibility with the Ad capsid and the secreted scFv. Importantly, we have designed our Zipper domains in pairs, such that only compatible heterodimers can associate, thus eliminating the possibility of aggregate formation. Zipper domains were incorporated into the C-terminus of Ad5 fiber and our knob-deleted chimeric fiber molecule, fiber-fibritin (FF). 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 FF 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 scFv 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.

129. Infectivity Enhancement of an Adenovirus Vector Via Genetic Incorporation of the Reovirus Spike Protein Sigma 1

During the last decade, human adenoviruses (Ad) have been exploited as vectors for gene transfer and gene therapy of various monogenic and polygenic diseases, with limited success. Early gene therapy efforts revealed that the clinical benefit of these gene delivery modalities was irrevocably linked to specific localization of the therapeutic agent. Many clinically relevant tissues are refractory to Ad5 infection, including several cancer tissue types, due to negligible CAR levels. Thus, development of novel Ad vectors demonstrating CAR-independent tropism may lead directly to therapeutic gain. Based on a clear understanding of native Ad cell recognition, development of genetically targeted vectors has rationally focused on the fiber knob domain, the primary determinant of Ad tropism. Reovirus serotype type 3 (T3) utilizes a fiber-like attachment protein, called sigma 1, to infect human cells. The sigma 1 molecule is comprised of an N-terminal tail region of alpha helix and eight beta-repeats, and a C-terminal globular head domain. Sigma 1 contains two receptor-binding domains (RBDs): one within the fiber tail that binds sialic acid (SA), and the other in the globular head that binds to junction adhesion molecule (JAM). The head RBD plays a key role in determining the tropism of T3 reoviruses, which have CAR-independent tropism. Based on these observations, we hypothesized that replacement of the Ad5 fiber with reovirus attachment protein sigma 1, a fiber-like molecule, would result in an Ad5 vector with a novel, CAR-independent tropism. We constructed a dual-fiber Ad5 vector, designated Ad5-sigma 1, encoding a luciferase reporter gene and two fibers: the first fiber is a chimeric fiber comprised of the tail domain of Ad5 fiber fused to the reovirus sigma 1 and the second fiber is the wild-type Ad5 fiber. Virus particles were rescued and purified and characterization of the dual-fiber Ad5-sigma 1 virions confirmed the presence of both fibers. Functionally, Ad5-sigma 1 was found to be able to utilize both receptors, CAR and JAM, for virus attachment to cells. In CAR positive cells, Ad5-sigma 1 gene transfer was reduced 80% by competition with recombinant Ad5 knob. Gene transfer was also inhibited by antibodies directed against JAM and sigma 1 in the L929 murine fibroblast cell line, known to express JAM and SA but not CAR. Ad5-sigma 1 provided 223-fold greater gene transfer versus the isogenic control vector in the L929 cells. In addition, Ad5-sigma 1 gene transfer was increased 6-fold in SA-positive, low-CAR chinese hamster ovary cells versus the isogenic control Ad5 vector. Furthermore, Ad5-sigma 1 provided 3.1- to 10.7- fold greater gene transfer versus control in a panel of ovarian cancer cell lines, known for relatively low CAR expression. We developed an infectivity enhanced Ad5 vector to address the key issue of limited Ad5 vector efficiency. The expanded tropism of the Ad5-sigma1 vector represents a crucial attribute for an Ad-based gene therapy vector specifically for ovarian cancer, and may represent an agent with future clinical utility.

Development of an adenoviral vector system with adenovirus serotype 35 tropism; efficient transient gene transfer into primary malignant hematopoietic cells.

A paucity of coxsackie adenovirus receptor (CAR) hampers the adenovirus serotype 5 (Ad5)-based vector-mediated gene transfer into malignant hematopoietic cells. Fiber-retargeted adenoviral vectors with species B tropism can potentially bypass the CAR requirement and facilitate efficient gene transfer into malignant hematopoietic cells. For feasible generation of fiber-retargeted adenoviral vectors, we have modified the versatile AdEasy system with a chimeric fiber gene encoding the Ad5 fiber tail domain and Ad35 fiber shaft and knob domains. An Ad5-based vector encoding the green fluorescent protein (GFP) gene under the control of the PGK promoter with Ad35 fiber receptor specificity was generated (Ad5F35-GFP). The Ad5F35-GFP vector-mediated gene transfer efficiency was compared with a fiber non-modified Ad5-GFP vector, which also encodes the GFP gene under the control of the PGK promoter. We demonstrated that a variety of Ad5-refractory malignant myeloid and B lymphoid cell lines were highly permissive to the Ad5F35-GFP vector infection. Importantly, primary chronic myeloid leukemic (CML) cells and chronic lymphocytic leukemia (CLL) B cells were superiorly transduced by the Ad5F35-GFP vector at a multiplicity of infection (MOI) of 100 compared with the Ad5-GFP vector. Our study will facilitate the generation of fiber-retargeted adenoviral vectors and enable transient genetic manipulation of primary malignant hematopoietic cells.