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Adenovirus (Ad)-based antiangiogenesis gene therapy is a promising approach for cancer treatment. Downregulation or loss of coxsackievirus and adenovirus receptor (CAR) is often detected in various human cancers, which hampers adenoviral gene therapy approaches. Cationic liposome-complexed adenoviral vectors have been proven useful in CAR-deficient cells to enhance therapeutic gene transfer in vivo. Here, we investigated the antitumor effects of recombinant adenovirus encoding endostatin (Ad-hE) encapsulated in cationic liposome (Ad-hE/Lipo) on CAR-deficient CT26 colon carcinoma murine models. In vitro, Ad-hE/Lipo enhanced adenovirus transfection in CAR-deficient cells (CT26), and endostatin gene expression was measured by both qualitative and quantitative detection. In addition, an antibody neutralizing assay indicated that neutralizing serum inhibited naked adenovirus 5 (Ad5) at rather higher dilution than the complexes of Ad5 and cationic liposomes (Ad5-CL), which demonstrated that Ad5-CL was more capable of protecting Ad5 from neutralization. In vivo, Ad-hE/Lipo treatment in the murine CT26 tumor model by intratumoral injection resulted in marked suppression of tumor growth and prolonged survival time, which was associated with a decreased number of microvessels and increased apoptosis of tumor cells. In conclusion, recombinant endostatin adenovirus encapsulated with cationic liposome effectively inhibited CAR-deficient tumor growth through an antiangiogenic mechanism in murine models without marked toxicity, thus showing a feasible strategy for clinical applications.

Glioblastoma multiforme (GBM) is the most aggressive human brain tumor, and is highly resistant to chemo- and radiotherapy. Selectively replicating oncolytic viruses represent a novel approach for the treatment of neoplastic diseases. Coxsackievirus-adenovirus receptor (CAR) is the primary receptor for adenoviruses, and loss or reduction of CAR greatly decreases adenoviral entry. Understanding the mechanisms regulating CAR expression and localization will contribute to increase the efficacy of oncolytic adenoviruses. Two glioma cell lines (U343MG and U373MG) were infected with the oncolytic adenovirus dl922-947. U373MG cells were more susceptible to cell death after viral infection, compared with U343MG cells. The enhanced sensitivity was paralleled by increased adenoviral entry and CAR mRNA and protein levels in U373MG cells. In addition, U373MG cells displayed a decreased ERK1/2 (extracellular signal-regulated kinase-1/2) nuclear-to-cytosolic ratio, compared with U343MG cells. Intracellular content of PED/PEA-15, an ERK1/2-interacting protein, was also augmented in these cells. Both ERK2 overexpression and genetic silencing of PED/PEA-15 by antisense oligonucleotides increased ERK nuclear accumulation and reduced CAR expression and adenoviral entry. Our data indicate that dl922-947 could represent an useful tool for the treatment of GBM and that PED/PEA-15 modulates CAR expression and adenoviral entry, by sequestering ERK1/2.

Selective replication-competent adenovirus serotype 5 vectors have been used for prostate cancer therapy. Unfortunately, gene transfer is inefficient because hormone-refractory metastatic prostate cancer cells have minimal coxsackievirus-adenovirus receptor expression. Vectors based on species B adenoviruses are attractive tools for use in human gene therapy because the viruses have low seroprevalence and they have efficient transduction capacity. Most species B adenoviruses use ubiquitously expressed complement-regulatory CD46 protein as a cellular receptor. Here we report the transduction efficacy and oncolytic capacity of a replication-competent Ad11p (RCAd11p) vector in human prostate cancer cells. Green fluorescent protein was efficiently expressed in a dose-dependent manner in PC-3 and DU 145 cells derived from metastasis of prostate cancer to bone and brain, respectively. However, transduction was less effective in LNCaP cells derived from prostate cancer metastasis to lymph nodes. The oncolytic capacity of the RCAd11p vector was 100 times higher in PC-3 cells than in the two other cell lines. The oncolysis was independent of the level of expression of p53 in the cells or on the absence of E1B55k expression in the vector. In vivo experiments revealed significant growth inhibition of PC-3 tumors in the xenograft mouse group treated with RCAd11p vector or Ad11pwt in comparison with the untreated control group. Thus, we have demonstrated that RCAd11p vector intrinsically possesses oncolytic properties, which were active in targeting tumor cells. Consequently, the novel RCAd11p vector has great potential for the treatment of incurable metastatic prostate disease.

Malignant gliomas remain refractory to adenovirus serotype 5 (Ad5) gene therapy because of the lack of the primary adenoviral receptor, the coxsackie-adenovirus receptor (CAR), on tumor cells. To bypass the dependence on CAR, we investigated the expression of adenovirus serotype 3 (Ad3) receptor, or CD46, on glioma cells. First, we analyzed the expression of CD46 by RT-PCR on primary and passaged glioma cells. We then performed immunofluorescence studies to examine protein expression of CAR and CD46 on the same tumor lines. Finally, we constructed a replication-defective Ad vector that binds to CD46 and contains a luciferase transgenic cassette in place of the deleted E1 region: Ad5/3 (containing tail/shaft domain of Ad5 and knob domain of Ad3). These vectors were analyzed in vitro and in vivo against malignant glioma and compared with wild-type Ad5 or control vector Ad3/5 (containing tail of Ad5, shaft of Ad3, and knob of Ad5). The chimeric vector Ad5/3 showed a significant increase in the transduction efficiency of glioma tumor cells. At the same time, blocking the CD46 receptor caused a 65% inhibition of adenoviral infection when using Ad5/3. Taken together, these results indicate that CD46 is overexpressed by malignant glioma. Retargeting to the Ad3 receptor enhances gene transfer and offers a novel target for gene therapy of malignant brain tumors.

Coxsackievirus and adenovirus receptor (CAR), alphav integrins, and heparan sulfate glycosaminoglycans (HSGs) are the tropism determinants of adenoviral (Ad) vectors in vivo. For the development of a targeted Ad vector, its broad tropism needs to be blocked (or reduced). We have previously developed Ad vectors with ablation of CAR, alphav integrin, and HSG binding by mutation of the FG loop in the fiber knob (deletion of T489, A490, Y491, and T492 of the fiber protein), deletion of the RGD motif of the penton base, and substitution of the fiber shaft domain for that derived from Ad type 35, respectively, and have shown that this triple-mutant Ad vector [Ad/deltaF(FG)deltaP-S35-L2] exhibits significantly lower transduction in mouse liver compared with the conventional Ad vector [Koizumi, N., Mizuguchi, H., Sakurai, F., Yamaguchi, T., Watanabe, Y., and Hayakawa, T. (2003). J. Virol. 77, 13062-13072]. In the present study, we optimized the fiber knob mutation for further reduced in vivo transduction and examined toxicity of the modified Ad vectors. Ad/deltaF(AB)deltaPS35- L2, a triple-mutant Ad vector containing a mutation of the AB loop in the fiber knob (R412S, A415G, E416G, and K417G), mediated approximately 15,000- and 500-fold lower mouse liver transduction by intravenous and intraperitoneal administration, respectively, than the conventional Ad vector, and mediated 10- fold lower mouse liver transduction than did Ad/deltaF(FG)deltaP-S35-L2. Ad/deltaF(AB)deltaP-S35-L2 also exhibited lower transduction of other organs compared with Ad/deltaF(FG)deltaP-S35-L2 and the conventional Ad vector. Levels of both liver serum enzymes (aspartate transferase [AST] and alanine transferase (ALT)] and interleukin (IL)-6 in mouse serum after intravenous administration of Ad/deltaF(AB)deltaP-S35-L2 were similar to those in the nontreatment mouse serum, whereas the conventional Ad vector led to high levels of AST, ALT, and IL-6. We therefore succeeded in further improving the mutant Ad vector, abolishing both viral natural tropism and toxicity. This new Ad vector appears to be a fundamental vector for targeted gene delivery.

Until recently, adenovirus-based gene therapy has been almost exclusively based on human adenovirus serotype 5 (Ad5). The aim of this study was to systematically compare the efficiency of transduction of primary muscle cells from various species by two adenoviral vectors from subgroups C and D. Transduction of a panel of myoblasts demonstrated a striking specificity of an Ad19a-based replication-defective E1-deleted vector (Ad19aEGFP) for human cells, whereas the Ad5-based vector had high affinity for nonhuman primate myoblasts. Transgene expression correlated well with cell-associated vector genomes. Up to 6.59% of the initially applied Ad19aEGFP vector particles were taken up by human myoblasts, as compared with 0.1% of the corresponding Ad5 vector. Remarkably, Ad19aEGFP but not Ad5EGFP efficiently transduced differentiated human myotubes, an in vitro model for skeletal muscle transduction. Uptake of Ad19aEGFP vector particles in human myotubes was 12-fold more efficient than that of Ad5EGFP. Moreover, both vectors demonstrated an early block at the level of vector uptake in mouse myoblasts and rat L6 cells. Investigation of the underlying mechanism for binding and uptake of the two vectors by human myoblasts showed high susceptibility for Ad19a to neuraminidase and wheat germ agglutinin (WGA) lectin, whereas Ad5-mediated transduction was dependent on binding to the coxsackie-adenovirus receptor (CAR) and sensitive to soluble RGD peptide and heparin. Our study offers insights into species-dependent factors that determine Ad tropism and, moreover, provides a basis for application of the novel Ad19a-based vector for gene transfer into human skeletal muscle.

Resistance to radiation and chemotherapy remains an obstacle to the treatment of brain tumors. We have demonstrated that the replication-deficient adenovirus d1520, which lacks the E1A 13S protein, replicates efficiently and exhibits oncolytic potential in multidrug-resistant cells with nuclear localization of the human transcription factor YB-1. However, besides others, key factors regarding oncolytic virotherapy are limited tumor transduction rate and low replication efficiency. The objective of this study was to determine whether the chemotherapeutic agent irinotecan, by enhancing nuclear localization of YB-1, and the histone deacetylase inhibitor trichostatin A, by upregulating coxsackievirus-adenovirus receptor (CAR) expression, could augment replication of and cell lysis by adenovirus dl520 in glioblastomas in vitro. We found that trichostatin A upregulated CAR expression and that irinotecan caused increased nuclear localization of YB-1 in both glioblastoma cell lines. Irinotecan alone, and trichostatin A alone, enhanced replication of and cell lysis by dl520. Importantly, when combining both agents, the replication efficiency (maximum, 27-fold) and induction of cytopathic effect (maximum, 3.8-fold) of dl520 were further augmented significantly. These results support the hypothesis that the enhanced oncolytic effect of dl520, after incubation with chemotherapeutic agents, is mediated by an increased accumulation of YB-1 in the nucleus (due to irinotecan) and by upregulation of CAR (due to trichostatin A). Thus, therapy combining virotherapy, chemotherapy, and histone deacetylase inhibitor treatment is a novel approach to enhance the oncolytic efficacy of dl520.

Human adenovirus-based vectors have emerged as a new promising vehicle for in vivo gene transfer-mediated therapy. However, the full potential of this methodology has not been fully realized because of the nonspecific tissue distribution of adenoviral vectors. Adenovirus infection is initiated by forming a complex between the fiber protein and a ubiquitously expressed host cell membrane protein, coxsackie B virus and adenovirus receptor (CAR). Therefore, ablating the adenovirus vector's ability to bind to the CAR is the first step in redirecting adenoviral tropism. To ablate CAR binding, we mutated the Bbeta sheet of the fiber knob, generating CAR-binding ablated replication-incompetent (dl-K420A-Z) and replication-competent (YKLK420A) adenoviral vectors. The in vitro transduction efficiency of dl-K420A-Z was significantly reduced in comparison to dl-LacZ carrying the wild-type fiber in CAR-positive cells but not in CAR-negative cells, suggesting that the mutation introduced in the Bbeta sheet of the fiber knob could disable the CAR-dependent transduction pathway. The in vivo transduction was also dramatically reduced in the liver and other organs for mice treated with dl-K420A-Z, compared with a cognate control vector, dl-LacZ. Concomitant with this attenuated gene transfer efficiency in vivo was a substantial reduction in the amount of general toxicity observed in the YKL-K420A-treated mice. Diminished toxicity was surmised from quantitative measurement of serum level of enzymes for liver and kidney function, hematologic chemistries, histopathology, and differences in lethality. Significant decrease in serum transaminases (alanine transferase [ALT] and aspartate transferase [AST]) was observed in mice treated with YKL-K420A. In addition, the lethality was lower in the YKLK420A- treated groups compared to the YKL-1-treated groups at all doses examined. Furthermore, the hepatopathologic analysis revealed that YKL-1 induced focal zonal necrosis and hepatocyte degeneration, while YKL-K420A induced mild spotty necrosis. In summary, this decreased vector tropism of CAR-binding ablated adenoviruses in normal tissues may increase the amount of virus available for infecting tumor cells and thus increase the antitumor efficacy with fewer unwanted side effects.

Adenovirus (Ad5) serotype 5 vectors are commonly used for gene transfer. Preclinical studies have shown that their application to systemic gene delivery, however, is limited by their highly efficient uptake in the liver, principally mediated by receptor-binding sites on the fiber shaft and knob domain. Using Ad to target other sites in vivo requires vectors that lack hepatic tropism. We therefore sought to exploit Ad family diversity to isolate vectors that possessed poor hepatic tropism. We pseudotyped the fibers from Ad16 (subgroup B; Ad5/16), Ad19p (subgroup D; Ad5/19p), and Ad37 (subgroup D; Ad5/37) onto Ad5 capsids and assessed infectivity profiles in vitro in multiple cell types and in vivo in rats. In rat, mouse, and human hepatocytes, Ad5/19p and Ad5/37 both possessed a striking lack of hepatic cell infectivity compared with Ad5. Both vectors were, however, able to transduce human vascular endothelial and smooth muscle cells with efficiencies equal to or greater than that of nonmodified Ad5. We evaluated liver uptake in 12-week-old male rats after intravenous injection. In contrast to a vector with the wild-type Ad5 fiber, Ad5, both Ad5/19p and Ad5/37 produced significantly less virion accumulation (measured at 1 hr and 5 days) and transgene expression in the liver. Thus, Ad5/19p and Ad5/37 may be useful platforms for the development of targeted Ad 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.

To assess the possibilities of retargeting adenovirus to activated endothelial cells, we conjugated bifunctional polyethylene glycol (PEG) onto the adenoviral capsid to inhibit the interaction between viral knob and coxsackie-adenovirus receptor (CAR). Subsequently, we introduced an alphav integrin-specific RGD peptide or E-selectin-specific antibody to the other functional group of the PEG molecule for the retargeting of the adenovirus to activated endothelial cells. In vitro studies showed that this approach resulted in the elimination of transgene transfer into CAR-positive cells, while at the same time specific transgene transfer to activated endothelial cells was achieved. PEGylated, retargeted adenovirus showed longer persistence in the blood circulation with area under plasma concentration-time curve (AUC) values increasing 12-fold compared to unmodified virus. Anti-E-selectin antibody-PEG-adenovirus selectively homed to inflamed skin in mice with a delayed-type hypersensitivity (DTH) inflammation, resulting in local expression of the reporter transgene luciferase. This is the first study showing the benefits of PEGylation on adenovirus behavior upon systemic administration. The approach described here can form the basis for further development of adenoviral gene therapy vectors with improved pharmacokinetics and increased efficiency and specificity of therapeutic gene transfer into endothelial cells in disease.

Adenoviral vectors used in gene therapy are predominantly derived from adenovirus serotype 5 (Ad5), which infects a broad range of cells. Ad5 cell entry involves interactions with the coxsackie-adenovirus receptor (CAR) and integrins. To assess these receptors in vivo, we mutated amino acid residues in fiber and penton that are involved in receptor interaction and showed that CAR and integrins play a minor role in hepatic transduction but that integrins can influence gene delivery to other tissues. These data suggest that an alternative entry pathway exists for hepatocyte transduction in vivo that is more important than CAR or integrins. In vitro data suggest a role for heparan sulfate glycosaminoglycans (HSG) in adenovirus transduction. The role of the fiber shaft in liver uptake was examined by introducing specific amino acid changes into a putative HSG-binding motif contained within the shaft or by preparing fiber shaft chimeras between Ad5 and Ad35 fibers. Results were obtained that demonstrate that the Ad5 fiber shaft can influence gene transfer both in vitro and to the liver in vivo. These observations indicate that the currently accepted two-step entry pathway, which involves CAR and integrins, described for adenoviral infection in vitro, is not used for hepatic gene transfer in vivo. In contrast, alpha(v) integrins influence gene delivery to the lung, spleen, heart, and kidney. The detargeted vector constructs described here may provide a foundation for the development of targeted adenoviral vectors.

Adenoviral (Ad) infection involves attachment mediated by the Ad fiber protein binding to the coxsackievirus-adenovirus receptor (CAR) of a target cell and internalization facilitated by the interaction of the Ad penton base protein with alpha(v) integrins. To understand the relative importance of the Ad binding and internalization steps for the transduction of fetal skeletal muscle, we used a panel of genetically modified vectors that specifically ablate the fiber-CAR interaction (AdL.F*), the penton base-alpha(v) integrin interaction (AdL.PB*), or both (AdL.PB*F*) to transduce embryonic day 16 (E-16) mouse muscle in vivo and primary E-16 muscle cells in vitro. Quantification of transgene expression and vector genome copies revealed a striking absence of E-16 muscle transduction by AdL.F* and AdL.PB*F*. In contrast, fetal muscle transduction with AdL.PB* was not significantly different than with the unmodified vector. Similar results were observed with in vitro Ad infection studies in primary E-16 muscle cells. From these data we conclude that the fiber-CAR interaction is important for the transduction of fetal muscle by Ad vectors. The high dependence on fiber-CAR binding will impact the development of strategies for Ad vector retargeting to achieve muscle-specific transduction in utero.

Adenoviral vectors based on serotype 5 (Ad5) have been widely used to deliver therapeutic genes to different organs and tissues. However, many tissues are poorly infected with Ad5 because of low-level expression of its primary receptor, coxsackievirus-adenovirus receptor (CAR). Two motifs, RGD and polylysine (pK7), have been shown to enhance Ad5 infection via CAR-independent pathways when incorporated into fiber separately. Because the two motifs bind to different cell surface proteins (RGD motif binds to integrins, and pK7 binds to heparan sulfate-containing receptors), we hypothesized that the two motifs function additively to improve gene transfer efficiency. In this study, we sought to improve infectivity of Ad5 by incorporating both RGD and pK7 motifs into fiber. We created an Ad5 vector containing an RGD motif in the HI loop and a pK7 motif at the C terminus of fiber (Ad5.RGD.pK7). Compared with unmodified and singly modified Ad5 vectors Ad5.RGD and Ad5.pK7, the doubly modified Ad5 demonstrated the highest infectivity in both CAR-positive and CAR-negative cells. The enhanced infectivity appeared to be mediated by additive effects of the two motifs. More importantly, Ad5.RGD.pK7 lost the natural CAR-dependent pathway while employing novel targeting mechanisms. This strategy thus may be used to overcome CAR deficiency and to achieve vector retargeting.

Recombinant adenoviral vectors have potential for the treatment of a variety of musculoskeletal defects and such gene therapy systems have been a recent research focus in orthopedic surgery. In studies reported here, two different adenovirus vectors have been compared for their ability to transduce human bone marrow mesenchymal stem cells (hBM-MSCs) and elicit bone formation in vivo. Vectors consisted either of standard adenovirus type 5 (Ad5) vector or a chimeric adenovirus type 5 vector that contains an adenovirus type 35 fiber (Ad5F35), which has been recently demonstrated to bestow a different cellular tropism, and a complete cDNA encoding human bone morphogenetic 2 (BMP2). Studies were also conducted to compare the transduction efficiency of these vectors using enhanced green fluorescent protein (GFP). hBM-MSCs transduced with Ad5F35 vectors had higher levels of transgene expression than those transduced with Ad5 vectors. The results also demonstrate that hBM-MSCs lack the coxsackie-adenovirus receptor (CAR), which is responsible for cellular adsorption of Ad5. Therefore, the data suggest that Ad5 virus adsorption to hBM-MSCs is inefficient. Ad5BMP2- or Ad5F35BMP2-transduced hBM-MSCs were also compared in an in vivo heterotopic bone formation assay. Mineralized bone was radiologically identified only in muscle that received the Ad5F35BMP2 transduced hBM-MSCs. In summary, Ad5F35BMP2 can efficiently transduce hBM-MSCs leading to enhanced bone formation in vivo.

The utility of recombinant adenoviral vectors (Adv) for gene therapy is limited by their low transduction efficiency and lack of specificity for target cells. The low transduction efficiency is often recognized as due to deficiency of the primary adenoviral receptor, the coxsackievirus-adenovirus receptor (CAR). In this paper, studies of CAR levels on human melanoma cell lines confirmed that low transduction efficiency was closely related to deficiency of the adenoviral receptor. To achieve CAR-independent gene transfer via Adv, we modified viral tropism via genetic alteration of the adenovirus type 5 (Ad5) fiber protein. Insertion of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob domain allowed the virus to use an alternative receptor, the integrin receptor, during the cell entry process. With this modified vector (Adv-F/RGD) transduction was increased 5- to 96-fold relative to a vector containing wild-type fiber (Adv-F/wt) in five human melanoma cells expressing integrins of the alpha(v)beta(3), alpha(v)beta(5) class, which are recognized by the RGD peptide motif. In contrast, no significant difference in transduction efficiency between Adv-F/RGD and Adv-F/wt was observed in 293 cells, which show high-level expression of CAR. In this study, we attempted to apply Adv-F/RGD for gene therapy for malignant melanoma. At the same multiplicity of infection, melanoma cells infected with Adv-F/RGD carrying human interleukin 2 (AxCAhIL2-F/RGD) produced a higher level of cytokine than cells infected with AxCAhIL2-F/wt. Treatment by intratumoral injection of AxCAhIL2-F/RGD was more effective than intratumoral injection of AxCAhIL2-F/wt in regressing tumors in a melanoma xenograft model. These data suggest that integrin-targeted adenoviral vectors may be a powerful tool in gene therapy for CAR-deficient melanomas.

Adenovirus vectors for gene therapy activate responses in the host that result in acute inflammation of transduced tissues. Our previous studies in vivo demonstrate that chemokines, including the C-C chemokine RANTES (regulated on activation, normal T cell expressed and secreted), contribute to the acute inflammation induced by adenovirus vectors. Various first-generation adenovirus vectors, including adCMV beta gal, were equally capable of inducing the expression of RANTES 3 hr after transduction in epithelial HeLa and REC cells. Deletional analysis of the human RANTES promoter revealed that induction by adCMV beta gal required the elements spanning base pairs -90 to -25 of the gene. Electrophoretic mobility shift assays demonstrated that nuclear extracts from adCMV beta gal-transduced HeLa cells bound to an NF-kappa B site at position -54. Overexpression of I-kappa B alpha suppressed adCMV beta gal induction of RANTES, confirming that this process was dependent on the nuclear translocation of NF-kappa B. The coxsackievirus-adenovirus receptor (CAR)-independent, serotype 3 adenovirus was equally capable of inducing the expression of RANTES in HeLa cells. This observation suggested that binding to CAR was not specifically required in adenovirus vector-induced RANTES expression. The use of RGD peptides to block adCMV beta gal interactions with alpha(v)-integrins reduced RANTES expression but also transduction efficiency. In CAR-deficient P815 cells, binding of adCMV beta gal to alpha(v)-integrins without efficient cell transduction did not result in increased RANTES expression. Expression of human CAR in P815 cells increased the binding and transduction efficiency of adCMV beta gal and resulted in RANTES expression in these cells. These results suggest that the induction of RANTES by adenovirus vectors is dependent on efficient interaction with its cell surface receptors and vector internalization. Understanding the biology of the host response to adenovirus vectors will impact the design of future generations of these agents aimed at reducing their immunogenicity and improving their safety.

Conditionally replicative adenovirus (CRAd) vectors are designed for specific oncolytic replication in tumor tissues with concomitant sparing of normal cells. As such, CRAds offer an unprecedented level of anticancer potential for malignancies that have been refractory to previous cancer gene therapy interventions. CRAd efficacy may, however, be compromised by inefficient dispersion of the replicating vector within the tumor tissue. To address this issue, we evaluated the utility of a fusogenic membrane glycoprotein (FMG), which induces the fusion of neighboring cellular membranes to form multinucleated syncytia. We hypothesized that the FMG-mediated syncytia would facilitate dispersion of the adenovirus (Ad) gene products and viral progeny. To test this, human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, which induce syncytia in the presence of CD4+ target cells, were expressed by an Ad (Ad5HIVenv) in permissive (CD4-positive) and nonpermissive (CD4-negative) cell lines. After validating this Ad-FMG model, the efficiency of Ad replication in the presence or absence of syncytia was evaluated. The results demonstrated that syncytium formation was compatible with Ad replication and dramatically increased the dispersion of virus gene products within the cytoplasm of the syncytia as well as viral particles in the nuclei of the syncytial mass. Moreover, progeny virions were released more efficiently from syncytia compared with nonsyncytial cells. These data demonstrate the utility of FMGs as a dispersion agent and suggest that FMGs can improve the efficacy of CRAd gene therapy.

Canine adenovirus type 2 (CAV-2) vectors may be attractive tools for gene transfer thanks to the lack of pre-existing immunity in humans, and because of the preferential transduction of neurons when the vector is injected into the brain and some innervated tissues. The coxsackievirus-adenovirus receptor (CAR) appears to play a major role during infection of most human serotypes, whereas the alpha(v)beta(3/5) integrins have been reported to play a significant auxiliary role. We showed that CAV-2 also attaches to and uses CAR to enter cells, but CAV-2 transduction could be notably different from that of the prototype human adenovirus serotype 5 (Ad5). Initially, the CAV-2 capsid appears to be 10-fold less negatively charged than Ad5. Second, the CAV-2 penton, hexon, and fiber proteins do not contain a known integrin-interacting motif. Because of its potential interest in the clinic, we analyzed the different steps of cellular trafficking and the propagation kinetics of CAV-2 vectors. We found that Ad5 and CAV-2 vectors have comparable kinetics of binding (10 min), internalization (10 min), endosomal escape (17 min), attachment to the nuclear membrane (35 min), and formation (18 hr) and release (34 hr) of functional virions. Surprisingly, the RGD(-) CAV-2 capsid also induced the reorganization of actin filaments in HeLa cells. Actin reorganization is thought to be dependent on alpha(v)beta(3/5) integrin stimulation.

High-capacity adenoviral (HC-Ad) vectors contain only the noncoding termini of the viral genome, can deliver large DNA fragments of up to 36 kb into target cells, and feature reduced toxicity and prolonged transgene expression in vivo. To enhance the potential of HC-Ad vectors to transduce specific cell types, we constructed a versatile infectious new helper virus plasmid that can be used readily to introduce peptide ligands into the HI loop of the fiber knob domain of Ad5-based HC-Ad vectors. Helper viruses with a 6x-His epitope or Arg-Gly-Asp (RGD) peptide insertion retained the full infectivity of the wild-type helper virus. The RGD-modified helper virus was used for production of a capsid-modified HC-Ad vector expressing beta-galactosidase. The RGD HC-Ad vector transduced the ovarian carcinoma cell lines SK-OV-3 and OVCAR-3 with 4- to 20-fold higher efficiency, compared to unmodified vectors. Transduction of both primary vascular smooth muscle cells as well as primary human endothelial cells was increased up to 15-fold with the RGD-modified vector. Competition experiments with recombinant knob protein and different RGD peptides indicated that the RGD-mediated transduction was Coxsackie and Adenovirus receptor (CAR)-independent and involved integrin alpha(v)beta(5). The use of fiber-modified helper viruses in the last amplification step of HC-Ad vector production allows for convenient and efficient targeting of these vectors towards different cell types. Targeting strategies will increase the spectrum of applications for HC-Ad vectors and will further add to their safety.

Recombinant adenoviruses expressing a therapeutic gene are currently used in clinical studies for treatment of advanced ovarian cancer. We therefore tested whether the expression level of primary (CAR) and secondary adenovirus receptors (integrins) was predictive of the efficacy of adenoviral gene transfer in ovarian cancer cells. Adenoviral transduction efficiency (ATE) was determined with an E1-deleted adenovirus type 5 expressing beta-galactosidase under a CMV promoter (AdGal). ATE was studied in relationship to the expression level of both CAR (coxsackie and adenovirus receptor) and integrins. A representative sample of 25 permanent human cell lines established from advanced ovarian cancer in our laboratory and the OV-2774 cell line were tested. Overall, ATE increased with increasing titers of AdGal. At a given titer of 50 infectious units per cell, transduction efficiency varied from 6 to 94% among the individual cell lines. All cell lines expressed CAR and integrin alpha(v)beta(5), but no relation between ATE and expression level of CAR or alpha(v)beta(5) integrin was observed. In contrast, cell lines with poor ATE, despite expressing high levels of CAR, lacked expression of integrins alpha(v)beta(3) and alpha(5)beta(1). Reconstitution of alpha(v)beta(3) integrin by reexpressing the beta(3) subunit significantly enhanced ATE of ovarian cancer cells. In ovarian cancer, neither integrins nor CAR alone appear to be potentially useful predictive markers for ATE by serotype 5 adenovirus in clinical gene therapy. A minimum level of CAR necessary for binding of adenoviruses was observed in all tested ovarian cancer cell lines. Loss of alpha(v)beta(3) integrin is frequently associated with advanced stages of ovarian cancer and can significantly reduce ATE.

Expression of the coxsackie-adenovirus receptor (CAR) is a critical determinant in cellular susceptibility to infection with adenovirus-based gene transfer vectors. This study is focused on the hypothesis that manipulation of the cytoplasmic tail and transmembrane regions of CAR can be used to change cell surface levels of CAR and, consequently, to alter the efficiency of Ad-mediated gene transfer. To accomplish this, Flag-tagged ([F]) human CAR ([F]CAR), [F]tailless-CAR (lacking the cytoplasmic tail), and [F]GPI-CAR (containing a GPI lipid anchor instead of the transmembrane and cytoplasmic regions) were exogenously expressed in CHO cells. Analysis of (125)I-labeled anti-Flag antibody binding to transfected cells revealed that [F]tailless-CAR and [F]GPI-CAR were expressed on the cell surface in 1.8- to 2.5-fold higher amounts than [F]CAR, while the total expression levels were similar. Infection with replication-deficient adenovirus encoding beta-galactosidase (Ad-betagal) demonstrated 1.5- to 2-fold higher levels of transgene expression in CHO cells expressing [F]tailless-CAR or [F]GPI-CAR, respectively, compared with cells containing [F]CAR. The form of CAR expressed did not affect the transport of fluorescent Cy3-Ad particles from the cell surface to the nuclear region. These observations indicate that transduction of target cells by Ad vectors can be optimized by increasing cell surface levels of CAR through functional deletion of the tail and membrane protein domains.

Adenovirus-mediated gene transfer is a novel treatment strategy for head and neck squamous cell carcinoma (HNSCC) that may improve the unacceptable morbidity and mortality associated with conventional treatment. Efficient adenoviral (AdV) infection largely depends on cellular expression of the human coxsackie and adenovirus receptor (hCAR); however, the relatively recent identification of this receptor precludes a comprehensive description of its tissue distribution. We have created tissue culture model systems that approximate the differentiation and three-dimensional structure of stratified squamous epithelium characteristic of head and neck mucosa. Using these systems, we have found that expression of hCAR in native and modeled normal oropharyngeal epithelium decreased as cells differentiated with the most superficial and differentiated cells expressing no detectable hCAR. In contrast, modeled stratified HNSCC cells, which did not differentiate morphologically and did not express cytokeratin markers of differentiation, had equivalent expression of hCAR in superficial and basal layers. The expression of hCAR in our models correlated not only with the undifferentiated state, but also with efficiency of AdV infection. Despite expression of hCAR in underlying basal and suprabasal cells, topical application of AdV to normal modeled epithelium resulted in inefficient transduction of the most superficial cell layer without any infection of underlying cells. These data suggest that in normal epithelium the overlying squamous cells act as a barrier preventing infection of underlying cells that would otherwise be easily infected. In modeled stratified HNSCC, transduction was much more efficient and occurred up to four cell layers deep, suggesting that unlike normal superficial epithelial cells, the superficial cells of stratified HNSCC do not act as an effective barrier to adenoviral infection. The distribution of hCAR in native tissue and the enhanced susceptibility of undifferentiated oropharyngeal epithelial cells, including undifferentiated cancer cells, to AdV infection has important implications for the development of AdV-based targeting strategies for the treatment of head and neck cancer or premalignancies.

Duchenne muscular dystrophy (DMD) and other inherited myopathies lead to progressive destruction of most skeletal muscles in the body, including those responsible for maintaining respiration. DMD is a fatal disorder caused by defects in the dystrophin gene. Recombinant adenovirus vectors (AdV) are considered a promising means for therapeutic delivery of a functional dystrophin gene to DMD muscles. If AdV-mediated dystrophin gene replacement in DMD is to be successful, development of a systemic delivery method for targeting the large number of diseased muscles will be required. In this study we investigated two major factors preventing efficient AdV-mediated gene transfer to skeletal muscles of adult animals after intravascular AdV administration: (1) an inability of AdV particles to breach the endothelial barrier and enter into contact with myofibers, and (2) a relatively nonpermissive myofiber population for AdV infection due at least in part to insufficient levels of the coxsackie/adenovirus attachment receptor (CAR). On the basis of established principles governing the transendothelial flux of macromolecules, we further hypothesized that an alteration in Starling forces (increased hydrostatic and decreased osmotic pressures) within the intravascular compartment would facilitate AdV transendothelial flux via convective transport. In addition, experimental muscle regeneration was employed to increase the prevalence of immature myofibers in which CAR expression is upregulated. Here we report that by employing the above-described strategy, high-level heterologous reporter gene expression was achievable in hindlimb muscles of normal rats as well as dystrophic (mdx) mice (genetic homolog of DMD) after a single intraarterial injection of AdV. Microsphere studies confirmed enhanced transport into muscle of fluorescent tracer particles in the size range of AdV, and there was a high concordance between CAR upregulation and myofiber transduction after intraarterial AdV delivery. Furthermore, in mdx mice examined 10 days after intraarterial AdV delivery, the aforementioned procedures had no adverse effects on the force-generating capacity of targeted muscles. These findings have implications for eventual AdV-mediated gene therapy of generalized skeletal muscle diseases such as DMD using a systemic intraarterial delivery approach.

Recombinant adenoviruses are currently being used as vectors for gene delivery to a wide variety of cells and tissues. Although generally efficacious for gene transfer in vitro, improvement in the efficiency of vector delivery in vivo may aid several gene therapy applications. One major obstacle is the lack of high-affinity viral receptors on the surface of certain cells that are targets for gene therapy. In principle, incorporation of avid, cell-specific ligands into the virion could markedly improve vector entry into the desired tissues. We have developed a strategy for addressing this issue in the lung by biopanning differentiated, ciliated airway epithelial cells against a phage display library. The peptide with the most effective binding was coupled to the surface of an adenovirus using bifunctional polyethylene glycol (PEG) molecules. The chemically modified adenoviral vector was able to effect gene transfer to well-differentiated human airway epithelial cells by an alternative pathway dependent on the incorporated peptide. Coupling of PEG to the surface of the virus also served to partially protect the virus from neutralizing antibodies in vitro. These experiments will aid in the design of improved adenoviral vectors with the capacity for more specific and efficient delivery of therapeutic genes to desired target tissues. We have used a novel method for enhancing gene delivery to target cells by coupling a biologically selected peptide to the surface of an adenovirus with bifunctional PEG molecules. Modification of the viral capsid by the addition of a peptide with binding preference for differentiated ciliated airway epithelia allowed gene delivery to those cells by a novel entry pathway. Incorporation of the CFTR gene in a similarly modified vector resulted in correction of defective Cl- transport in well-differentiated epithelial cultures established from human cystic fibrosis (CF) donors. The presence of PEG molecules on the surface of the virus served, in addition, to reduce antibody neutralization. Modification of adenoviruses with PEG/peptide complexes can serve to partially overcome the barrier of inefficient gene transfer in some cell types and some of the adverse immunological responses associated with gene delivery by these vectors.

Skeletal muscle fibers are infected efficiently by adenoviral vectors only in neonatal animals. This lack of tropism for mature skeletal muscle may be partly due to inefficient binding of adenoviral particles to the cell surface. We evaluated in developing mouse muscle the expression levels of two high-affinity receptors for adenovirus, MHC class I and the coxsackie and adenovirus receptor (CAR). The moderate levels of MHC class I transcripts that were detected in quadriceps, gastrocnemius, and heart muscle did not vary between postnatal day 3 and day 60 adult tissue. A low level of CAR expression was detected on postnatal day 3 in quadriceps and gastrocnemius muscles, but CAR expression was barely detectable in adult skeletal muscle even by reverse transcriptase-polymerase chain reaction. In contrast, CAR transcripts were moderately abundant at all stages of heart muscle development. Ectopic expression of CAR in C2C12 mouse myoblast cells increased their transducibility by adenovirus at all multiplicities of infection (MOIs) tested as measured by lacZ reporter gene activity following AVCMVlacZ infection, with an 80-fold difference between CAR-expressing cells and control C2C12 cells at an MOI of 50. Primary myoblasts ectopically expressing CAR were injected into muscles of syngeneic hosts; following incorporation of the exogenous myoblasts into host myofibers, an increased transducibility of adult muscle fibers by AVCMVlacZ was observed in the host. Expression of the lacZ reporter gene in host myofibers coincided with CAR immunoreactivity. Furthermore, sarcolemmal CAR expression was markedly increased in regenerating muscle fibers of the dystrophic mdx mouse, fibers that are susceptible to adenovirus transduction. These analyses show that CAR expression by skeletal muscle correlates with its susceptibility to adenovirus transduction, and that forced CAR expression in mature myofibers dramatically increases their susceptibility to adenovirus transduction.