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Reovirus σ1 fiber incorporated into adenovirus serotype 5 enhances infectivity via a CAR-independent pathway
Abstract
Adenovirus serotype 5 (Ad5) has been used for gene therapy with limited success because of insufficient infectivity in cells with low expression of the primary receptor, the coxsackie and adenovirus receptor (CAR). To enhance infectivity in tissues with low CAR expression, tropism expansion is required via non-CAR pathways. Serotype 3 Dearing reovirus utilizes a fiber-like sigma1 protein to infect cells expressing sialic acid and junction adhesion molecule 1 (JAM1). We hypothesized that replacement of the Ad5 fiber with sigma1 would result in an Ad5 vector with CAR-independent tropism. We therefore constructed a fiber mosaic Ad5 vector, designated as Ad5-sigma1, encoding two fibers: the sigma1 and the wild-type Ad5 fiber. Functionally, Ad5-sigma1 utilized CAR, sialic acid, and JAM1 for cell transduction and achieved maximum infectivity enhancement in cells with or without CAR. Thus, we have developed a new type of Ad5 vector with expanded tropism, possessing fibers from Ad5 and reovirus, that exhibits enhanced infectivity via CAR-independent pathway(s).
... Accordingly, we suggest that HAdV- 5-based vectors that incorporate the HAdV-3 fiber protein and the native HAdV-5 fiber protein will exhibit expanded tropism, resulting in efficient transduction of cells expressing either high or low levels of CAR. We have previously developed HAdV-5 vectors that incorporate two distinct fiber proteins — called fiber-mosaic HAdV vectors — using two different methods: co-infection of cells with two different HAdV 'parent' vectors (Takayama et al., 2003) and genetic insertion of a second fiber gene into the HAdV-5 genome (Pereboeva et al., 2004Pereboeva et al., , 2007 Tsuruta et al., 2005 Tsuruta et al., , 2007 ). However, when using the coinfection method, the possibility exists that not all viral progeny incorporate both fiber types, due to variability in the initial coinfection step. ...
... However, when using the coinfection method, the possibility exists that not all viral progeny incorporate both fiber types, due to variability in the initial coinfection step. Using the second method, we genetically engineered fiber-mosaic HAdV vectors to encode a second fiber gene, including the bacteriophage T4 fibritin domain (Pereboeva et al., 2004), the bacteriophage T4 fibritin containing the 1.3S subunit of Propionibacterium shermanii transcarboxylase (Pereboeva et al., 2007), or the reovirus cellular attachment protein sigma 1 (Tsuruta et al., 2005). These additional fibers were co-expressed with either the wild type HAdV-5 fiber gene, or a chimeric HAdV-3 fiber gene in the case of sigma 1 (Tsuruta et al., 2007). ...
... These additional fibers were co-expressed with either the wild type HAdV-5 fiber gene, or a chimeric HAdV-3 fiber gene in the case of sigma 1 (Tsuruta et al., 2007). These studies showed that a second fiber protein was able to function as a ligand for its respective cellular receptor and provide expanded vector tropism (Pereboeva et al., 2007; Tsuruta et al., 2005 Tsuruta et al., , 2007). Since tumors represent a heterogeneous cell population (Heppner, 1984Heppner, , 1993 ), this fibermosaic system could thus be suitable for targeting multiple types of cells with distinct receptor expression profiles within tumors. ...
Human adenovirus serotype 5 (HAdV-5) attaches to its primary receptor, the coxsackie and adenovirus receptor (CAR) as the first step of infection. However, CAR expression decreases as tumors progress, thereby diminishing the utility of HAdV-5-based vectors for cancer therapy. In contrast, many aggressive tumor cells highly express CD46, a cellular receptor for HAdV-3. We hypothesized that a mosaic HAdV vector, containing two kinds of fiber proteins, would provide extensive transduction in a heterogeneous population of tumor cells with varying expression levels of HAdV receptors. We therefore generated a fiber-mosaic HAdV vector displaying both a chimeric HAdV-3 fiber and the HAdV-5 fiber protein. We verified the structural integrity of purified viral particles and confirmed that the fiber-mosaic HAdV vector has expanded tropism. We conclude that the use of fiber-mosaic HAdV vectors is a promising approach for transducing a heterogeneous cell population with different expression levels of adenovirus receptors.
... One more interesting strategy to broaden tropism and provide maximum enhanced transduction via utilization of multiple receptors is "dual fiber mosaicism" by expressing two separate fibers with distinct receptor-binding capabilities on the same viral particle (Fig. 2) (Tsuruta et al., 2005). Takayama et al. generated a dual-knob mosaic virus expressing both wild type Ad5 and chimeric Ad5/3 fibers on the same virion by coinfection of HEK293 cells with Ad5 and Ad5/3 viruses. ...
... Tsuruta et al. constructed a dual fiber-mosaic Ad5-based vector bearing fibers of wild type Ad5 and serotype 3 Dearing reovirus, σ1 attachment protein, which binds sialic acid and junction adhesion molecule 1 (JAM1). The Ad5-5/σ1 vector showed increased transduction of CAR-negative cell lines and primary human ovarian carcinoma cells (Tsuruta et al., 2005). In the follow-up study, Tsuruta et al. constructed an Ad5-based vector encoding both the 5/3 and 5/σ1 chimeric fibers. ...
The cellular internalization (infection of cells) of adenovirus 5 (Ad5) is mediated by the initial attachment of the globular knob domain of the capsid fiber protein to the cell surface coxsackievirus and adenovirus receptor (CAR), then followed by the interaction of the virus penton base proteins with cellular integrins. In tumors, there is a substantial intra- and intertumoral variability in CAR expression. The CAR-negative cells generally exhibit very low infectability. Since the fiber knob is a primary mediator of Ad5 binding to the cell surface, improved infectivity of Ad5-based vectors as oncolytic agents may be achieved via genetic modifications of this domain. The strategies to modify or broaden tropism and increase transduction efficiency of Ad5-based vectors include: 1) an incorporation of a targeting peptide into the fiber knob domain (the HI loop and/or C-terminus); 2) fiber knob serotype switching, or pseudotyping, by constructing chimeric fibers consisting of the knob domain derived from an alternate serotype (e.g., Ad5/3 or Ad5/35 chimeras), which binds to receptor(s) other than CAR (e.g., desmoglein 2/DSG2 and/or CD46); 3) "fiber complex mosaicism", an approach of combining serotype chimerism with peptide ligand(s) incorporation (e.g., Ad5/3-RGD); 4) "dual fiber mosaicism" by expressing two separate fibers with distinct receptor-binding capabilities on the same viral particle (e.g., Ad5-5/3 or Ad5-5/σ1); 5) fiber xenotyping by replacing the knob and shaft domains of wild-type Ad5 fiber protein with fibritin trimerization domain of T4 bacteriophage or σ1 attachment protein of reovirus. Other genetic approaches to increase the CAR-independent transduction efficiency include insertion of a targeting peptide into the hypervariable region of the capsid protein hexon or fusion to the C-terminus of pIX. Finally, we consider a yet unsolved molecular mechanism of liver targeting by Ad5-based vectors (CAR-, integrin-, fiber shaft KKTK motif-, and hepatic heparan sulfate glycosaminoglycans-independent, but fiber-, hexon- and blood factor X-dependent).
... Ad5-Sigma vector has a chimeric fiber with the tail and shaft from wild-type Ad5 fiber and the knob domain of serotype 3 Dearing reovirus called sigma 1 protein. This fiber modification enhances infectivity for certain types of cells via utilization of a coxsackie virus and adenovirus receptor (CAR)-independent cell entry mechanism [19,20]. Ad5-MSLN vector is an Ad5 vector that has its original promoter replaced by mesothelin (MSLN) promoter [21]. ...
Aim of the study:
Adenovector encoding tissue plasminogen activator (tPA) was shown to reduce experimental peritoneal adhesion. We investigated the targeting potential of our modified adenovector, its ability to reduce adhesions and the epigenetic role of histone methyltransferase EZH2 in adhesion formation.
Materials and methods:
Control lacZ, nonmodified tPA or modified tPA vectors were instilled in the peritoneal cavity after injury in de novo adhesions or after lysis of adhesions in recurrent adhesions. Adhesion severity was scored and adhesions and liver tissues were examined for adenovirus E4 gene and tPA mRNA expression. Levels of tPA, plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-β1 (TGF-β1), and EZH2 expression were measured.
Results:
E4 transcripts were detected in adhesions of nonmodified and modified and in livers of nonmodified but not in livers of modified de novo adhesions. Both nonmodified (p = 0.021) and modified vectors (p = 0.036) reduced the severity of de novo adhesions compared to lacZ vector. Levels of tPA in nonmodified (p = 0.021) and modified adhesions (p = 0.001) were elevated while PAI-1 (p = 0.013 and p = 0.001, respectively) and TGF-β1 levels (p = 0.002 and p = 0.016, respectively) were reduced compared with lacZ group. All vectors were not expressed in recurrent adhesions and severity score were not different among groups. EZH2 levels were elevated in de novo nontreated (p = 0.001) and was further increased in recurrent (p = 0.001) nontreated adhesions compared with noninjured peritoneum.
Conclusion:
Modified adenovirus successfully targeted de novo adhesions but not liver tissues and reduced the severity of de novo adhesions. EZH2 is involved in the development and progression of peritoneal adhesions.
... The strategy of re-engineering virus cell-attachment proteins (transductional targeting) has worked well for oncolytic adenovirus to overcome limited expression of its receptor, coxsackievirus and adenovirus receptor (CAR), on tumor cells. In fact, since the adenovirus fiber and reovirus σ1 cell-attachment proteins show structural similarity, the JAM-A binding head domain of reovirus was successfully fused to the adenovirus fiber tail to re-target adenovirus [93][94][95][96][97]. As a proof-of-concept for feasibility of transductional re-targeting in reovirus, van den Wollenberg (2008) added a hexahistidine (His) tag to the C-terminus of σ1 [98], and then expressed a single-chain His-tag-specific antibody fragment on the surface of JAM-A-deficient U118 glioblastoma cells. ...
Viruses that specifically replicate in tumor over normal cells offer promising cancer therapies. Oncolytic viruses (OV) not only kill the tumor cells directly; they also promote anti-tumor immunotherapeutic responses. Other major advantages of OVs are that they dose-escalate in tumors and can be genetically engineered to enhance potency and specificity. Unmodified wild type reovirus is a propitious OV currently in phase I-III clinical trials. This review summarizes modifications to reovirus that may improve potency and/or specificity during oncolysis. Classical genetics approaches have revealed reovirus variants with improved adaptation towards tumors or with enhanced ability to establish specific steps of virus replication and cell killing among transformed cells. The recent emergence of a reverse genetics system for reovirus has provided novel strategies to fine-tune reovirus proteins or introduce exogenous genes that could promote oncolytic activity. Over the next decade, these findings are likely to generate better-optimized second-generation reovirus vectors and improve the efficacy of oncolytic reotherapy.
... Ad5-Sigma is an Ad vector with fibers of Ad5 and reovirus, and the receptor-binding molecule of serotype T3D (type 3Dearing) reovirus called the σ1 protein that binds sialic acid and junction adhesion molecule 1 which determine the T3D reovirus tropism in enhancing the infectivity of the virus vector and gene transfer ranging from 2.3-to 45-fold in all tested cell lines [20]. Our data demonstrate that Ad5-Sigma-luc consistently exhibited the highest gene transfer among all fiber-modified viruses in human adhesion cells. ...
Postoperative abdominal/pelvic peritoneal adhesions are a major source of morbidity (bowel obstruction, infertility, ectopic gestation as well as chronic pelvic pain) in women. In this study, we screened various transduction and transcription modifications of adenovirus (Ad) to identify those that support maximal Ad-mediated gene delivery to human adhesion fibroblasts, which in turn would enhance the efficacy of this novel treatment/preventative strategy for postoperative adhesions. We transduced primary cultures of human peritoneal adhesion fibroblasts with fiber-modified Ad vectors Ad5-RGD-luc, Ad5-Sigma-luc, Ad5/3-luc and Ad5-CAV2-luc as well as transcriptional targeting viruses Ad5-survivin-luc, Ad5-heparanase-luc, Ad5-mesothelin (MSLN)-CRAd-luc and Ad5-secretory leukoprotease inhibitor (SLPI)-luc, and compared their activity to wild-type Ad5-luc. At 48 h, luciferase activity was measured and normalized to the total protein content in the cells. Among the fiber-modified Ad vectors, Ad5-Sigma-luc and among the transcriptional targeting modified Ad vectors, Ad5-MSLN-CRAd-luc showed significantly increased expression levels of luciferase activity at 5, 10 and 50 plaque forming units/cell in adhesion fibroblast cells compared with wild-type Ad5-luc (p < 0.05). Specific modifications of Ad improve their gene delivery efficiency towards human peritoneal adhesion fibroblasts. Developing a safe localized method to prevent/treat postoperative adhesion formation would have a major impact on women health.
... This was first shown by the incorporation of the fiber-like σ1 reovirus attachment protein into the Ad fiber. This allowed for effective transduction of target cells expressing junctional adhesion molecule (Tsuruta et al., 2005). ...
Cancer gene therapy approaches have benefited greatly from the utilization of molecular-based therapeutics. Of these, adenovirus-based interventions hold much promise as a platform for targeted therapeutic delivery to tumors. However, a barrier to this progression is the lack of native adenovirus receptor expression on a variety of cancer types. As such, any adenovirus-based cancer therapy must take into consideration retargeting the vector to nonnative cellular surface receptors. Predicated upon the knowledge gained in native adenovirus biology, several strategies to transductionally retarget adenovirus have emerged. Herein, we describe the biological hurdles as well as strategies utilized in adenovirus transductional targeting, covering the progress of both adapter-based and genetic manipulation-based targeting. Additionally, we discuss recent translation of these targeting strategies into a clinical setting.
... Novel " xenotyping " strategies involve the substitution of Ad5 knob proteins with those of non-human adenoviruses such as canine adenovirus (CAV-1 and CAV-2) [258] or members of the genus Atadenovirus, ovine atadenovirus type 7 (OAdV7) [260] and bovine atadenovirus [260]. Additionally, a successful fiber mosaic virus has been constructed by incorporating the trimeric σ1 spike protein from Reovirus into Ad5 [261,262]. This approach was made technically possible by the high degree of structural similarity between the receptor-binding determinants of these two distinct viral families. ...
Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and "bridging" interactions. "Bridging" interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of "bridging interactions" such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated "stealth" vectors which avoid these interactions. Simultaneous retargeting and detargeting can be achieved by combining multiple genetic and/or chemical modifications.
... The resultant mesothelin promoter -based viruses were AdMSLN-CRAdwt and AdMSLNCRAd5/3. Wild-type Ad5 (Ad5wt) was used as a ubiquitous replication control vector, whereas Ad5Luc1 (E1-deleted 14) and AdCMVSDF (E1-deleted nonreplicative vector, created by pShuttle-CMV and pAdEasy-1 in the AdEasy Vector System, Qbiogene, Inc.) were used as replication-incompetent control viruses with wild-type fiber. These control vectors were propagated in 293 cells and purified using a standard protocol (13). ...
Recently, virotherapy has been proposed as a new therapeutic approach for ovarian cancer. Conditionally replicative adenoviruses (CRAd) may contain tumor-specific promoters that restrict virus replication to cancer cells. Mesothelin, a cell surface glycoprotein, is overexpressed in ovarian cancer but not in normal ovarian tissues. The purpose of this study was to explore the therapeutic utility of a mesothelin promoter-based CRAd in a murine model of ovarian cancer, using noninvasive in vivo imaging.
We constructed a mesothelin promoter-based CRAd with a chimeric Ad5/3 fiber (AdMSLNCRAd5/3) that contains an Ad5 tail, Ad5 shaft, and an Ad3 knob. Previously, a chimeric Ad5/3 fiber has shown improved infectivity in many ovarian cancer cells. Viral replication and oncolysis were assessed in a panel of ovarian cancer cell lines. To test the oncolytic efficacy of AdMSLNCRAd5/3 in a murine model, bioluminescence imaging of tumor luciferase activity and survival analysis were done.
AdMSLNCRAd5/3 achieved up to a 10,000-fold higher cell killing effect and up to 120-fold higher levels of viral replication in all human ovarian cancer cells, compared with wild-type Ad5. AdMSLNCRAd5/3 significantly inhibited tumor growth as confirmed by in vivo imaging (P < 0.05). Survival with AdMSLNCRAd5/3 was significantly enhanced when compared with no virus or with a wild-type Ad5-treated group (P < 0.05).
The robust replication, oncolysis, and in vivo therapeutic efficacy of AdMSLNCRAd5/3 showed that this CRAd is a promising candidate for treating ovarian cancer. Importantly, we have applied in vivo imaging that has allowed repeated and longitudinal measurements of tumor growth after CRAd treatment.
... To overcome these challenges, alternative strategies based on fiber knob replacement rather than modification have been proposed. In these approaches, the knob of the Ad5 fiber is deleted and trimerization of the knobless protein restored by fusing it with a trimerization moiety, such as the coiled-coil domain of the retrovirus envelope glycoprotein (53), the neck region peptide of human lung surfactant D (32,48), or reovirus sigma-1 protein (35,47,52). ...
Efforts to develop adenovirus vectors suitable for genetic interventions in humans have identified three major limitations of the most frequently used vector prototype, human adenovirus serotype 5 (Ad5). These limitations--widespread preexisting anti-Ad5 immunity in humans, the high rate of transduction of normal nontarget tissues, and the lack of target-specific gene delivery--justify the exploration of other Ad serotypes as vector prototypes. In this paper, we describe the development of an alternative vector platform using simian Ad serotype 24 (sAd24). We found that sAd24 virions formed unstable complexes with blood coagulation factor X and, because of that, transduced the liver and other organs at low levels when administered intravenously. The overall pattern of biodistribution of sAd24 particles was similar, however, to that of Ad5, and the intravenously injected sAd24 was cleared by Kupffer cells, leading to their depletion. We modified the virus's fiber protein to design a Her2-specific derivative of sAd24 capable of infecting target human tumor cells in vitro. In the presence of neutralizing anti-Ad5 antibodies, Her2-mediated infection with targeted sAd24 compared favorably to that with the Ad5-derived vector. When used to target Her2-expressing tumors in animals, this fiber-modified vector achieved a higher level of gene transfer to metastasis-containing murine lungs than to tumor-free lungs. In aggregate, these studies provide important insights into sAd24 biology, identify its advantages and limitations as a vector prototype, and are thus essential for further development of an sAd24-based gene delivery platform.
... However, clinical trials utilizing Ad5 vectors have provided disappointing results111213, mainly due to inefficient gene delivery to human cancer cells141516. One explanation for this observation is that the expression level of the coxsackie virus and adenovirus receptor (CAR), which mediates cell attachment via the fiber protein of the Ad5 vector [17], is low in many cancers [14,18]. For example, CAR expression is decreased in specimens of prostate tumors at varying degrees of progression when compared with normal prostate tissue [19]. ...
We have developed a range of adenoviral (Ad) vectors based on human adenovirus serotype 5 (HAdV-5) displaying the fiber shaft and knob domains of species B viruses (HAdV-3, -11, or -35). These species B Ads utilize different cellular receptors than HAdV-5 for infection. We evaluated whether Ad vectors displaying species B fiber shaft and knob domains (Ad5F3Luc1, Ad5F11Luc1, and Ad5F35Luc1) would efficiently infect cancer cells of distinct origins, including prostate cancer.
The fiber chimeric Ad vectors were genetically generated and compared with the original Ad vector (Ad5Luc1) for transductional efficiency in a variety of cancer cell lines, including prostate cancer cells and primary prostate epithelial cells (PrEC), using luciferase as a reporter gene.
Prostate cancer cell lines infected with Ad5F3Luc1 expressed higher levels of luciferase than Ad5Luc1, as well as the other chimeric Ad vectors. We also analyzed the transductional efficiency via monitoring of luciferase activity in prostate cancer cells when expressed as a fraction of the gene transfer in PrEC cells. In the PC-3 and DU145 cell lines, the gene transfer ratio of cancer cells versus PrEC was once again highest for Ad5F3Luc1.
Of the investigated chimeric HAdV-5/species B vectors, Ad5F3Luc1 was judged to be the most suitable for targeting prostate cancer cells as it showed the highest transductional efficiency in these cells. It is foreseeable that an Ad vector incorporating the HAdV-3 fiber could potentially be used for prostate cancer gene therapy.
... Comparison of Ad pK7, AdRGD, and Ad5/3 revealed that while different modifications to the knob work better in some cell lines, there is an increase in viral infectivity in all the modified vectors compared to the Ad5 knob alone [47]. Using the σ1 protein, the receptor-binding molecule of serotype 3 bearing reovirus, in fusion with Ad5 fiber, a recombinant Ad was created that binds to sialic acid and JAM1 molecules in human cell lines including glioma [49]. This fusion Ad 5-Sigma vector was also found to have increased infectivity in a panel of glioma cells compared to wild type Ad5 [47]. ...
Glioblastoma multiforme is the most common form of primary brain cancer. In the past decade, virotherapy of tumors has gained credence, particularly in glioma management, as these tumors are not completely resectable and tend to micro-metastasize. Adenoviral vectors have an advantage over other viral vectors in that they are relatively non-toxic and do not integrate in the genome. However, the lack of coxsackie and adenovirus receptors on surface of gliomas provides for inefficient transduction of wild-type adenoviral vectors in these tumors. By targeting receptors that are overexpressed in gliomas, modified adenoviral constructs have been shown to efficiently infect glioma cells. In addition, by taking advantage of tumor-specific promoter elements, oncolytic adenoviral vectors offer the promise of selective tumor-specific replication. This dual targeting strategy has enabled specificity in both laboratory and pre-clinical settings. This review examines current trends in adenoviral virotherapy of gliomas, with an emphasis on targeting modalities and future clinical applications.
... Use of a j1-based attachment protein as an AdV targeting platform confirms recent reports that used j1 to confer reovirus tropism to an AdV [38,39]. Notably, the AdVTs with reovirus tropism were produced in the presence of complementing fiber, whereas AdG.L.Tail-T(ii)-MH was efficiently produced in the absence of complementing fiber. ...
Targeting adenovirus vectors (AdV's) for selective transduction of specific cell types requires ablation of native adenovirus tropism and introduction of a unique target-binding moiety. To bring these requirements within reach, we developed a novel strategy to target AdV's genetically that relies on replacement of the entire adenovirus fiber protein with a fusion molecule comprising the virion-anchoring domain of fiber and the oligomerization domain of reovirus attachment protein sigma1. The chimeric molecule forms trimers, is transported to the nucleus, and assembles onto the adenovirus capsid. In contrast to previously reported genetically targeted vectors, the AdV presented herein propagates efficiently without a requirement for complementing fiber. Due to ablation of the native adenovirus tropism, the infectivity of this AdV was at least 35-fold reduced on 293 cells. Importantly, a His tag incorporated into the chimeric attachment protein conferred His-tag-dependent tropism to the AdV, which resulted in a 12- to 40-fold greater transduction efficiency on two different cell lines expressing a His-tag-binding receptor. In addition, the infection efficiency was strongly reduced by preincubation with a His-tag-specific Ab. Thus, this sigma1-based chimeric attachment molecule provides a promising new platform for the generation of truly targeted AdV's.
... 253,254 Particularly interesting are results with a fiber mosaic Ad5 vector encoding two fibers: the s1 and the wt Ad5 fiber. 255 This mosaic transduces some primary OS cell lines even more efficiently than AdCMVlucRGD (V Ternovoi, unpublished data, 2005). ...
The clinical outcome for osteosarcoma (OS) remains discouraging despite efforts to optimize treatment using conventional modalities including surgery, radiotherapy and chemotherapy. Novel therapeutic approaches based on our expanding understanding of the mechanisms of tumor cell killing have the potential to alter this situation. Tumor suppressor gene therapy aims to restore the function of a tumor suppressor gene lost or functionally inactivated in cancer cells. One such molecule, the p53 tumor suppressor gene plays a critical role in safeguarding the integrity of the genome and preventing tumorigenesis. Introduction of wild-type (wt) p53 into transformed cells has been shown to be lethal for most cancer cells in vitro, but clinical trials of p53 gene replacement have had limited success. Analysis of these clinical trials highlighted the insufficient efficacy of current vectors and low proapoptotic activity of wt p53 as a single agent in vivo. In this review, a contemporary summarization of the current status of adenovirus-mediated p53 gene therapy of OS is presented. Advancement in our understanding of p53 tumor suppressor activity, the molecular biology of chemoresistant OS, and recent advances in tumor targeting with adenoviral vectors are also addressed. Based on these parameters, prospects for future investigations are proposed.
... Specifically, Dmitrev et al (13) reported that construction of modified adenoviral vectors containing the RGD peptide in the HI loop region, which targets the integrins αvß3 and αvß5 (14) instead of CAR, increased in gene transfer to ovarian cancer cell lines (30-600-fold) and to primary ovarian cancer cells obtained from patients (2-3-fold). Recently, many other approaches have been reported which include targeting Ad to the serotype 3 receptor with a chimeric fiber protein (15,16), targeting Ad to tumor cells with the non-human canine Ad type 1 or 2 knob (17), targeting Ad to a heparin sulfatecontaining receptor with an Ad fiber incorporating polylysine (pK7) (18) and targeting Ad to the junction adhesion molecule 1 (JAM1) with an Ad fiber incorporating reovirus sigma 1 fiber (19). ...
Conventional cancer treatments are not adequate for the majority of most patients stricken with squamous cell carcinomas of the head and neck (SCCHN). Conditionally replicating adenoviruses (CRAds) represent a promising new modality for treating of neoplastic diseases, including SCCHN. Specifically, CRAd agents infect tumor cells and selectively replicate within them, thus causing their death while sparing surrounding normal cells in the host. Oncolysis results from the replicative life cycle of the virus, which lyses infected tumor cells and releases viral progeny for propagation of infection and resultant lysis of neighboring cancer cells, sparing normal host cells. However, to date there have been two main limitations to successful clinical application of these CRAd agents: poor infectivity and poor tumor specificity. Here we report the construction of a CRAd agent, CRAd-CXCR4.F5/3, in which the adenovirus E1 gene is driven by a tumor-specific CXCR4 promoter, and the viral infectivity is enhanced by a fiber modification, F5/3, containing an Ad3 knob chimeric fiber protein. As expected, this agent improved both of the viral infectivity and tumor specificity as evaluated in established SCCHN tumor cell lines and in primary tumor tissues from multiple patients. As an added benefit, the activity of the CXCR4 promoter was low in human liver as described previously. Based on these data, the CRAd-CXCR4.F5/3 is a promising novel CRAd agent for SCCHN targeting with low host toxicity.
Human adenovirus (Ad) has been used extensively to develop gene transfer vectors for vaccine and gene therapy applications. A major factor limiting the efficacy of the current generation of Ad vectors is their inability to accomplish specific gene delivery to the cells of interest. Transductional targeting strategies seek to redirect virus binding to the appropriate cellular receptor to increase infection efficiency in selected cell types to achieve therapeutic intervention. These efforts mainly focused on incorporating targeting ligands by means of chemical conjugation or genetic modification of Ad capsid proteins and using bispecific adapter molecules to mediate virus recognition of target cells. This review summarizes current progress in Ad tropism modification maneuvers that embody genetic capsid modification and adapter-based approaches that have encouraging implications for further development of advanced vectors suitable for clinical translation.
The objective of the study was to explore the influence of junctional adhesion molecule A (JAM-A) gene decoration on proliferation and differentiation of human epidermal stem cells (hEpSCs). JAM-A gene and JAM-A interference gene lentivirus eukaryotic expression vectors were established. The recombinant lentivirus was introduced into hEpSCs to observe and detect viral transfection by fluorescence microscopy and Western blot, respectively. After confirmation of successful introduction of the target gene, cell growth curves were mapped out by cytometry to detect cell proliferation in different groups. The expression of hEpSCs labeled molecules was detected by immunofluorescence, and cell safety was detected by teratoma test in all groups. (1) Fluorescence microscopy showed that in the JAM-A over-expression (JAM-A(ov) EpSCs) group, the green fluorescence was mainly distributed in the cell membrane; in the JAM-A interference (JAM-A(kd) EpSCs) group and blank vector (GFP EpSCs) group, all cell bodies were luminous. Western blot showed that JAM-A protein was up-regulated in JAM-A(ov) EpSCs and down-regulated in JAM-A(kd) EpSCs. (2) Growth curves showed that hEpSCs entered the quick-growing phase 4 days after inoculation and reached the platform phase at day 7. JAM-A(ov) EpSCs proliferated more slowly than GFP EpSCs, while JAM-A(kd) EpSCs proliferated significantly faster than GFP EpSCs. (3) Immunofluorescence showed that the expression of transient amplification epidermal marker keratin 14, hEpSCs marker keratin I9 and β-integrin was down-regulated in JAM-A(kd) EpSCs group as compared to that in the GFP EpSCs group, and the expression of epidermal terminal differentiation marker K10 was negative in the JAM-A(kd) EpSCs group. There was no significant difference in the expression of specific molecules between JAM-A(ov) EpSCs and hEpSCs. (4) The result of teratoma test was negative in all groups. The proliferative ability of hEpSCs was increased markedly after down-regulation of JAM-A. Cells presented initial differentiation, but retained their stem cell characteristics without evidence of tumorigenesis.
Conditional replicative, oncolytic adenoviruses are genetically modified to replicate primarily in malignant cells with oncogenetic defects, selectively killing cancer cells in the process. Viral progenies released within the tumor microenvironment can penetrate, infect, and destroy additional cell layers of the solid tumor mass. Much excitement has been generated over the clinical successes of ONYX-015 adenovirus treatment for advanced head and neck cancer, pancreatic carcinoma, and metastatic colorectal carcinoma at the locoregional level. However, follow-up trials with systemic viral administration have not met initial expectations. This review examines recent basic science developments that may potentially overcome the two key limitations of systemic virotherapy, namely, suboptimal tumor oncolysis and pharmacokinetic shortcomings due to rapid viral clearance. Late-generation constructs incorporate alternative viral backbone modifications and antitumor transgenes to enhance oncolytic potency. Furthermore, altered viral tropism, through masking or substitution of viral surface components, is likely to promote tumor-selective targeting and safety. Merits of these approaches in the clinical arena are discussed. Drug Dev. Res. 66:260–277, 2006. © 2006 Wiley-Liss, Inc.
Addition of exogenous peptide sequences on viral capsids is a powerful approach to study the process of viral infection or to retarget viruses toward defined cell types. Until recently, it was not possible to manipulate the genome of mammalian reovirus and this was an obstacle to the addition of exogenous sequence tags onto the capsid of a replicating virus. This obstacle has now been overcome by the availability of the plasmid-based reverse genetics system. In the present study, reverse genetics was used to introduce different exogenous peptides, up to 40 amino acids long, at the carboxyl-terminal end of the σ1 outer capsid protein. The tagged viruses obtained were infectious, produce plaques of similar size, and could be easily propagated at high titers. However, attempts to introduce a 750 nucleotides-long sequence failed, even when it was added after the stop codon, suggesting a possible size limitation at the nucleic acid level.
Virotherapy, the use of oncolytic properties of viruses for eradication of tumor cells, is an attractive strategy for treating cancers resistant to traditional modalities. Adenoviruses can be genetically modified to selectively replicate in and destroy tumor cells through exploitation of molecular differences between normal and cancer cells. The lytic life cycle of adenoviruses results in oncolysis of infected cells and spreading of virus progeny to surrounding cells. In this study, we evaluated different strategies for improving safety and efficacy of oncolytic virotherapy against human ovarian adenocarcinoma. We examined the antitumor efficacy of Ad5/3-Δ24, a serotype 3 receptor-targeted pRb-p16 pathway-selective oncolytic adenovirus, in combination with conventional chemotherapeutic agents. We observed synergistic activity in ovarian cancer cells when Ad5/3-Δ24 was given with either gemcitabine or epirubicin, common second-line treatment options for ovarian cancer. Our results also indicate that gemcitabine reduces the initial rate of Ad5/3-Δ24 replication without affecting the total amount of virus produced. In an orthotopic murine model of peritoneally disseminated ovarian cancer, combining Ad5/3-Δ24 with either gemcitabine or epirubicin resulted in greater therapeutic benefit than either agent alone. Another useful approach for increasing the efficacy of oncolytic agents is to arm viruses with therapeutic transgenes such as genes encoding prodrug-converting enzymes. We constructed Ad5/3-Δ24-TK-GFP, an oncolytic adenovirus encoding the thymidine kinase (TK) green fluorescent protein (GFP) fusion protein. This novel virus replicated efficiently on ovarian cancer cells, which correlated with increased GFP expression. Delivery of prodrug ganciclovir (GCV) immediately after infection abrogated viral replication, which might have utility as a safety switch mechanism. Oncolytic potency in vitro was enhanced by GCV in one cell line, and the interaction was not dependent on scheduling of the treatments. However, in murine models of metastatic ovarian cancer, administration of GCV did not add therapeutic benefit to this highly potent oncolytic agent. Detection of tumor progression and virus replication with bioluminescence and fluorescence imaging provided insight into the in vivo kinetics of oncolysis in living mice. For optimizing protocols for upcoming clinical trials, we utilized orthotopic murine models of ovarian cancer to analyze the effect of dose and scheduling of intraperitoneally delivered Ad5/3-Δ24. Weekly administration of Ad5/3-Δ24 did not significantly enhance antitumor efficacy over a single treatment. Our results also demonstrate that even a single intraperitoneal injection of only 100 viral particles significantly increased the survival of mice compared with untreated animals. Improved knowledge of adenovirus biology has resulted in creation of more effective oncolytic agents. However, with more potent therapy regimens an increase in unwanted side-effects is also possible. Therefore, inhibiting viral replication when necessary would be beneficial. We evaluated the antiviral activity of chlorpromazine and apigenin on adenovirus replication and associated toxicity in fresh human liver samples, normal cells, and ovarian cancer cells. Further, human xenografts in mice were utilized to evaluate antitumor efficacy, viral replication, and liver toxicity. Our data suggest that these agents can reduce replication of adenoviruses, which could provide a safety switch in case of replication-associated side-effects. In conclusion, we demonstrate that Ad5/3-Δ24 is a useful oncolytic agent for treatment of ovarian cancer either alone or in combination with conventional chemotherapeutic drugs. Insertion of genes encoding prodrug-converting enzymes into the genome of Ad5/3-Δ24 might not lead to enhanced antitumor efficacy with this highly potent oncolytic virus. As a safety feature, viral activity can be inhibited with pharmacological substances. Clinical trials are however needed to confirm if these preclinical results can be translated into efficacy in humans. Promising safety data seen here, and in previous publications suggest that clinical evaluation of the agent is feasible. Ei saatavilla
Novel therapeutic approaches for endometriosis based on molecular strategies may prove to be useful. Conditionally replicative adenoviruses (CRAds) are designed to exploit key differences between target and normal cells. The wild-type adenovirus (Adwt) promoter can be replaced by tissue-specific promoters, allowing viral replication only in target cells. Viral infectivity can be enhanced by altering Ad tropism via fiber modification. We investigated whether CRAds can be used to target endometriosis and determined the most efficient transcriptional- and transductional-targeting strategy.
An in vitro study was carried out using human endometriotic cell lines, 11Z (epithelial) and 22B (stromal), normal human ovarian surface epithelial cell line (NOSE006) and primary human endometriosis cells. A total of 9 promoters and 12 Ad tropism modifications were screened by means of a luciferase reporter assay. From this screening data, three CRAds (CRAd-S-pK7, CRAd-S-RGD, CRAd-S-F5/3sigma1, all incorporating the survivin promoter but with different fiber modifications) were selected to perform experiments using Adwt and a replication-deficient virus as controls. CRAds were constructed using a plasmid recombination system. Viral-binding capacity, rates of entry and DNA replication were evaluated by quantitative real-time PCR of viral genome copy. Cell-killing effects were determined by crystal violet staining and a cell viability assay for different concentrations of viral particles per cell.
Comparison of promoters demonstrated that the survivin promoter exhibited the highest induction in both endometriotic cell lines. Among the fiber-modified viruses, the polylysine modification (pK7) showed the best infection enhancement. CRAd-S-pK7 was validated as the optimal CRAd to target endometriosis in terms of binding ability, entry kinetics, DNA replication and cell-killing effect. CRAd-S-pK7 also exhibited a high level of DNA replication in primary endometriosis cells.
CRAd-S-pK7 has the best infection and cell-killing effect in the context of endometriosis. It could prove to be a useful novel method to target refractory cases of endometriosis.
Cancer gene therapy approaches will derive considerable benefit from adenovirus (Ad) vectors capable of self-directed localization to neoplastic disease or immunomodulatory targets in vivo. The ablation of native Ad tropism coupled with active targeting modalities has demonstrated that innate gene delivery efficiency may be retained while circumventing Ad dependence on its primary cellular receptor, the coxsackie and Ad receptor. Herein, we describe advances in Ad targeting that are predicated on a fundamental understanding of vector/cell interplay. Further, we propose strategies by which existing paradigms, such as nanotechnology, may be combined with Ad vectors to form advanced delivery vehicles with multiple functions.
Adenovirus serotype 5 (Ad5) has been used for gene therapy with limited success due to insufficient infectivity in cells with low expression of the primary receptor, the coxsackie and adenovirus receptor (CAR). Evidence that adenovirus serotype receptors other than CAR may be of use was presented in previous studies that showed that the Ad3 receptor is expressed at high levels in ovarian cancer cells. We hypothesized that combined use of unique chimeric fibers in the context of novel mosaic adenovirus vectors would enhance infectivity via non-CAR pathways in ovarian cancer cells.
We constructed and characterized Ad5 vectors that use Ad3 knob and reovirus fibers to generate a mosaic fiber virion. Serotype 3 Dearing reovirus uses a fiber-like sigma 1 protein to infect cells expressing sialic acid and junction adhesion molecule 1. We therefore constructed a mosaic fiber Ad5 vector, designated Ad5/3-sigma 1, encoding two fibers: a sigma 1 chimeric fiber and the chimeric Ad5/3 fiber composed of an Ad3 knob.
Functionally, Ad5/3-sigma 1 used sialic acid, junction adhesion molecule 1, and Ad3 receptor for cell transduction and achieved maximum infectivity enhancement in ovarian cancer cells with low CAR expression. Furthermore, Ad5/3-sigma 1 achieved infectivity enhancement in primary tissue slices of human ovarian tumor.
We have developed a new type of Ad5 vector with the novel tropism, possessing fibers from Ad3 and reovirus, which exhibits enhanced infectivity via CAR-independent pathway(s). In addition, the flexible genetic platform of vector allows different combination of fiber variants that can be incorporated within the same particle.
Malignant glioma continues to be a major target for gene therapy and virotherapy due to its aggressive growth and the current lack of effective treatment. However, these approaches have been hampered by inefficient infection of glioma cells by viral vectors, particularly vectors derived from serotype 5 adenoviruses (Ad5). This results from limited cell surface expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on tumor cells. To circumvent this problem, Ad fiber pseudotyping, the genetic replacement of either the entire fiber or fiber knob domain with its structural counterpart from another human Ad serotype that recognizes a cellular receptor other than CAR, has been shown to enhance Ad infectivity in a variety of tumor types, including human glioma. Here, we have extended the paradigm of genetic pseudotyping to include fiber domains from non-human or "xenotype" Ads for infectivity enhancement of human glioma cell populations. In this study, we evaluated the gene transfer efficiency of a panel of Ad vectors which express one of five different "xenotype" fiber knob domains, including those derived from murine, ovine, porcine and canine species, in both human glioma cell lines as well as primary glioma tumor cells from patients. Adenovirus vectors displaying either canine Ad or porcine Ad fiber elements had the highest gene transfer to both glioma cell lines and primary tumor cells. The correlation between the viral infectivity of modified adenovirus vectors and expression of human CAR and CD46 (an adenovirus type B receptor) on the surfaces of tumor cells was also analyzed. Taken together, human adenovirus vectors modified with "xenotype" fiber elements could be excellent candidates to target human glioma.
Adenoviruses have been isolated from a large number of different species (mammalian and fowl) and over 100 different serotypes have been reported, some 43 of them human. The human adenoviruses, particularly types 2, 5, and 12, have been the most extensively characterized, and these viruses have served as valuable tools in the study of the molecular biology of DNA replication, transcription, RNA processing, and protein synthesis in mammalian cells. Seeref. 1 for a general review.
Mammalian reoviruses, prototype members of the Reoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins—ς1, μ1, and ς3—to enter host cells. ς1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of ς1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by “recoating” genome-containing core particles that lacked ς1, μ1, and ς3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to ς1. The recoated particles bound to and infected cultured cells in a ς1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant ς1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the ς1 protein. Additional experiments showed that recoated particles containing ς1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound ς1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of ς1 with respect to its structure, assembly into particles, and roles in entry.
While adenovirus (Ad) gene delivery vectors are useful in many gene therapy applications, their broad tropism means that they cannot be directed to a specific target cell. There are also a number of cell types involved in human disease which are not transducible with standard Ad vectors, such as Epstein-Barr virus (EBV)-transformed B lymphocytes. Adenovirus binds to host cells via the viral fiber protein, and Ad vectors have previously been retargeted by modifying the fiber gene on the viral chromosome. This requires that the modified fiber be able to bind to the cell in which the vector is grown, which prevents truly specific vector targeting. We previously reported a gene delivery system based on a fiber gene-deleted Ad type 5 (Ad5) vector (Ad5.betagal.DeltaF) and packaging cells that express the viral fiber protein. Expression of different fibers in packaging cells will allow Ad retargeting without modifying the viral chromosome. Importantly, fiber proteins which can no longer bind to the producer cells can also be used. Using this approach, we generated for the first time pseudotyped Ad5.betagal.DeltaF particles containing either the wild-type Ad5 fiber protein or a chimeric fiber with the receptor-binding knob domain of the Ad3 fiber. Particles equipped with the chimeric fiber bound to the Ad3 receptor rather than the coxsackievirus-adenovirus receptor protein used by Ad5. EBV-transformed B lymphocytes were infected efficiently by the Ad3-pseudotyped particles but poorly by virus containing the Ad5 fiber protein. The strategy described here represents a broadly applicable method for targeting gene delivery to specific cell types.
Many serotype 3 reoviruses bind to two different host cell molecules, sialic acid and an unidentified protein, using discrete receptor-binding domains in viral attachment protein, final sigma1. To determine mechanisms by which these receptor-binding events cooperate to mediate cell attachment, we generated isogenic reovirus strains that differ in the capacity to bind sialic acid. Strain SA+, but not SA-, bound specifically to sialic acid on a biosensor chip with nanomolar avidity. SA+ displayed 5-fold higher avidity for HeLa cells when compared with SA-, although both strains recognized the same proteinaceous receptor. Increased avidity of SA+ binding was mediated by increased k(on). Neuraminidase treatment to remove cell-surface sialic acid decreased the k(on) of SA+ to that of SA-. Increased k(on) of SA+ enhanced an infectious attachment process, since SA+ was 50-100-fold more efficient than SA- at infecting HeLa cells in a kinetic fluorescent focus assay. Sialic acid binding was operant early during SA+ attachment, since the capacity of soluble sialyllactose to inhibit infection decreased rapidly during the first 20 min of adsorption. These results indicate that reovirus binding to sialic acid enhances virus infection through adhesion of virus to the cell surface where access to a proteinaceous receptor is thermodynamically favored.
Adenoviral (Ad) vectors have been widely used in the context of cancer gene therapy approaches. Their utility in these contexts, however, has frequently been limited by tumor cell resistance to Ad infection. The basis of this resistance has been defined recently as resulting from a deficiency of the primary adenovirus receptor, coxsackie adenovirus receptor. As a means to circumvent this limitation, a variety of tropism modification strategies have allowed coxsackie adenovirus receptor-independent gene delivery via the Ad vector. These advanced generation adenovirus vectors exhibit enhanced infectivity, which can allow direct therapeutic gain. Such vectors may allow improvements in efficacy in the context of ongoing human clinical gene therapy approaches for cancer.
Exploiting the lytic life cycle of viruses has gained recent attention as an anticancer strategy (oncolysis). To explore the utility of adenovirus (Ad)-mediated oncolysis for rhabdomyosarcoma (RMS), we tested RMS cell lines for Ad gene transduction and infection. RMS cells were variably transduced by Ad. Compared with control cells, RMS cells were less sensitive or even resistant to oncolysis by wild-type virus. RMS cells expressed the Ad internalization receptors, alpha(v) integrins, but had low or undetectable expression of the major attachment receptor, coxsackievirus-Ad receptor (CAR). Mutant Ads with ablated CAR binding exhibited only 5-20% of transgene expression in RMS cells seen with a wild-type vector, suggesting that residual or heterogeneous CAR expression mediated the little transduction that was detectable. Immunohistochemical analysis of archived clinical specimens showed little detectable CAR expression in five embryonal and eight alveolar RMS tumors. Stable transduction of the cDNA for CAR enabled both efficient Ad gene transfer and oncolysis for otherwise resistant RMS cells, suggesting that poor CAR expression is the limiting feature. Gene transfer to RMS cells was increased >2 logs using Ads engineered with modified fiber knobs containing either an integrin-binding RGD peptide or a polylysine peptide in the exposed HI loop. The RGD modification enabled increased oncolysis for RMS cells by a conditionally replicative Ad, Ad delta24RGD, harboring a retinoblastoma-binding mutation in the E1A gene. Thus, the development of replication-competent vectors targeted to cell surface receptors other than CAR is critical to advance the use of Ad for treating RMS.
Gene delivery efficiency in clinical cancer gene therapy trials with recombinant adenoviruses (Ads) based on serotype 5 (Ad5) has been limited partly because of variable expression of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human primary cancer cells. As a means of circumventing CAR deficiency, Ad vectors have been retargeted by creating chimeric fibers possessing knob domains of alternate Ad serotypes. In this study, we have constructed an Ad5-based vector, Ad5/3luc1, with a chimeric fiber protein featuring a knob domain derived from Ad3. This virus is retargeted to the Ad3 receptor and, therefore, has different tissue tropism. A novel knob binding assay was used to measure expression of CAR and the Ad3 receptor. Further, to evaluate the correlation of receptor expression and infectivity by Ad, a panel of ovarian cancer cell lines and purified primary cancer cells were infected with Ad5luc1 and Ad5/3luc1 at 50, 200, and 1000 viral particles/cell. Our results confirm that Ad5/3luc1 is retargeted to the Ad3 receptor. Furthermore, the Ad3 receptor is present at higher levels than CAR on ovarian adenocarcinoma cells. Also, the amount of binding to primary receptor appears to be the major factor determining the efficiency of transgene expression. The Ad5/3 chimera displays enhanced infectivity for ovarian cancer cell lines and purified primary tumor cells, which could translate into increased efficacy in clinical trials.
The use of adenovirus (Ad) vectors for cancer gene therapy applications is currently limited by several factors, including broad Ad tropism associated with the widespread expression of coxsackievirus and adenovirus receptor (CAR) in normal human tissues, as well as limited levels of CAR in tumor cells. To target Ad to relevant cell types, we have proposed using soluble CAR (sCAR) ectodomain fused with a ligand to block CAR-dependent native tropism and to simultaneously achieve infection through a novel receptor overexpressed in target cells. To confer Ad targeting capability on cancer cells expressing the c-erbB-2/HER-2/neu oncogene, we engineered a bispecific adapter protein, sCARfC6.5, that consisted of sCAR, phage T4 fibritin polypeptide, and C6.5 single-chain fragment variable (scFv) against c-erbB-2 oncoprotein. Incorporation of fibritin polypeptide provided trimerization of sCAR fusion proteins that, compared with monomeric sCAR protein, resulted in augmented affinity to Ad fiber knob domain and in increased ability to block CAR-dependent Ad infection. We demonstrated that sCARfC6.5 protein binds to cellular c-erbB-2 oncoprotein and mediates efficient Ad targeting via a CAR-independent pathway. As illustrated in cancer cell lines that overexpress c-erbB-2, targeted Ad, complexed with sCARfC6.5 adapter protein, provided from 1.5- to 17-fold enhancement of gene transfer compared with Ad alone and up to 130-fold increase in comparison with untargeted Ad complexed with sCARf control protein. The use of recombinant trimeric sCAR-scFv adapter proteins may augment Ad vector potency for targeting cancer cell types.
Reovirus selectively replicates in and destroys cancer cells with an activated Ras signaling pathway. In this study, we evaluated the feasibility of using reovirus (serotype 3, strain Dearing) as an antihuman colon and ovarian cancer agent. In in vitro studies, reovirus infection in human colon and ovarian cell lines was assessed by cytopathic effect as detected by light microscopy, [(35)S]Methionine labeling of infected cells for viral protein synthesis and progeny virus production by plaque assay. We observed that reovirus efficiently infected all five human colon cancer cell lines (Caco-2, DLD-1, HCT-116, HT-29, and SW48) and four human ovarian cancer cell lines (MDAH2774, PA-1, SKOV3, and SW626) which were tested, but not a normal colon cell line (CCD-18Co) or a normal ovarian cell line (NOV-31). We also observed that the Ras activity in the human colon and ovarian cancer cell lines was elevated compared with that in normal colon and ovarian cell lines. In animal models, intraneoplastic as well as i.v. inoculation of reovirus resulted in significant regression of established s.c. human colon and ovarian tumors implanted at the hind flank. Histological studies revealed that reovirus infection in vivo was restricted to tumor cells, whereas the surrounding normal tissue remained uninfected. Additionally, in an i.p. human ovarian cancer xenograft model, inhibition of ascites tumor formation and the survival of animals treated with live reovirus was significantly greater than of control mice treated with UV-inactivated reovirus. Reovirus infection in ex vivo primary human ovarian tumor surgical samples was also confirmed, further demonstrating the potential of reovirus therapy. These results suggest that reovirus holds promise as a novel agent for human colon and ovarian cancer therapy.
The efficacy of adenovirus (Ad)-based gene therapy might be significantly improved if viral vectors capable of tissue-specific gene delivery could be developed. Previous attempts to genetically modify the tropism of Ad vectors have been only partially successful, largely due to the limited repertoire of ligands that can be incorporated into the Ad capsid. Early studies identified stringent size limitations imposed by the structure of the Ad fiber protein on ligands incorporated into its carboxy terminus and thus limited the range of potential ligand candidates to short peptides. We have previously identified the HI loop of the fiber knob domain as a preferred site for the incorporation of targeting ligands and hypothesized that the structural properties of this loop would allow for the insertion of a wide variety of ligands, including large polypeptide molecules. In the present study we have tested this hypothesis by deriving a family of Ad vectors whose fibers contain polypeptide inserts of incrementally increasing lengths. By assessing the levels of productivity and infectivity and the receptor specificities of the resultant viruses, we show that polypeptide sequences exceeding by 50% the size of the knob domain can be incorporated into the fiber with only marginal negative consequences on these key properties of the vectors. Our study has also revealed a negative correlation between the size of the ligand used for vector modification and the infectivity and yield of the resultant virus, thereby predicting the limits beyond which further enlargement of the fiber knob would not be compatible with the virion's integrity.
The coxsackievirus and adenovirus receptor (CAR) is a membrane glycoprotein with a cytoplasmic domain, a transmembrane domain and an extracellular region consisting of two immunoglobulin-like domains, an amino-terminal immunoglobulin variable (IgV)-related domain (D1), which is distal to the cell surface, and a proximal IgC2 domain (D2). The coxsackievirus and adenovirus receptor has been shown to exhibit tumour suppression activity in human bladder and prostate cancer cells. In the current paper, we demonstrate that CAR is a tumour suppressor in glioma cells and that the extracellular D2 domain is not required for this inhibitory effect. This finding provides a biological basis for the observation that expression of CAR is downregulated in malignant glioma cells. This suggests that strategies to redirect adenoviruses to achieve CAR-independent infection will be necessary to realise the full potential of adenoviral vectors for cancer gene therapy.
Mammalian reoviruses are nonenveloped viruses that contain a segmented double-stranded RNA genome. Most mammalian species, including humans, serve as hosts for reovirus infection, but reovirus-induced disease is restricted to the very young (reviewed in reference 61). Reovirus infections of newborn mice have been used as a tractable experimental system for studies of viral pathogenesis. The segmented genome of these viruses has allowed the genetic basis for complex viral phenotypes to be determined by analysis of reassortant viruses containing mixtures of gene segments derived from parental strains that exhibit biological polymorphisms of interest. One of the best characterized models of reovirus pathogenesis is infection of the murine central nervous system (CNS), in which serotype 1 (T1) and serotype 3 (T3) reoviruses display markedly different patterns of disease (Table (Table11).
TABLE 1.
Properties of reovirus pathogenesis attributable to σ1 protein
After oral inoculation of newborn mice, reovirus is taken up by intestinal M cells (72) and undergoes primary replication in lymphoid tissue of the Peyer's patches. The virus then invades the CNS, yet T1 and T3 strains use different routes of dissemination and manifest distinct pathological consequences. T1 reovirus spreads to the CNS hematogenously and infects ependymal cells (62, 69), resulting in hydrocephalus (68). In contrast, T3 reovirus spreads to the CNS neurally and infects neurons (38, 62, 69), causing lethal encephalitis (58, 68). Analysis of reassortant viruses obtained by coinfecting cells with prototype strains T1 Lang (T1L) and T3 Dearing (T3D) demonstrated that the pathways of viral spread in the host (62) and tropism for neural tissues (24, 69) segregate with the viral S1 gene, which encodes the viral attachment protein σ1 (33, 67). T1L × T3D reassortant viruses were also used to show that serotype-specific differences in virus binding to primary cultures of ependymal cells and neurons are determined by the S1 gene (24, 59). These studies suggest that σ1 dictates the CNS cell types that serve as targets for reovirus infection, presumably by its capacity to bind receptors expressed by specific CNS cells.
Reovirus pathogenesis is not restricted to the CNS, and σ1 is not the sole determinant of reovirus virulence. Reovirus infection causes pathology and physiologic dysfunction in a wide range of organs and tissues, including the hepatobiliary system, the myocardium, lungs, and endocrine tissues (reviewed in reference 65). Of these, myocarditis (54) has become a particularly well-established experimental model of reovirus-induced disease. Myocarditis caused by reovirus infection is unusual in comparison to other viral etiologies of myocarditis in that the pathogenesis is not immune mediated. Instead, reovirus cytopathicity is a direct cause of myocyte injury, which results from a complex interplay of the interferon (1, 42, 55) and apoptosis (22) pathways. Efficiency of viral RNA synthesis is a key factor in determining the extent of myocardial injury (51). Accordingly, viral gene segments encoding proteins involved in viral transcription and genome replication play important roles in determining strain-specific differences in the capacity of reovirus to induce myocarditis (52, 53, 55).
Adenovirus (Ad) targeting is a novel approach for the design and administration of therapeutic agents wherein the agent is rationally designed to localize and restrict transgene expression to the site of disease in a self-directed manner, usually via exploitation of unique biophysical and genetic properties specific to the diseased tissue. The ablation of promiscuous native Ad tropism coupled with active targeting modalities has demonstrated that innate gene delivery efficiency may be retained while circumventing Ad dependence on its primary cellular receptor, the coxsackie and adenovirus receptor (CAR), to achieve CAR-independent vector tropism. Herein, we describe advances in Ad targeting that are predicated not only on fundamental understanding of vector/cell interplay, but also on the specific transcriptional profiles of target tissues. Further, targeting is discussed in the context of improving the safety and efficacy of clinical approaches utilizing adenoviral vectors and replication competent oncolytic agents. In summary, existing results suggest a critical linkage between targeted agents and increases in therapeutic utility.
The utility of adenovirus (Ad) vectors for gene transduction can be limited by receptor specificity. We developed a gene-delivery vehicle in which the potent Ad5 vector was genetically reengineered to display the mucosal-targeting sigma1 protein of reovirus type 3 Dearing (T3D). A sigma1 construct containing all but a small virion-anchoring domain was fused to the N-terminal 44 aa of Ad5 fiber. This chimeric attachment protein Fibtail-T3Dsigma1 forms trimers and assembles onto Ad virions. Fibtail-T3Dsigma1 was recombined into the Ad5 genome, replacing sequences encoding wild-type fiber. The resulting vector, Ad5-T3Dsigma1, expresses Fibtail-T3Dsigma1 and infects Chinese hamster ovary cells transfected with human or mouse homologs of the reovirus receptor, junctional adhesion molecule 1 (JAM1), but not the coxsackievirus and Ad receptor. Treatment of Caco-2 intestinal epithelial cells with either JAM1-specific antibody or neuraminidase reduced transduction by Ad5-T3Dsigma1, and their combined effect decreased transduction by 95%. Ad5-T3Dsigma1 transduces primary cultures of human dendritic cells substantially more efficiently than does Ad5, and this transduction depends on expression of JAM1. These data provide strong evidence that Ad5-T3Dsigma1 can be redirected to cells expressing JAM1 and sialic acid for application as a vaccine vector.
Reoviruses infect cells that manifest an activated Ras-signaling pathway, and have been shown to effectively destroy many different types of neoplastic cells, including those derived from brain, breast, colon, ovaries, and prostate. In this study, we investigated the reovirus as a potential therapeutic agent against lymphoid malignancies. A total of 9 lymphoid cell lines and 27 primary human lymphoid malignancies, as well as normal lymphocytes and hematopoietic stem/progenitor cells, were tested for susceptibility to reovirus infection. For in vitro studies, the cells were challenged with reovirus (serotype 3 Dearing), and viral infection was assessed by cytopathic effects, viability, viral protein synthesis, and progeny virus production. We present evidence of efficient reovirus infection and cell lysis in the diffuse large B-cell lymphoma cell lines and Burkitt lymphoma cell lines Raji and CA46 but not Daudi, Ramos, or ST486. Moreover, when Raji and Daudi cell lines were grown subcutaneously in severe combined immunodeficient/nonobese diabetic (SCID/NOD) mice and subsequently injected with reovirus intratumorally or intravenously, significant regression was observed in the Raji-induced, but not the Daudi-induced, tumors, which is consistent with the in vitro results. Susceptibility to reovirus infection was also detected in 21 of the 27 primary lymphoid neoplasias tested but not in the normal lymphocytes or hematopoietic stem/progenitor cells. Our results suggest that reovirus may be an effective agent against several types of human lymphoid malignancies.
The reovirus σ1 protein (HA) is the polypeptide responsible for specific cell surface binding and hemagglutination. In order to define the biochemical basis of these properties, we have isolated a number of hybridomas secreting monoclonal antibodies directed against the serotype 1 and 3 HA proteins. Using neutralization and hemagglutination inhibition (HI) testing, we have been able to define at least four distinct antigenic regions (epitopes) on the type 3 HA. One class of antibodies had exclusively neutralizing activity; a second class had only HI activity. One antibody had neutralizing and HI activity and the fourth class of monoclonal antibodies had no detectable neutralization or HI activity. Thus there appears to be marked functional specialization within regions of the reovirus type 3 HA.
The adenovirion has been shown to contain at least nine different polypeptides demonstrable by dissociation and acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Each polypeptide of adenovirus type 2 is chemically distinct by isotopic ratio analysis, and they all contain lysine, arginine, tryptophan, valine, and threonine. The molecular weights of these polypeptides determined by gel electrophoresis range from 120,000 for the largest and most prominent component to 7500 for the smallest. Comparison of nontumorigenic type 2 with tumorigenic types 7A and 12 by double-isotope labeling revealed a generally similar peptide pattern for all types. However, there were distinct differences between the corresponding peptides of all three types. These results imply extensive differences in the genes for most of the capsid proteins.
To expand the utility of recombinant adenovirus vectors for gene therapy applications, methods to alter native viral tropism to achieve cell-specific transduction would be beneficial. To this end, we are pursuing genetic methods to alter the cell recognition domain of the adenovirus fiber. To incorporate these modified fibers into mature virions, we have developed a method based on homologous DNA recombination between two plasmids. A fiber-deleted, propagation-defective rescue plasmid has been designed for recombination with a shuttle plasmid encoding a variant fiber gene. Recombination between the two plasmids results in the derivation of recombinant viruses containing the variant fiber gene. To establish the utility of this method, we constructed a recombinant adenovirus containing a fiber gene with a silent mutation. In addition, we generated an adenovirus vector containing chimeric fibers composed of the tail and shaft domains of adenovirus serotype 5 and the knob domain of serotype 3. This modification was shown to alter the receptor recognition profile of the virus containing the fiber chimera. Thus, this two-plasmid system allows for the generation of adenovirus vectors containing variant fibers. This method provides a rapid and facile means of generating fiber-modified recombinant adenoviruses. In addition, it should be possible to use this system in the development of adenovirus vectors with modified tropism to allow cell-specific targeting.
The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.
Adenovirus-based gene therapy vectors now in use cannot be targeted to specific cell types in vivo and are immunogenic, properties which limit their clinical utility. Improved vectors lacking the genes for viral structural proteins may overcome these limitations. We have developed cell lines which stably express the adenovirus type 5 (Ad5) fibre protein in its native trimeric form. These cells can complement an Ad5 mutant with a defect in the fibre gene, and are capable of incorporating the Ad5 fibre into particles of a different Ad serotype. As the fibre protein is responsible for the initial binding of virus to cells, packaging cell lines expressing different or modified fibre proteins will be useful in studying the mechanism by which adenovirus infects different cell types.
In vivo cancer gene therapy approaches for squamous cell carcinoma of the head and neck (SCCHN) based on adenoviral vector-mediated gene delivery have been limited by the suboptimal efficacy of gene transfer to tumor cells. We hypothesized that this issue was due to deficiency of the primary adenoviral receptor, the coxsackie-adenovirus receptor (CAR), on the tumor targets. Studies of CAR levels on SCCHN cell lines confirmed that their relative refractoriness to the adenoviral vector was based on this deficiency. To circumvent this deficiency, we applied an adenoviral vector targeted to a tumor cell marker characteristic of SCCHN. In this regard, integrins of the alpha2beta1 and alpha3beta1 class are frequently overexpressed in SCCHN. Furthermore, these integrins recognize the RGD peptide motif. On this basis, we applied an adenoviral vector genetically modified to contain such a peptide within the HI loop of the fiber protein as a means to alter viral tropism. Studies confirmed that the CAR-independent gene delivery achieved via this strategy allowed enhanced gene transfer efficiencies to SCCHN tumor cells. Importantly, this strategy could achieve preferential augmentation of gene transfer in tumor cells compared with normal cells. The ability to achieve enhanced and specific gene transfer to tumor cells via adenoviral vectors has important implications for gene therapy strategies for SCCHN and for other neoplasms in general.
Efficient gene transfer by recombinant adenovirus (Ad) vectors depends on expression of CAR and alpha(v) integrin on target cells. Because Ad may also infect nearby nontarget cells expressing these receptors, such as peritoneal mesothelial cells after i.p. injection, we hypothesized that targeting Ad gene delivery to a receptor overexpressed on most ovarian carcinoma cells, such as TAG-72, would enhance the selectivity of Ad gene transfer when used in this context. A monoclonal antibody that has been investigated clinically for immunotherapy and immunodetection of ovarian carcinomas, namely CC49, was used to construct a bispecific conjugate with the Fab fragment of a neutralizing anti-knob mAb to target Ad binding via TAG-72. This conjugate facilitated TAG-72-specific, CAR-independent Ad reporter gene transfer to both ovarian cancer cell lines and primary ovarian cancer cells cultured from malignant ascites fluid. Fab-CC49 was very selective for tumor cells, augmenting Ad gene transfer to primary ovarian cancer cells 2- to 28-fold relative to untargeted Ad, while also decreasing gene transfer to autologous cultured mesothelial cells 4- to 9-fold. These data suggest that targeting Ad via TAG-72 may improve the selectivity of Ad gene transfer for ovarian tumors 8- to 252-fold on i.p. vector injection. These results also define the requirements for a candidate target receptor in the rational design of a targeted Ad vector for ultimate clinical utility, one that selectively infects tumor cells and spares normal cells on i.p. injection. Such a vector may increase gene transfer and decrease the toxicity of Ad vectors, which would improve the therapeutic index of cytotoxic gene therapy for ovarian cancer in clinical trials.
Virus attachment to cells plays an essential role in viral tropism and disease. Reovirus serotypes 1 and 3 differ in the capacity to target distinct cell types in the murine nervous system and in the efficiency to induce apoptosis. The binding of viral attachment protein sigma1 to unidentified receptors controls these phenotypes. We used expression cloning to identify junction adhesion molecule (JAM), an integral tight junction protein, as a reovirus receptor. JAM binds directly to sigma1 and permits reovirus infection of nonpermissive cells. Ligation of JAM is required for reovirus-induced activation of NF-kappaB and apoptosis. Thus, reovirus interaction with cell-surface receptors is a critical determinant of both cell-type specific tropism and virus-induced intracellular signaling events that culminate in cell death.
Primary ovarian cancer cells obtained from fresh tumor have many advantages over established cell lines. Therefore, a procedure for the specific and efficient purification of such neoplastic cells is critical. We report an effective immunomagnetic method for the isolation of tumor cells from the ascitic fluid of patients diagnosed with ovarian adenocarcinoma.
This procedure incorporates the use of monoclonal antibody (mAb) CC49, which recognizes the tumor-associated glycoprotein 72 (TAG-72). TAG-72 is highly expressed on ovarian tumor cell surfaces with little or no reactivity with normal tissues. Also used in this protocol are immunomagnetic beads, which bind to the CC49 mAb via a secondary antibody. When ovarian cancer cells adhere to the magnetic beads, a magnetic field is used to separate the tumor cells from all other cellular components.
Using ascitic fluid from five patients, we found that preparations before purification contained between 38 and 52% neoplastic cells. Using our method, we produced preparations that were between 63 and 96% pure for cancer cells, thus obtaining an average increase in tumor cell enrichment of 86%.
We, therefore, believe this method is preferable for producing high yields of pure ovarian neoplastic cells. We are now employing this technique in our laboratory to provide a stringent and pure template for our studies on gene transfer to primary ovarian cancer cells.
Reovirus attaches to cellular receptors with the sigma1 protein, a fiber-like molecule protruding from the 12 vertices of the icosahedral virion. The crystal structure of a receptor-binding fragment of sigma1 reveals an elongated trimer with two domains: a compact head with a new beta-barrel fold and a fibrous tail containing a triple beta-spiral. Numerous structural and functional similarities between reovirus sigma1 and the adenovirus fiber suggest an evolutionary link in the receptor-binding strategies of these two viruses. A prominent loop in the sigma1 head contains a cluster of residues that are conserved among reovirus serotypes and are likely to form a binding site for junction adhesion molecule, an integral tight junction protein that serves as a reovirus receptor. The fibrous tail is mainly responsible for sigma1 trimer formation, and it contains a highly flexible region that allows for significant movement between the base of the tail and the head. The architecture of the trimer interface and the observed flexibility indicate that sigma1 is a metastable structure poised to undergo conformational changes upon viral attachment and cell entry.
The efficiency of cancer gene therapy with recombinant adenoviruses based on serotype 5 (Ad5) has been limited partly because of variable, and often low, expression by human primary cancer cells of the primary cellular-receptor which recognizes the knob domain of the fiber protein, the coxsackie and adenovirus receptor (CAR). As a means of circumventing CAR deficiency, Ad vectors have been retargeted by utilizing chimeric fibers possessing knob domains of alternate Ad serotypes. We have reported that ovarian cancer cells possess a primary receptor for Ad3 to which the Ad3 knob binds independently of the CAR-Ad5 knob interaction. Furthermore, an Ad5-based chimeric vector, designated Ad5/3, containing a chimeric fiber proteins possessing the Ad3 knob, demonstrates CAR-independent tropism by virtue of targeting the Ad3 receptor. Based on these findings, we hypothesized that a mosaic virus possessing both the Ad5 knob and the Ad3 knob on the same virion could utilize either primary receptor, resulting in expanded tropism. In this study, we generated a dual-knob mosaic virus by coinfection of 293 cells with Ad5-based and Ad5/3-based vectors. Characterization of the resultant virions confirmed the incorporation of both Ad5 and Ad3 knobs in the same particle. Furthermore, this mosaic virus was able to utilize either receptor, CAR and the Ad3 receptor, for virus attachment to cells. Enhanced Ad infectivity with the mosaic virus was shown in a panel of cell lines, with receptor profiles ranging from CAR-dominant to Ad3 receptor-dominant. Thus, this mosaic virus strategy may offer the potential to improve Ad-based gene therapy approaches by infectivity enhancement and tropism expansion.
Genetic modification of the adenovirus (Ad) capsid is one of the successful strategies to achieve viral retargeting. However, it has been widely recognized that structural constraints imposed by viral proteins limit the number and nature of incorporated targeting ligands and often hamper viral propagation. To address this issue, we propose a genetic fiber-mosaic virus (having two distinct fibers in one viral particle) as a means to facilitate fiber modifications. Fiber-mosaic virus having tandem fibers: a wild type (wt) fiber and second adjunctive fiber, will utilize natural viral entry for the conventional propagation of the vectors whereas, adjunctive fiber will serve multiple potential purposes such as targeting, purification, or imaging of viral particles via genetic incorporation of the corresponding functional moieties. We generated the mosaic adenovirus vector encoding two fibers: wild-type and adjunctive fiber--Fiber-Fibritin (FF) and confirmed incorporation of FF in the mosaic viral particles. We investigated binding specificity of the mosaic virus and the possible interference of the two fibers during virus life cycle. Fiber-mosaic Ad attained new binding properties provided by the second fiber, while preserving the binding ability attributed to the wt fiber. Our results suggest that the two fibers being presented and structurally separated on the viral particle may also function separately as binding counterparts for virus attachment. Therefore, the mosaic setting will allow more flexibility in Ad retargeting approaches.