Adenovirus entry into host cells: a role for alpha(v) integrins.

The mechanism(s) by which nonenveloped viruses enter host cells is poorly understood. The recent identification of cell-surface alpha(v) integrins as receptors for adenovirus internalization has shed much light on this process. In addition, analysis of alpha(v) integrins as internalization receptors for adenovirus has provided further insights into the biology of integrins.

Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization.

Human adenovirus type 2 (Ad2) enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins alpha v beta 3 and alpha v beta 5. While both alpha v integrins promote virus internalization, alpha v beta 5 is involved in the subsequent event of membrane permeabilization. Cells transfected with the beta 5 or beta 3 subunit, expressing either alpha v beta 5 and alpha v beta 3, respectively, were capable of supporting Ad2 infection to varying degrees. In this case, cells expressing alpha v beta 5 were significantly more susceptible to Ad2-induced membrane permeabilization, as well as to Ad2 infection, than cells expressing alpha v beta 3. Adenovirus-mediated gene delivery was also more efficient in cells expressing alpha v beta 5. These results suggest that the interaction of alpha v beta 5 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virus-mediated membrane permeabilization and suggest a novel biological role for integrin alpha v beta 5 in the infectious pathway of a human adenovirus.

Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing adenoviridae.

The human adenovirus serotype 5 (Ad5) is used widely for applications in human gene therapy. Cellular attachment of Ad5 is mediated by binding of the carboxyl-terminal knob of its fiber coat protein to the Coxsackie adenovirus receptor (CAR) protein. However, Ad5 binding to CAR hampers the development of adenovirus vectors capable of specifically targeting (diseased) tissues or organs. Through sequence analysis and mutagenesis, a conserved receptor-binding region was identified on the side of three divergent CAR-binding knobs. The feasibility of simultaneous CAR ablation and redirection of an adenovirus to a new receptor is demonstrated.

CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts.

Primary fibroblasts are not efficiently transduced by subgroup C adenovirus (Ad) vectors because they express low levels of the high-affinity Coxsackie virus and adenovirus receptor (CAR). In the present study, we have used primary human dermal fibroblasts as a model to explore strategies by which Ad vectors can be designed to enter cells deficient in CAR. Using an Ad vector expressing the human CAR cDNA (AdCAR) at high multiplicity of infection, primary fibroblasts were converted from being CAR deficient to CAR sufficient. Efficiency of subsequent gene transfer by standard Ad5-based vectors and Ad5-based vectors with alterations in penton and fiber was evaluated. Marked enhancement of binding and transgene expression by standard Ad5 vectors was achieved in CAR-sufficient fibroblasts. Expression by AdDeltaRGDbetagal, an Ad5-based vector lacking the arginine-glycine-aspartate (RGD) alphaV integrin recognition site from its penton base, was achieved in CAR-sufficient, but not CAR-deficient, cells. Fiber-altered Ad5-based vectors, including (a) AdF(pK7)betagal (bearing seven lysines on the end of fiber) (b) AdF(RGD)betagal (bearing a high-affinity RGD sequence on the end of fiber), and (c) AdF9sK betagal (bearing a short fiber and Ad9 knob), demonstrated enhanced gene transfer in CAR-deficient fibroblasts, with no further enhancement in CAR-sufficient fibroblasts. Together, these observations demonstrate that CAR deficiency on Ad targets can be circumvented either by supplying CAR or by modifying the Ad fiber to bind to other cell-surface receptors.

Adenovirus targeted to heparan-containing receptors increases its gene delivery efficiency to multiple cell types.

Adenovirus (Ad) is used as a vector for gene delivery in therapies involving genetic disease, vascular disease, and cancer. The first step for efficient gene transfer is effective virus binding to the target cells. We have found that Ad-mediated gene delivery to multiple cell types is much less efficient compared to epithelial-derived cells. Low gene delivery to nonepithelial cell types was directly correlated to a deficiency of the cellular receptor which mediates Ad binding. To overcome this inefficiency we constructed a new virus, AdPK, that contains a heparin-binding domain that targets the virus to broadly expressed, heparan-containing cellular receptors. AdPK delivers genes to multiple cell types at markedly higher efficiencies than unmodified Ad. Viruses with enhanced attachment characteristics significantly improve gene transfer efficiency and may expand the tissues amenable to efficient Ad-mediated gene therapy.

Fiber knob modifications overcome low, heterogeneous expression of the coxsackievirus-adenovirus receptor that limits adenovirus gene transfer and oncolysis for human rhabdomyosarcoma cells.

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.

Adenoviral vectors for gene transfer.

Adenoviruses began to be developed into highly effective gene expression vectors in the early 1980s. Recently, the increased interest in utilizing this transfer system in vivo has posed new problems for heterologous gene-transfer, spurring a renewed effort in the field of vector development toward solving the structural, immunological and targeting problems posed by gene therapy applications.

Polylysine modification of adenoviral fiber protein enhances muscle cell transduction.

Adenoviral vectors (ADVs) are used widely for gene delivery to different tissues including muscle. One particularly promising use for ADVs is in the transfer of the dystrophin gene to the muscle of patients with Duchenne muscular dystrophy (DMD). However, studies in different animal models of DMD suggest that ADVs inefficiently transduce mature skeletal muscle. In this article we test whether AdZ.F(pK7), a genetically modified ADV that expresses a polylysine moiety on the end of the fiber protein, could enhance transduction of muscle cells and circumvent the maturation-dependent loss of muscle infectivity by ADVs. The efficiency of transduction was tested at different levels of muscle maturation. In vitro, AdZ.F(pK7) showed a higher level of transduction at all stages of differentiation including myoblasts, myotubes, and single muscle fibers. In vivo, mature skeletal muscle was transduced fourfold better by AdZ.F(pK7) than by the unmodifled vector (AdZ.F). Together, these observations demonstrate improved ADV transduction of skeletal muscle by modifying ADV tropism, and provide a proof-of-principle that modification of ADVs to target muscle-specific molecules could result in tissue-specific transfer of skeletal muscle tissue as well.

Transduction of bone marrow cells by the AdZ.F(pK7) modified adenovirus demonstrates preferential gene transfer in myeloma cells.

Adenoviral vectors can efficiently infect myeloma cell lines, but transduction of fresh myeloma cells performed at low multiplicity of infections (MOIs) showed only partial efficacy. The modified adenoviral vector AdZ.F(pK7), through binding of polylysines to heparan sulfate-containing receptors, could increase virus adsorption and gene transfer efficiency in myeloma cells, which express heparan sulfate-containing receptors. Thus, we investigated the ability of AdZ.F(pK7) vector to achieve efficient gene transfer in primary cultured fresh myeloma cells. Transduction of 16 primary cultured myeloma samples showed that gene transfer was much more efficient with AdZ.F(pK7) than with control AdZ.F. Both addition of soluble heparin and cell treatment with heparinase I dramatically inhibited gene transfer in myeloma cells by AdZ.F(pK7) but had no effect with AdZ.F, while addition of recombinant fiber protein inhibited AdZ.F but not AdZ.F(pK7), confirming that AdZ.F(pK7) gene transfer in myeloma cells is mediated by the targeting of heparan sulfates. AdZ.F(pK7) transduction of bone marrow cells showed that myeloma cells and hematopoietic progenitor AC133-, CD34-, and CD33-positive cells were efficiently transduced at an MOI of 100, but that only myeloma cells were significantly transduced at an MOI of 12. Thus, AdZ.F(pK7) vector seems to be well suited for immunological approaches of gene therapy or bone marrow-purging applications in multiple myeloma.

Transduction and apoptosis induction in the rat prostate, using adenovirus vectors.

Proapoptotic adenovirus vectors offer great promise for the treatment of cancer and nonmalignant conditions. Benign prostate hyperplasia (BPH) is a common nonmalignant enlargement of the prostate that involves epithelial, stromal, and smooth muscle components of the gland. We tested the hypothesis that an adenovirus vector expressing Fas ligand can be used to induce apoptosis in the prostate. We analyzed the efficiency of transduction and apoptosis induction in primary cultures of human prostate cells after adenovirus-mediated gene transfer. Efficient transduction was observed in primary prostate epithelial cells. Stromal and smooth muscle cells were more difficult to transduce, as no coxsackie-adenovirus receptor (CAR) expression was detectable on these cells. However, transduction was achieved in these cells when the multiplicity of infection was increased to 100 focal-forming units per cell, or when the vectors were delivered as calcium phosphate precipitates. Infection of all three primary prostate cell types with an adenovirus vector that expresses Fas ligand (AdFasL/G) resulted in rapid apoptosis. Direct injection of the rat prostate with an adenovirus vector carrying luciferase resulted in substantial luciferase expression. TUNEL analysis demonstrated that AdFasL/G administration induced low-level apoptosis in prostatic epithelial cells throughout the gland. As a first step toward enhancing the efficiency of prostate transduction in vivo, we tested an adenovirus vector that was engineered to have an expanded tropism. This vector, AdZ.F2K(pK7), was 10- to 500-fold more efficient than unmodified vectors in transducing prostate epithelial, smooth muscle, and stromal cells in culture. Moreover, AdZ.F2K(pK7) was more efficient than an unmodified vector at transducing the rat prostate in vivo, although the effect was dose dependent.

Adenovirus-mediated transduction of intestinal cells in vivo.

The intestinal tract has many features that make it an attractive target for therapeutic gene transfer. In this study, replication-defective adenoviral vectors were used to explore parameters that may be important in administering gene therapy vectors to the intestine. After surgically accessing the intestine, an E1-, E3-deleted adenoviral vector encoding beta-galactosidase (beta-Gal) was directly injected into various regions of the small and large intestine of rats and rabbits. Significant transduction of the tissue was observed and histochemical staining was used to identify enterocytes as the primary targets of gene transfer. Expression of beta-Gal did not differ substantially when the virus was administered to the duodenum, ileum, or colon. When the vector was directly administered to segments of the distal ileum containing a Peyer's patch, transgene expression was approximately 10-fold higher than in segments lacking a Peyer's patch. In the Peyer's patches, a high level of expression was localized to epithelial cells, potentially M cells, overlying the lymphoid follicle domes. Transduction of these cells could have application in DNA-mediated oral vaccination. Administration of an adenoviral vector encoding a secreted alkaline phosphatase to the lumen resulted in expression and secretion of this gene product into the circulation. This finding demonstrates the potential of enterocytes to serve as heterotopic sites for the synthesis of heterologous gene products that would be secreted into the lumen of the intestinal tract or into the bloodstream.

Circumvention of anti-adenovirus neutralizing immunity by administration of an adenoviral vector of an alternate serotype.

Effective gene transfer and expression following repetitive administration of adenoviral (Ad) vectors in experimental animals is limited by anti-Ad neutralizing antibodies. Knowing that anti-Ad humoral immunity is serotype-specific, we hypothesized that anti-Ad neutralizing immunity could be circumvented using Ad vectors of different serotypes (Ad2, Ad5) within the same subgroup (C) to transfer and express beta-glucuronidase (beta glu) in the lung. Sprague-Dawley rats received an intratracheal administration of either Ad2 beta glu or Ad5 beta glu, and, 14 days later, repeat administration of either the same vector or a vector of a different serotype. Analysis of serum and bronchoalveolar lavage fluid following initial vector administration demonstrated systemic and local serotype-specific neutralizing antibodies. For both the Ad2 and Ad5 vectors, beta glu expression 24 hr following the second administration of the same serotype was < 30% of that of naive animals. In contrast, beta glu expression 24 hr following second administration of a different serotype Ad vector was similar to expression at 24 hr of naive animals receiving a single administration (Ad5 beta glu followed by Ad2 beta glu, as well as Ad2 beta glu followed by Ad5 beta glu; p > 0.2 both comparisons). Although the alternative serotype bypassed anti-Ad neutralizing immunity, persistence of expression was reduced compared to that following administration to naive animals. Compatible with this observation, systemic administration of the same vectors to C57B1/6 mice demonstrated induction of cytotoxic T lymphocytes directed against the beta glu transgene, as well as products of the Ad genome. Interestingly, intratracheal administration of vectors with different serotypes and different transgenes to rats resulted in longer expression (but still not normalized) compared to that achieved with vectors of different serotypes but the same transgene. These observations demonstrate that alternate use of Ad vectors from different serotypes within the same subgroup can circumvent anti-Ad humoral immunity to permit effective gene transfer after repeat administration, although the chronicity of expression is limited, likely by cellular immune process directed against both the transgene and viral gene products expressed by the vector.

Modifications of the fiber in adenovirus vectors increase tropism for malignant glioma models.

Recombinant adenovirus (Ad) vectors provide a means of local, therapeutic gene delivery to a wide range of neoplasms. Ad-mediated gene therapy trials in malignant glioma models have been limited by the need for high viral titers and multiple dosages. In an attempt to improve Ad vector gene transfer, we studied human (U87, D54) and rodent (GL261, C6) malignant glioma cell lines transfected with various doses of unmodified Ad vectors (AdZ), Ad vectors that contain an alteration of the fiber-coat protein and that direct virus binding to heparan sulfate receptors (AdZ.F(pK7)), and Ad vectors with modifications of the fiber-coat protein that direct virus binding to alpha1, integrin cellular receptors (AdZ.F(RGD)). AdZ.F(pK7) increased the frequency of cells expressing the reporter gene, beta-galactosidase, and improved transduction by 2- to 20-fold compared with AdZ in U87, D54, and GL261 cells. In U87, D54, GL261, and C6 tumors, AdZ.F(pK7) increased gene transfer by 10- to 100-fold compared with AdZ. AdZ.F(RGD) increased gene expression in C6 xenografts compared with AdZ, but had reduced transduction compared with the C6 xenografts of AdZ in all other glioma tumors. These findings suggest that the increased tropisms resulting from alterations of the Ad vector fiber-coat protein as in AdZ.F(pK7) and AdZ.F(RGD) offer a feasible approach to improving in vitro and in vivo transduction efficiencies in certain malignant glioma cell lines.

Optimization of growth methods and recombinant protein production in BTI-Tn-5B1-4 insect cells using the baculovirus expression system.

A novel insect cell line from Trichoplusia ni, BTI-Tn 5B1-4 (Tn 5), was compared to Spodoptera frugiperda, Sf 9, cells for production of two recombinant secreted proteins: truncated Epstein-Barr viral attachment protein (EBV gp105) and truncated, soluble tissue factor (sTF). Under optimum conditions for both cell lines, Tn 5 cells produced 28-fold more secreted sTF than Sf 9 cells, respectively, on a per cell basis. The total production of gp105 was similar for the two cell lines. However, Tn5 cells secreted gp105 much more efficiently, resulting in 5-fold higher levels in the extracellular medium. Despite these increases, Tn 5 cells are attachment-dependent, and protein production is sensitive to the cell density (cells/cm2), unlike the Sf9 cell line which can be easily grown and scaled up in cell suspension cultures without significantly affecting its per cell production. Thus, protein production from Tn 5 cells above 0.1 L scales was optimized with respect to cell density using standard techniques for the growth of attachment-dependent cells. Roller bottles precoated with DEAE-based microcarriers and suspension cultures employing collagen-coated microcarriers were found to be effective ways of culturing Tn 5 cells. Predetermined optimal cell densities were used to produce EBV gp105 in microcarrier-coated roller bottles or in suspension cultures using collagen-coated microcarriers at concentrations close to those observed in tissue culture flasks.

A simple model to predict the effectiveness of molecules that block attachment of human rhinoviruses and other viruses.

The binding of viruses to cell surfaces is often mediated by cell surface receptors. The use of soluble receptors, such as intracellular adhesion molecule-1 (ICAM-1) for human rhinovirus (HRV), CD4 for human immunodeficiency virus (HIV), and CR2 for Epstein-Barr virus, for in vivo antiviral therapy is under serious investigation. A number of synthetic compounds that affect HRV attachment and uncoating (termed WIN compounds) are also being studied. However, the mechanism behind the dose-response effect of these agents in preventing infection has not been clearly demonstrated. In addition to simple blocking (by receptors) or inactivation of binding sites (by WIN compounds) on the virus surface, other mechanisms of inhibition have been proposed and demonstrated, including cooperative inactivation of neighboring sites, receptor-induced viral attachment protein (VAP) shedding, virus particle inactivation, and inhibition of multivalent virus binding. We present a simple mathematical model to predict the effect of these molecules on virus infection by incorporating only the blocking or site inactivation step of the blocking molecule and its resulting effect on attachment. The ability of the model to reproduce the response of a virus to a dose of blocking molecules is used to distinguish the role of blockers in inhibiting attachment from the other mechanisms of viral inactivation that have been proposed. The model includes both the reversible attachment of the virus to its cellular receptor and to soluble receptors or synthetic molecules (blockers).(ABSTRACT TRUNCATED AT 250 WORDS)

Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system.

Eight cell lines derived from the insects Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae, and Estigmene acrea were evaluated for recombinant beta-galactosidase and infectious virus production following infection with the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Production was assessed on a specific (per cell and per microgram of uninfected cellular protein) and on a volumetric (per milliliter) basis. Cell density was found to be an important factor in comparing the cell lines due to a density-dependent inhibition of specific protein and virus production that appeared to result from cell-cell contact. After infection of cells at low-density specific beta-galactosidase production per cell would drop between 3- and 6-fold in five of the eight cell lines when plated on tissue culture plates at near-confluent and confluent cell densities. The cell lines Sf 21 and Sf 9 were least sensitive to cell density. After accounting for cell density effects and differences in cell size, two cell lines, BTI Tn 5B1-4 and BTI TnM, were identified that were superior to the other cell lines, including Sf 21 and Sf 9, in beta-galactosidase production. Optimal volumetric and specific beta-galactosidase production from Tn 5B1-4 and TnM cells was 2-fold and 5-fold higher, respectively, in both cell lines than the optimal production from Sf 9 or Sf 21 cells. The Tn 5B1-4 cell line also had the highest viability of all the cell lines at 3 days postinfection and could be adapted to serum-free media.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative recombinant protein production of eight insect cell lines.

A recombinant Autographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived from Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae and Estigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, the T. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than the S. frugiperda-derived Sf9 or Sf2l cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.

Multiscale kinetic modeling of liposomal Doxorubicin delivery quantifies the role of tumor and drug-specific parameters in local delivery to tumors.

Nanoparticle encapsulation has been used as a means to manipulate the pharmacokinetic (PK) and safety profile of drugs in oncology. Using pegylated liposomal doxorubicin (PLD) vs. conventional doxorubicin as a model system, we developed and experimentally validated a multiscale computational model of liposomal drug delivery. We demonstrated that, for varying tumor transport properties, there is a regimen where liposomal and conventional doxorubicin deliver identical amounts of doxorubicin to tumor cell nuclei. In mice, typical tumor properties consistently favor improved delivery via liposomes relative to free drug. However, in humans, we predict that some tumors will have properties wherein liposomal delivery delivers the identical amount of drug to its target relative to dosing with free drug. The ability to identify tumor types and/or individual patient tumors with high degree of liposome deposition may be critical for optimizing the success of nanoparticle and liposomal anticancer therapeutics.CPT: Pharmacometrics & Systems Pharmacology (2012) 1, e15; doi:10.1038/psp.2012.16; advance online publication 21 November 2012.

Targeting adenovirus.

The use of targeted viral vectors to localize gene transfer to specific cell types holds many advantages over conventional, non-targeted vectors currently used in gene therapy. The resulting improvements in gene localization from targeted adenovirus vectors are likely to reduce immunogenicity and toxicity, increase safety, and enable the systemic administration of these vectors for multiple indications including cancer, cardiovascular disease, and inflammatory disease. Recent advances in the biological understanding of adenovirus structure and adenovirus receptor interactions have fueled the rapid development of targeted adenovirus vectors. Two basic requirements are necessary to create a targeted adenovirus vector: interaction of adenovirus with its native receptors must be removed and novel, tissue-specific ligands must be added to the virus. Two general approaches have been used to achieve these basic requirements. In the 'two-component' approach, a bispecific molecule is complexed with the adenovirus. The bispecific component simultaneously blocks native receptor binding and redirects virus binding to a tissue-specific receptor. In the 'one-component' approach the adenovirus is genetically modified to ablate native receptor interactions and a novel ligand is genetically incorporated into one of the adenovirus coat proteins. Two-component systems offer great flexibility in rapidly validating the feasibility of targeting via a particular receptor. One-component systems offer the best advantages in producing a manufacturable therapeutic and in more completely ablating all native adenovirus receptor interactions. The coming challenges for targeted adenovirus vectors will be the demonstration that the technology performs in vivo. Ultimately, or in parallel, 'receptor-targeting' technology can be combined with improved adenovirus backbones and with 'transcriptional targeting' approaches to create adenovirus which deliver genes selectively, safely, and with little immune response.