Oncogenic driver genes and the inflammatory microenvironment dictate liver tumor phenotype.

UNLABELLED: The majority of hepatocellular carcinoma develops in the background of chronic liver inflammation caused by viral hepatitis and alcoholic or nonalcoholic steatohepatitis. However, the impact of different types of chronic inflammatory microenvironments on the phenotypes of tumors generated by distinct oncogenes is largely unresolved. To address this issue, we generated murine liver tumors by constitutively active AKT-1 (AKT) and ß-catenin (CAT), followed by induction of chronic liver inflammation by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and carbon tetrachloride. Also, the impact of DDC-induced chronic liver inflammation was compared between two liver tumor models using a combination of AKT-CAT or AKT-NRAS(G12V) . Treatment with DDC and carbon tetrachloride significantly facilitated the adenoma-to-carcinoma conversion and accelerated the growth of AKT-CAT tumors. Furthermore, DDC treatment altered the morphology of AKT-CAT tumors and caused loss of lipid droplets. Transcriptome analysis of AKT-CAT tumors revealed that cellular growth and proliferation were mainly affected by chronic inflammation and caused up-regulation of Cxcl16, Galectin-3, and Nedd9, among others. Integration with transcriptome profiles from human hepatocellular carcinomas further demonstrated that AKT-CAT tumors generated in the context of chronic liver inflammation showed enrichment of poor prognosis gene sets or decrease of good prognosis gene sets. In contrast, DDC had a more subtle effect on AKT-NRAS(G12V) tumors and primarily enhanced already existent tumor characteristics as supported by transcriptome analysis. However, it also reduced lipid droplets in AKT-NRAS(G12V) tumors. CONCLUSION: Our study suggests that liver tumor phenotype is defined by a combination of driving oncogenes but also the nature of chronic liver inflammation. (Hepatology 2016;63:1888-1899).

Genetic engineering of CHO cells for viral resistance to minute virus of mice.

Contamination by the parvovirus minute virus of mice (MVM) remains a challenge in Chinese hamster ovary (CHO) biopharmaceutical production processes. Although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. We evaluated engineering a CHO parental cell line (CHOZN® GS-/- ) to create a new host cell line that is resistant to MVM infection by modifying the major receptors used by the virus to enter cells. Attachment to a cell surface receptor is a key first step in the infection cycle for many viruses. While the exact functional receptor for MVM binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated. In this work, we used the zinc finger nuclease gene editing technology to validate the role of sialic acid on the cell surface in the binding and internalization of the MVM virus. Our approach was to systematically mutate genes involved in cell surface sialylation and then challenge each cell line for their ability to resist viral entry and propagation. To test the importance of sialylation, the following genes were knocked out: the CMP-sialic acid transporter, solute carrier family 35A1 (Slc35a1), the core 1-ß-1,3-galactosyltransferase-1 specific chaperone (Cosmc), and mannosyl (α-1,3-)-glycoprotein ß-1,2-N-acetylglucosaminyltransferase (Mgat1) as well as members of the sialyltransferase family. Slc35a1 is responsible for transporting sialic acid into the Golgi. Knocking out function of this gene in a cell results in asialylated glycan structures, thus eliminating the ability of MVM to bind to and enter the cell. The complete absence of sialic acid on the Slc35a1 knockout cell line led to complete resistance to MVM infection. The Cosmc and Mgat1 knockouts also show significant inhibition of infection likely due to their effect on decreasing cell surface sialic acid. Previously in vitro glycan analysis has been used to elucidate the precise sialic acid structures required for MVM binding and internalization. In this work, we performed the sequential knockout of various sialyltransferases that add terminal sialic acid to glycans with different linkage specificities. Cell lines with modifications of the various genes included in this study resulted in varying effects on MVM infection expanding on the knowledge of MVM receptors. MVM resistant host cell lines were also tested for the production of model recombinant proteins. Our data demonstrate that resistance against the MVM virus can be incorporated into CHO production cell lines, adding another level of defense against the devastating financial consequences of MVM infection without compromising recombinant protein yield or quality. Biotechnol. Bioeng. 2017;114: 576-588. © 2016 Wiley Periodicals, Inc.

The CXCR4-tropic human immunodeficiency virus envelope promotes more-efficient gene delivery to resting CD4+ T cells than the vesicular stomatitis virus glycoprotein G envelope.

Current gene transfer protocols for resting CD4(+) T cells include an activation step to enhance transduction efficiency. This step is performed because it is thought that resting cells are resistant to transduction by lentiviral-based gene therapy vectors. However, activating resting cells prior to transduction alters their physiology, with foreseeable and unforeseeable negative consequences. Thus, it would be desirable to transduce resting CD4(+) T cells without activation. We recently demonstrated, contrary to the prevailing belief, that wild-type human immunodeficiency virus (HIV) integrates into resting CD4(+) T cells. Based on that finding, we investigated whether a commonly used, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped lentiviral gene therapy vector could also integrate into resting CD4(+) T cells. To investigate this, we inoculated resting CD4(+) T cells with lentiviral particles that were pseudotyped with VSV-G or CXCR4-tropic HIV Env and assayed binding, fusion, reverse transcription, and integration. We found that the VSV-G-pseudotyped lentiviral vector failed to fuse to resting CD4(+) T cells while HIV Env-pseudotyped lentiviral vectors fused, reverse transcribed, and integrated in resting cells. Our findings suggest that HIV Env could be used effectively for the delivery of therapeutic genes to resting CD4(+) T cells and suggest that fusion may be the critical step restricting transduction of resting CD4(+) T cells by lentiviral gene therapy vectors.

Adenovirus and Oxaliplatin cooperate as agnostic sensitizers for immunogenic cell death in colorectal carcinoma.

Treatments with cytotoxic agents or viruses may cause Immunogenic Cell Death (ICD) that immunize tumor-bearing hosts but do not cause complete regression of tumor. We postulate that combining two ICD inducers may cause durable regression in immunocompetent mice. ICD was optimized in vitro by maximizing calreticulin externalization in human colorectal carcinoma (CRC) cells by exposure to mixtures of Oxaliplatin (OX) and human adenovirus (AdV). Six mm diameter CT26 or 4T1 carcinomas in flanks of BALB/c mice were injected once intratumorally (IT) with OX, AdV or their mixture. Tumor growth, Tumor-Infiltrating Lymphocytes (TIL), nodal cytotoxicity, and rejection of a viable cell challenge were measured. Tumors injected IT once with an optimum mixture of 80 µM OX - AdV 25 Multiplicity of Infection (MOI) in PBS buffer were 17-29% the volume of control tumors. When buffer was changed from PBS to 5% dextrose in water (D5W), volumes of tumors injected IT with 80 µM OX-AdV 25 MOI were 10% while IT OX or AdV alone were 32% and 40% the volume of IT buffer-treated tumors. OX-AdV IT increased CD3+ TIL by 4-fold, decreased CD8+ PD-1+ TIL from 79% to 19% and induced cytotoxicity to CT26 cells in draining node lymphocytes while lymphocytes from CT26-bearing untreated mice were not cytotoxic. OX-AdV IT in D5W caused complete regression in 40% of mice. Long-term survivors rejected a contralateral challenge of CT26. The buffer for Oxaliplatin is critical. The two ICD inducer mixture is promising as an agnostic sensitizer for carcinomas like colorectal carcinoma.

Mode of transmission affects the sensitivity of human immunodeficiency virus type 1 to restriction by rhesus TRIM5alpha.

Rhesus TRIM5alpha (rhTRIM5alpha), but not human TRIM5alpha (huTRIM5alpha), potently inhibits human immunodeficiency virus (HIV) infection and is thus a potentially valuable therapeutic tool. Primary human CD4 T cells engineered to express rhTRIM5alpha were highly resistant to cell-free HIV type 1 (HIV-1) infection. However, when cocultured with unmodified T cells, rhTRIM5alpha-expressing cells became highly permissive to HIV-1 infection. Physical separation of rhTRIM5alpha-expressing cells and unmodified cells revealed that rhTRIM5alpha efficiently restricts cell-free but not cell-associated HIV transmission. Furthermore, we observed that HIV-infected human cells could infect rhesus CD4 T cells by cell-to-cell contact, but the infection was self-limiting. Subsequently, we noted that a spreading infection ensued when HIV-1-infected rhTRIM5alpha-expressing human cells were cultured with huTRIM5alpha- but not rhTRIM5alpha-expressing cells. Our results suggest that cell-associated HIV transmission in humans is blocked only when both donor and recipient cells express rhTRIM5alpha. These studies further define the role of rhTRIM5alpha in cell-free and cell-associated HIV transmission and delineate the utility of rhTRIM5alpha in anti-HIV therapy.

Generation and selection of targeted adenoviruses embodying optimized vector properties.

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

High efficiency transduction of dendritic cells by adenoviral vectors targeted to DC-SIGN.

Dendritic cells (DCs) are a central element in the development of antigen-specific immune responses. The lack of a specific and efficient technique for the in vivo delivery of antigens to DCs remains a major obstacle limiting a vaccine's ability to induce an effective immune response. The efficacy of adenoviral (Ad) vectors in this regard can be enhanced through alterations in vector tropism such that DC-targeted transduction is achieved. Here, the efficiency of DC transduction by Ad vectors retargeted to DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN) was studied and compared to that of Ad vectors retargeted through CD40. A comparable and significant enhancement of gene transfer to monocyte derived DCs (MDDCs) was accomplished by means of an Ad vector harboring the Fc-binding domain of Staphylococcus aureus protein A in combination with antibodies to DC-SIGN or to CD40 or with fused complexes of human Ig-Fc with their natural ligands, i.e., ICAM-3 or CD40L, respectively. Whereas CD40-targeted Ad transduction resulted in a more profound phenotypic DC maturation, DC-SIGN- and CD40-targeted Ad both induced similar levels of IL-12 secretion. These data demonstrate the usefulness of DC-SIGN as a DC-restricted targeting motif for Ad-mediated vaccination strategies.

Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

Heterologous HIV-based lentiviral/adenoviral vectors immunizations result in enhanced HIV-specific immunity.

Viral vectors are considered as one of the major means for the induction of strong immune responses against recombinant antigens by genetic immunization. Among these, lentiviral vectors are particularly attractive vehicles, as they can infect a wide variety of cells and can transduce replicating as well as non-replicating cells. We have engineered VRX1023, an HIV-1-based lentiviral vector (LV) vaccine candidate, to deliver HIV-1 Gag, Pol and Rev antigens under control of the native LTR promoter. While VRX1023 has been shown to elicit strong cell-mediated and humoral immunity as a stand-alone vaccine, we report here its combination in a heterologous prime-boost approach. Its combination with an adenovirus serotype 5 (Ad5)-based vector in the mouse model increased the frequency and polyfunctionality of HIV-specific CD4+ and CD8+ T cells. Homologous prime-boost regimens induced high levels of anti-vector neutralizing antibodies in Ad5-immunized mice, whereas the VSV-G-pseudotyped VRX1023 LV elicited low levels of anti-lentiviral vector neutralization. In addition, the heterologous prime-boost strategy resulted in a 5-fold reduction in Ad5-specific vector neutralization as compared to Ad5 homologous immunization. In conclusion, this study demonstrates that LV and Ad5 vector candidates can be combined in a heterologous immunization regimen, yielding dramatically improved immunogenicity while overcoming anti-vector immunity. These findings may have implications for the development of HIV vaccine regimens in populations with elevated Ad5 seroprevalence or when repeated vector administrations are required.

An HIV-based lentiviral vector as HIV vaccine candidate: Immunogenic characterization.

Recently developed viral-vectored HIV vaccine candidates, despite achieving high levels transgene expression and inducing high magnitude immune responses to HIV, have faced limitations related to anti-vector immunity. In contrast, lentiviral vectors (LV) have been shown to be less sensitive to anti-vector neutralizing activity, while displaying desirable characteristics, such as transduction of non-dividing cells, including antigen-presenting cells, and long-term transgene expression. We have developed VRX1023, an HIV-based LV expressing HIV Gag, Pol and Rev under the control of the native HIV LTR. In mice, this vector induced significant mucosal and systemic cellular and humoral responses against HIV after sub-cutaneous injection. Similarly to other viral vectors, this LV candidate can be effectively used in DNA prime, LV boost strategies, where it elicited as high as 21% HIV Gag-specific CD8 responses as measured by intracellular cytokine staining. Moreover, anti-vector immunity is not an obstacle to repeated LV administrations, as shown by improved anti-HIV responses compared to single LV immunization. In head to head comparisons with Ad5 vectors expressing the same vaccine payload, VRX1023 elicited higher and more persistent cellular and antibody responses to HIV than its adenoviral counterpart. In preparation for clinical use, manufacturing scale-up of a highly purified VRX1023 vector lot following cGMP was successfully achieved without altering the robust immunogenicity observed with the research-grade vector. VRX1023, in addition to competing favorably with existing vectors such as Ad5 for anti-HIV immune responses, demonstrates unique features likely to address some of the pitfalls of current vector-based HIV vaccine strategies.

Lentiviral vectors for HIV disease prevention and treatment.

HIV/AIDS has posed major challenges to the scientific community, both in terms of treatment and prevention. Current drug regimens, while efficacious, are expensive, inaccessible to major parts of the world, induce major side effects, and cannot prevent escape mutants due to lack of compliance and drug fatigue. In the vaccine field, recent setbacks related to the interruption and cancellation of major advanced clinical trials using adenoviral vectors have highlighted the need for new and innovative strategies. Unique features of HIV-based lentiviral vectors (LVs) and the current progress in the LV-based platform development make them an attractive alternative for the further LV-based HIV vaccine development. In preclinical studies, they have demonstrated a high degree of immunogenicity, while overcoming pitfalls faced by other viral vectors. These findings, combined with recent progress in large scale LV production/purification, make this strategy worth considering for further vaccine development.

A genetically engineered adenovirus vector targeted to CD40 mediates transduction of canine dendritic cells and promotes antigen-specific immune responses in vivo.

Targeting viral vectors encoding tumor-associated antigens to dendritic cells (DCs) in vivo is likely to enhance the effectiveness of immunotherapeutic cancer vaccines. We have previously shown that genetic modification of adenovirus (Ad) 5 to incorporate CD40 ligand (CD40L) rather than native fiber allows selective transduction and activation of DCs in vitro. Here, we examine the capacity of this targeted vector to induce immune responses to the tumor antigen CEA in a stringent in vivo canine model. CD40-targeted Ad5 transduced canine DCs via the CD40-CD40L pathway in vitro, and following vaccination of healthy dogs, CD40-targeted Ad5 induced strong anti-CEA cellular and humoral responses. These data validate the canine model for future translational studies and suggest targeting of Ad5 vectors to CD40 for in vivo delivery of tumor antigens to DCs is a feasible approach for successful cancer therapy.

Targeted and shielded adenovectors for cancer therapy.

Conditionally replicative adenovirus (CRAd) vectors are novel vectors with utility as virotherapy agents for alternative cancer therapies. These vectors have already established a broad safety record in humans and overcome some of the limitations of non-replicative adenovirus (Ad) vectors. In addition, one potential problem with these vectors, attainment of tumor or tissue selectivity has widely been addressed. However, two confounding problems limiting efficacy of these drug candidates remains. The paucity of the native Ad receptor on tumor tissues, and host humoral response due to pre-existing titers of neutralizing antibodies against the vector itself in humans have been highlighted in the clinical context. The well-characterized CRAd, AdDelta24-RGD, is infectivity enhanced, thus overcoming the lack of coxsackievirus and adenovirus receptor (CAR), and this agent is already rapidly progressing towards clinical translation. However, the perceived host humoral response potentially will limit gains seen from the infectivity enhancement and therefore a strategy to blunt immunity against the vector is required. On the basis of this caveat a novel strategy, termed shielding, has been developed in which the genetic modification of a virion capsid protein would provide uniformly shielded Ad vectors. The identification of the pIX capsid protein as an ideal locale for genetic incorporation of shielding ligands to conceal the Ad vector from pre-existing neutralizing antibodies is a major progression in the development of shielded CRAds. Preliminary data utilizing an Ad vector with HSV-TK fused to the pIX protein indicates that a shield against neutralizing antibodies can be achieved. The utility of various proteins as shielding molecules is currently being addressed. The creation of AdDelta24S-RGD, an infectivity enhanced and shielded Ad vector will provide the next step in the development of clinically and commercially feasible CRAds that can be dosed multiple times for maximum effectiveness in the fight against cancers in humans.

A single-component CD40-targeted adenovirus vector displays highly efficient transduction and activation of dendritic cells in a human skin substrate system.

Dendritic cell (DC) based tumor vaccination usually involves the administration of ex vivo generated autologous DC. Transduction of DC by viral vectors in vivo has been proposed as a more standardized and easily clinically applicable approach. Previously, we have reported that an Ad5 vector targeted to CD40 via genetic capsid incorporation of CD40L achieves selective transduction of DC in vitro. In the present study, we evaluate the ability of this vector to deliver transgenes in a stringent human substrate system. We report the capacity of this CD40-targeted vector to infect, with high efficiency, cutaneous DC resident in human skin explants, while simultaneously inducing their activation and maturation. This latest generation of single-component, fully targeted vectors should make feasible the clinical testing of in vivo DC-targeted vaccines.

Genetically targeted adenovirus vector directed to CD40-expressing cells.

The success of gene therapy depends on the specificity of transgene delivery by therapeutic vectors. The present study describes the use of an adenovirus (Ad) fiber replacement strategy for genetic targeting of the virus to human CD40, which is expressed by a variety of diseased tissues. The tropism of the virus was modified by the incorporation into its capsid of a protein chimera comprising structural domains of three different proteins: the Ad serotype 5 fiber, phage T4 fibritin, and the human CD40 ligand (CD40L). The tumor necrosis factor-like domain of CD40L retains its functional tertiary structure upon incorporation into this chimera and allows the virus to use CD40 as a surrogate receptor for cell entry. The ability of the modified Ad vector to infect CD40-positive dendritic cells and tumor cells with a high efficiency makes this virus a prototype of choice for the derivation of therapeutic vectors for the genetic immunization and targeted destruction of tumors.

Targeting of adenovirus via genetic modification of the viral capsid combined with a protein bridge.

A potential barrier to the development of genetically targeted adenovirus (Ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to Ad capsid proteins due to their nuclear localization during assembly of the virion. To overcome this problem, we developed a new targeting strategy, which combines genetic modifications of the Ad capsid with a protein bridge approach, resulting in a vector-ligand targeting complex. The components of the complex associate by virtue of genetic modifications to both the Ad capsid and the targeting ligand. One component of this mechanism of association, the Fc-binding domain of Staphylococcus aureus protein A, is genetically incorporated into the Ad fiber protein. The ligand is comprised of a targeting component fused with the Fc domain of immunoglobulin, which serves as a docking moiety to bind to these genetically modified fibers during the formation of the Ad-ligand complex. The modular design of the ligand solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Our study shows that targeting ligands incorporating the Fc domain and either an anti-CD40 single-chain antibody or CD40L form stable complexes with protein A-modified Ad vectors, resulting in significant augmentation of gene delivery to CD40-positive target cells. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.