Inhibition of 14q32 MicroRNAs miR-329, miR-487b, miR-494, and miR-495 increases neovascularization and blood flow recovery after ischemia.

RATIONALE: Effective neovascularization is crucial for recovery after cardiovascular events. OBJECTIVE: Because microRNAs regulate expression of up to several hundred target genes, we set out to identify microRNAs that target genes in all pathways of the multifactorial neovascularization process. Using www.targetscan.org, we performed a reverse target prediction analysis on a set of 197 genes involved in neovascularization. We found enrichment of binding sites for 27 microRNAs in a single microRNA gene cluster. Microarray analyses showed upregulation of 14q32 microRNAs during neovascularization in mice after single femoral artery ligation. METHODS AND RESULTS: Gene silencing oligonucleotides (GSOs) were used to inhibit 4 14q32 microRNAs, miR-329, miR-487b, miR-494, and miR-495, 1 day before double femoral artery ligation. Blood flow recovery was followed by laser Doppler perfusion imaging. All 4 GSOs clearly improved blood flow recovery after ischemia. Mice treated with GSO-495 or GSO-329 showed increased perfusion already after 3 days (30% perfusion versus 15% in control), and those treated with GSO-329 showed a full recovery of perfusion after 7 days (versus 60% in control). Increased collateral artery diameters (arteriogenesis) were observed in adductor muscles of GSO-treated mice, as well as increased capillary densities (angiogenesis) in the ischemic soleus muscle. In vitro, treatment with GSOs led to increased sprout formation and increased arterial endothelial cell proliferation, as well as to increased arterial myofibroblast proliferation. CONCLUSIONS: The 14q32 microRNA gene cluster is highly involved in neovascularization. Inhibition of 14q32 microRNAs miR-329, miR-487b, miR-494, and miR-495 provides a promising tool for future therapeutic neovascularization.

Design, synthesis and biological evaluation of novel antagonist compounds of Toll-like receptors 7, 8 and 9.

Oligonucleotides containing an immune-stimulatory motif and an immune-regulatory motif act as antagonists of Toll-like receptor (TLR)7 and TLR9. In the present study, we designed and synthesized oligonucleotide-based antagonists of TLR7, 8 and 9 containing a 7-deaza-dG or arabino-G modification in the immune-stimulatory motif and 2'-O-methylribonucleotides as the immune-regulatory motif. We evaluated the biological properties of these novel synthetic oligoribonucleotides as antagonists of TLRs 7, 8 and 9 in murine and human cell-based assays and in vivo in mice and non-human primates. In HEK293, mouse and human cell-based assays, the antagonist compounds inhibited signaling pathways and production of a broad range of cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-12, IL-6, interferon (IFN)-α, IL-1ß and interferon gamma-induced protein (IP)-10, mediated by TLR7, 8 and 9. In vivo in mice, the antagonist compounds inhibited TLR7- and TLR9-mediated cytokine induction in a dose- and time-dependent fashion. Peripheral blood mononuclear cells (PBMCs) obtained from antagonist compound-treated monkeys secreted lower levels of TLR7-, 8- and 9-mediated cytokines than did PBMCs taken before antagonist administration. The antagonist compounds described herein provide novel agents for the potential treatment of autoimmune and inflammatory diseases.

Sequential induction of type I and II interferons mediates a long-lasting gene induction in the liver in response to a novel toll-like receptor 9 agonist.

BACKGROUND & AIMS: The toll-like receptor 9 (TLR9) agonist IMO-2125 is currently evaluated in clinical trials for chronic hepatitis C therapy. The aim of this study was to investigate the in vivo mode of action of a closely related compound, referred to as immunomodulatory oligonucleotide (IMO). METHODS: We analyzed the Jak-STAT pathway activation and induction of interferon-stimulated genes in the liver of wild type, interferon-α/ß receptor-deficient and interferon-γ-deficient mice, after administration of IMO. RESULTS: IMO induced a prolonged activation of the Jak-STAT pathway and upregulation of interferon-stimulated genes in the mouse liver. Contrary to the response observed after interferon-α injection, the signalling induced by IMO was not abrogated following repeated administration. At early time points after IMO injection, STAT1 phosphorylation and interferon-stimulated gene induction required a functional interferon-α/ß receptor, whereas at the later time points, the activation was type I interferon-independent. Microarray analysis revealed that IMO induced a broad transcriptional response in the mouse liver. This included upregulation of cytokine and chemokine genes responsible for recruitment of IFN-γ producers, such as T cells and natural killer cells. Interferon-γ-deficient mice showed a transient response to IMO, demonstrating the central role of interferon-γ in sustained activation of Jak-STAT pathway by IMO. CONCLUSIONS: The bimodal kinetics of response to IMO in the mouse liver are driven by the sequential endogenous production of type I and II interferons. The lack of refractoriness to IMO, combined with the long-lasting induction of interferon-stimulated genes, reveals a favourable pharmacodynamics profile of this novel TLR9 agonist for the treatment of chronic viral hepatitis.

Phase 1 studies of the safety and immunogenicity of electroporated HER2/CEA DNA vaccine followed by adenoviral boost immunization in patients with solid tumors.

BACKGROUND: DNA electroporation has been demonstrated in preclinical models to be a promising strategy to improve cancer immunity, especially when combined with other genetic vaccines in heterologous prime-boost protocols. We report the results of 2 multicenter phase 1 trials involving adult cancer patients (n=33) with stage II-IV disease. METHODS: Patients were vaccinated with V930 alone, a DNA vaccine containing equal amounts of plasmids expressing the extracellular and trans-membrane domains of human HER2, and a plasmid expressing CEA fused to the B subunit of Escherichia coli heat labile toxin (Study 1), or a heterologous prime-boost vaccination approach with V930 followed by V932, a dicistronic adenovirus subtype-6 viral vector vaccine coding for the same antigens (Study 2). RESULTS: The use of the V930 vaccination with electroporation alone or in combination with V932 was well-tolerated without any serious adverse events. In both studies, the most common vaccine-related side effects were injection site reactions and arthralgias. No measurable cell-mediated immune response (CMI) to CEA or HER2 was detected in patients by ELISPOT; however, a significant increase of both cell-mediated immunity and antibody titer against the bacterial heat labile toxin were observed upon vaccination. CONCLUSION: V930 vaccination alone or in combination with V932 was well tolerated without any vaccine-related serious adverse effects, and was able to induce measurable immune responses against bacterial antigen. However, the prime-boost strategy did not appear to augment any detectable CMI responses against either CEA or HER2. TRIAL REGISTRATION: Study 1 - ClinicalTrials.gov, NCT00250419; Study 2 - ClinicalTrials.gov, NCT00647114.

Blocking toll-like receptors 7 and 9 reduces postinterventional remodeling via reduced macrophage activation, foam cell formation, and migration.

OBJECTIVE: The role of toll-like receptors (TLRs) in vascular remodeling is well established. However, the involvement of the endosomal TLRs is unknown. Here, we study the effect of combined blocking of TLR7 and TLR9 on postinterventional remodeling and accelerated atherosclerosis. METHODS AND RESULTS: In hypercholesterolemic apolipoprotein E*3-Leiden mice, femoral artery cuff placement led to strong increase of TLR7 and TLR9 presence demonstrated by immunohistochemistry. Blocking TLR7/9 with a dual antagonist in vivo reduced neointimal thickening and foam cell accumulation 14 days after surgery by 65.6% (P=0.0079). Intima/media ratio was reduced by 64.5% and luminal stenosis by 62.8%. The TLR7/9 antagonist reduced the arterial wall inflammation, with reduced macrophage infiltration, decreased cytoplasmic high-mobility group box 1 expression, and altered serum interleukin-10 levels. Stimulation of cultured macrophages with TLR7 and TLR9 ligands enhanced tumor necrosis factor-α expression, which is decreased by TLR7/9 antagonist coadministration. Additionally, the antagonist abolished the TLR7/9-enhanced low-density lipoprotein uptake. The antagonist also reduced oxidized low-density lipoprotein-induced foam cell formation, most likely not via decreased influx but via increased efflux, because CD36 expression was unchanged whereas interleukin-10 levels were higher (36.1 ± 22.3 pg/mL versus 128.9 ± 6.6 pg/mL; P=0.008). CONCLUSIONS: Blocking TLR7 and TLR9 reduced postinterventional vascular remodeling and foam cell accumulation indicating TLR7 and TLR9 as novel therapeutic targets.

Design of synthetic oligoribonucleotide-based agonists of Toll-like receptor 3 and their immune response profiles in vitro and in vivo.

Double-stranded RNA of viral origin and enzymatically synthesized poly I:C act as agonists of TLR3 and induce immune responses. We have designed and synthesized double-stranded synthetic oligoribonucleotides (dsORNs) which act as agonists of TLR3. Each strand of dsORN contains two distinct segments, namely an alignment segment composed of a heteronucleotide sequence and an oligo inosine (I) or an oligo cytidine (C) segment. We report here the results of studies of dsORNs containing varying lengths and compositions of alignment and oligo I/oligo C segments. dsORNs of 50-mer length with a 15-mer alignment segment and a 35-mer oligo I/oligo C segment form stable duplexes under physiological conditions and induce TLR3-mediated immune responses. dsORNs activated the IRF3 signaling pathway in J774 cells, induced production of cytokines, including IFN-ß, IFN-α, IP-10, IL-12 and IL-6, in murine and human cell-based assays and also induced multiple cytokines following systemic administration in mice and non-human primates.

A vaccine targeting telomerase enhances survival of dogs affected by B-cell lymphoma.

Canine cancers occur with an incidence similar to that of humans and share many features with human malignancies including histological appearance, tumor genetics, biological behavior, and response to conventional therapies. As observed in humans, the telomerase reverse transcriptase (TERT) activity is largely confined to tumor tissues and absent in the majority of normal dog tissues. Therefore, dog TERT (dTERT) can constitute a valid target for translational cancer immunotherapy. We have evaluated the ability of adenovirus serotype 6 (Ad6) and DNA electroporation (DNA-EP) to induce immune responses against dTERT in dogs affected by malignant lymphoma (ML). The vaccine was combined with standard chemotherapy regimen [cyclophosphamide, vincristine, prednisone (COP)]. dTERT-specific immune response was induced in 13 out of 14 treated animals (93%) and remained detectable and long-lasting with the absence of autoimmunity or other side effects. Most interestingly, the survival time of vaccine/Chemo-treated dogs was significantly increased over historic controls of Chemo-treated animals (>97.8 versus 37 weeks, respectively, P = 0.001). Our results show that Ad6/DNA-EP-based cancer vaccine against dTERT overcomes host immune tolerance, should be combined with chemotherapy, induces long-lasting immune responses, and significantly prolongs the survival of ML canine patients. These data support further evaluation of this approach in human clinical trials.

Growth delay of human bladder cancer cells by Prostate Stem Cell Antigen downregulation is associated with activation of immune signaling pathways.

BACKGROUND: Prostate stem cell antigen (PSCA) is a glycosylphosphatidylinositol (GPI) anchored protein expressed not only in prostate but also in pancreas and bladder cancer as shown by immunohistochemistry and mRNA analysis. It has been targeted by monoclonal antibodies in preclinical animal models and more recently in a clinical trial in prostate cancer patients. The biological role played in tumor growth is presently unknown. In this report we have characterized the contribution of PSCA expression to tumor growth. METHODS: A bladder cell line was engineered to express a doxycycline (dox) regulated shRNA against PSCA. To shed light on the PSCA biological role in tumor growth, microarray analysis was carried out as a function of PSCA expression. Expression of gene set of interest was further analyzed by qPCR RESULTS: Down regulation of the PSCA expression was associated with reduced cell proliferation in vitro and in vivo. Mice bearing subcutaneous tumors showed a reduced tumor growth upon treatment with dox, which effectively induced shRNA against PSCA as revealed by GFP expression. Pathway analysis of deregulated genes suggests a statistical significant association between PSCA downregulation and activation of genes downstream of the IFNalpha/beta receptor. CONCLUSIONS: These experiments established for the first time a correlation between the level of PSCA expression and tumor growth and suggest a role of PSCA in counteracting the natural immune response.

Treatment of mammary carcinomas in HER-2 transgenic mice through combination of genetic vaccine and an agonist of Toll-like receptor 9.

PURPOSE: Oligodeoxynucleotides containing unmethylated CpG dinucleotides induce innate and adaptive immunity through Toll-like receptor 9 (TLR9). In the present study, we have examined the ability of a novel agonist of TLR9, called immunomodulatory oligonucleotide (IMO), to enhance effects of a HER-2/neu plasmid DNA electroporation/adenovirus (DNA-EP/Ad) vaccine. EXPERIMENTAL DESIGN: BALB/NeuT mice were treated with DNA-EP vaccine alone, IMO alone, or the combination of two agents starting at week 13, when all mice showed mammary neoplasia. Tumor growth and survival were documented. Antibody and CD8+ T-cell responses were determined. Peptide microarray analysis of sera was carried out to identify immunoreactive epitopes. Additionally, microCT and microPET imaging was carried out in an advanced-stage tumor model starting treatment at week 17 in BALB/NeuT mice. RESULTS: The combination of DNA-EP and IMO resulted in significant tumor regression or delay to tumor progression. 2-Deoxy-2-[18F]fluoro-D-glucose microPET and microCT imaging of mice showed reduced tumor size in the DNA-EP/IMO combination treatment group. Mice treated with the combination produced greater antibody titers with IgG2a isotype switch and antibody-dependent cellular cytotoxicity activity than did mice treated with DNA-EP vaccine. An immunogenic B-cell linear epitope, r70, within the HER-2 dimerization domain was identified through microarray analysis. Heterologous DNA-EP/Ad vaccination combined with IMO increased mice survival. CONCLUSION: The combination of HER-2/neu genetic vaccine and novel agonist of TLR9 had potent antitumor activity associated with antibody isotype switch and antibody-dependent cellular cytotoxicity activities. These results support possible clinical trials of the combination of DNA-EP/Ad-based cancer vaccines and IMO.

Coadministration of telomerase genetic vaccine and a novel TLR9 agonist in nonhuman primates.

The human telomerase reverse transcriptase (hTERT) is an attractive target for human cancer vaccination because its expression is reactivated in most human tumors. We have evaluated the ability of DNA electroporation (DNA-EP) and adenovirus serotype 6 (Ad6) to induce immune responses against hTERT in nonhuman primates (NHPs) (Macaca mulatta). Vaccination was effective in all treated animals, and the adaptive immune response remained detectable and long lasting without side effects. To further enhance the efficacy of the hTERT vaccine, we evaluated the combination of hTERT vaccine and a novel TLR9 agonist, referred to as immunomodulatory oligonucleotide (IMO). Monkeys were dosed weekly with IMO concurrently with the vaccine regimen and showed increases in cytokine secretion and activation of natural killer (NK) cells compared with the group that received vaccine alone. Using a peptide array, a specific profile of B-cell reactive epitopes was identified when hTERT vaccine was combined with IMO. The combination of IMO with hTERT genetic vaccine did not impact vaccine-induced TERT-specific cell-mediated immunity. Our results show that appropriate combination of a DNA-EP/Ad6-based cancer vaccine against hTERT with IMO induces multiple effects on innate and adaptive immune responses in NHPs.

Comparative expression pathway analysis of human and canine mammary tumors.

BACKGROUND: Spontaneous tumors in dog have been demonstrated to share many features with their human counterparts, including relevant molecular targets, histological appearance, genetics, biological behavior and response to conventional treatments. Mammary tumors in dog therefore provide an attractive alternative to more classical mouse models, such as transgenics or xenografts, where the tumour is artificially induced. To assess the extent to which dog tumors represent clinically significant human phenotypes, we performed the first genome-wide comparative analysis of transcriptional changes occurring in mammary tumors of the two species, with particular focus on the molecular pathways involved. RESULTS: We analyzed human and dog gene expression data derived from both tumor and normal mammary samples. By analyzing the expression levels of about ten thousand dog/human orthologous genes we observed a significant overlap of genes deregulated in the mammary tumor samples, as compared to their normal counterparts. Pathway analysis of gene expression data revealed a great degree of similarity in the perturbation of many cancer-related pathways, including the 'PI3K/AKT', 'KRAS', 'PTEN', 'WNT-beta catenin' and 'MAPK cascade'. Moreover, we show that the transcriptional relationships between different gene signatures observed in human breast cancer are largely maintained in the canine model, suggesting a close interspecies similarity in the network of cancer signalling circuitries. CONCLUSION: Our data confirm and further strengthen the value of the canine mammary cancer model and open up new perspectives for the evaluation of novel cancer therapeutics and the development of prognostic and diagnostic biomarkers to be used in clinical studies.

Impaired interferon type I signalling in the liver modulates the hepatic acute phase response in hepatitis C virus transgenic mice.

BACKGROUND/AIMS: The immunomodulatory active hepatitis C virus (HCV) has been shown to interfere with antiviral interferon (IFN) type I functions. The aim of the study was to determine whether further basic innate immunologic functions are influenced by HCV. METHODS: The acute phase response (APR) was induced in HCV transgenic (tg) mice and C57BL/6J control mice using lipopolysaccharide. Activation of transcription factors, mRNA expression and production of cytokines and acute phase proteins (APP) were determined. IFN type I and tumor necrosis factor (TNF) alpha signalling were investigated after polyI:C or TNF-alpha treatment. RESULTS: HCV tg mice showed an attenuated APR: hepatic activation of nuclear factor kappa B (NFkappaB) and interferon-stimulated gene factor 3 (ISGF3), hepatic expression of interleukin (IL) 6, IL-10, and IFN-gamma mRNA, serum concentrations of IL-6 and IFN-gamma and production of type II acute phase proteins were reduced compared to wild-type mice. While no differences in NFkappaB activation could be detected after TNF-alpha injection, HCV tg mice showed reduced activation of ISGF3 and reduced transactivation of IFN target genes after polyI:C treatment. CONCLUSIONS: Besides antiviral defence mechanisms, interruption of IFN type I signalling by HCV modulates the APR which is aimed at a variety of pathogens.

Characterization of the NOD/scid-[Tg]DR1 mouse expressing HLA-DRB1*01 transgene: a model of SCID-hu mouse for vaccine development.

OBJECTIVE: We previously showed enhanced engraftment of human T cells in the transgenic NonObese Diabetic/severe combined immunodeficient (NOD/scid)-DR1 mice, compared to NOD/scid mice. We now characterize their immunobiology, innate immunity, and intrahepatic neonatal engraftment of cord blood mononuclear cells (CBMNC), and test immune responses of these chimeric mice to an experimental cancer vaccine. METHODS: Fluorescence in situ hybridization analysis, blood biochemistry, hematology, and fluorescein-activated cell sorting analyses of cellular subsets were performed on NOD/scid-DR1 mice, in comparison to parental NOD/scid mice. Innate immunity and lifespan were examined. Histology of engrafted tissues and short-term intrahepatic engraftment of CBMNC were performed. Intracellular interferon-gamma (IFN-gamma) production was assessed in mice immunized with cancer vaccine. RESULTS: The DR1 transgene was located on chromosome 5 and no significant changes were observed in blood chemistry, peripheral blood counts, lymphoid subsets, natural killer cell and lipopolysaccharide response, and antigen presentation in the NOD/scid-DR1 mice, compared to NOD/scid mice. Interestingly, NOD/scid-DR1 mice had a significantly longer lifespan (approximately 14 months) than NOD/scid mice (approximately 8.5 months). Engraftment with human cord blood cells resulted in slight changes in the architecture/structure of spleens. No correlation was found between DR1 genotype of the donor CBMNC and extent of engraftment of human T cells. Enhanced engraftment of human cells was observed with intrahepatic injections of CBMNC in neonatal NOD/scid DR1 mice. Intracellular IFN-gamma was detected in human cells, when chimeric mice were immunized with a cancer vaccine. CONCLUSION: NOD/scid-DR1 mice were similar in most of the physiological parameters as the NOD/scid mice, with the exception of longer lifespan. Intrahepatic engraftment of neonatal mice is the preferred protocol of xenotransplantation in this model and the engrafted human cells can respond to a cancer vaccine.

DNA and adenoviral vectors encoding carcinoembryonic antigen fused to immunoenhancing sequences augment antigen-specific immune response and confer tumor protection.

A panel of vectors was constructed to encode carcinoembryonic antigen (CEA) fused at its C-terminal end to various polypeptides, so as to compare their immunogenicity by plasmid DNA immunization and adenovirus injection in wild-type and CEA transgenic (CEA.tg) mice. Fusions between CEA and the minimized domain of tetanus toxin fragment C (CEA-DOM) or the Fc portion of IgG1 (CEA-FcIgG) were identified as highly immunogenic and elicited significant CEA-specific antibody and CD8+ T cell responses. CEA.tg mice were protected from tumor growth on challenge with MC38-CEA tumor cells only when immunized with repeated injections of plasmid pV1J/CEA-DOM followed by Ad/CEA-DOM. Depletion of T-regulatory cells resulted in an increased immune response and antitumor effect with DNA plus adenovirus immunization. In addition, this protective effect was abrogated if the NK, CD4+, or CD8+ cell population from immunized mice was depleted before tumor challenge. Passive transfer studies demonstrated that CD4+ and CD8+ T cells and antibodies contributed to the antitumor effect, thus suggesting that a genetic vaccine based on the use of plasmid DNA and adenoviral vectors encoding CEA fused to immunoenhancing sequences augments CEA-specific immune responses and effectively protects from tumor development.

Individual mouse analysis of the cellular immune response to tumor antigens in peripheral blood by intracellular staining for cytokines.

Among the experimental animal models, mice remain the most widely used for the evaluation of immunotherapeutic strategies. Vaccines against parasites and viral antigens are commonly administered to the appropriate mouse strain which also allows testing of the therapeutic effect. Similarly, in mice transgenic for human tumor associated antigens (TAA), cancer vaccines must lead to breakage of immune tolerance to elicit a significant effect on the tumor. However, one of the major drawbacks in the monitoring of cellular immune responses induced by vaccination is that functional immunological assays require suppression of the animals to collect the spleen or lymph nodes for analysis. Here, we report the application of a rapid intracellular staining (ICS) method to quantify antigen-specific T cells responses in small volumes of murine blood. Genetic vaccination with plasmid DNA followed by electroporation (DNA-EP) and the use of adenoviral vectors (Ad) encoding CEA as a model target antigen were applied to different strains of mice. Optimal blood volume, number of lymphocytes, sensitivity and reproducibility of intracellular staining for IFN-gamma were determined both in non-tolerant/wild type mice as well as in tolerant CEA transgenic mice upon restimulation of PBMCs with CEA peptides. Groups of vaccinated mice were then sacrificed and PBMCs and splenocytes from individual animals were compared for intracytoplasmic detection of IFN-gamma and TNF-alpha. A significant correlation was observed between splenic and blood immune responses. Finally, the cellular immune response was followed over time in groups of vaccinated mice. The kinetics of IFN-gamma producing effectors were measured after priming and successive boosting with adenoviral vectors. We show that intracellular staining for mouse PBMCs is a rapid and simple method to measure antigen-specific immune responses. It does not require animal euthanasia and mirrors the response observed in lymphoid organs such as the spleen.

Superior Immunologic and Therapeutic Efficacy of a Xenogeneic Genetic Cancer Vaccine Targeting Carcinoembryonic Human Antigen.

We have generated a xenogeneic vaccine against human carcinoembryonic antigen (hCEACAM-5 or commonly hCEA) using as immunogen rhesus CEA (rhCEA). RhCEA cDNA was codon-usage optimized (rhCEAopt) and delivered by sequential DNA electro-gene-transfer (DNA-EGT) and adenoviral (Ad) vector. RhCEAopt was capable to break tolerance to CEA in hCEA transgenic mice and immune responses were detected against epitopes distributed over the entire length of the protein. Xenovaccination with rhCEA resulted in the activation of CD4+ T-cell responses in addition to self-reactive CD8+ T-cells, the development of high-titer antibodies against hCEA, and significant antitumor effects upon challenge with hCEA+ tumor cells. The superior activity of rhCEAopt compared with hCEAopt was confirmed in hCEA/HHD double-transgenic mice, where potent CD8+ T-cell responses against specific human HLA A*0201 hCEA epitopes were detected. Our data show that xenogeneic gene-based vaccination with rhCEA is a viable approach to break tolerance against CEA, thus suggesting further development in the clinical setting.

Hyaluronidase increases electrogene transfer efficiency in skeletal muscle.

Electrogene transfer (EGT) of plasmid DNA into skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. We report here that preinjecting hyaluronidase (HYAse) significantly increases the gene transfer efficiency of muscle EGT. Three constructs encoding mouse erythropoietin (pCMV/mEPO), secreted alkaline phosphatase (pCMV/SeAP), and luciferase (pGGluc) were electroinjected intramuscularly in BALB/c mice and rabbits with and without HYAse pretreatment. Preinjection 1 or 4 hr before EGT increased EPO gene expression by about 5-fold in mice and maintained higher gene expression than plasmid EGT alone. A similar increment in gene expression was observed on pretreatment with HYAse and electroinjection of pCMV/mEPO into rabbit tibialis muscle. The increment of gene expression in rabbits reached 17-fold on injection of plasmid pCMV/SeAP and 24-fold with plasmid pGGluc. Injection of a plasmid encoding beta-galactosidase (pCMV/beta gal/NLS) and subsequent staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside indicated that HYAse increased the tissue area involved in gene expression. No irreversible tissue damage was observed on histological analysis of treated muscles. HYAse is used in a variety of clinical applications, and thus the combination of HYAse pretreatment and muscle EGT may constitute an efficient gene transfer method to achieve therapeutic levels of gene expression.

Expression of a new woodchuck IFN-alpha gene by a helper-dependent adenoviral vector in woodchuck hepatitis virus-infected primary hepatocytes.

Recombinant interferon-alpha (rIFN-alpha) is currently used in the treatment of viral hepatitis either alone or in combination with small molecules. However, this treatment is not very efficacious, and more effective protocols are needed. To this end, we have explored the woodchuck hepatitis system, validated as an infection model for vaccination and antiviral studies against human hepatitis B virus (HBV) infection. The lack of a woodchuck IFN-alpha (WoIFN-alpha) homolog has prevented study of viral inhibition, which may be instrumental in understanding the IFN-alpha-induced antiviral pathways responsible for HBV clearance in humans. We have, therefore, cloned two WoIFN-alpha homologs from the woodchuck genome, which showed high similarity to the human IFN-alpha (HuIFN-alpha) gene at both nucleotide and amino acid levels. WoIFN-alpha showed a species-specific activity without any efficacy on human or mouse cells. In agreement with this antiviral activity, induction of Mx protein was observed in woodchuck cells only on WoIFN-alpha treatment. The antiviral efficacy of a WoIFN-alpha gene transfer was explored using a helper-dependent adenoviral (Ad) vector (HD-WoIFN) as a delivery vehicle. This treatment resulted in the reduction of woodchuck hepatitis viral proteins in primary hepatocytes from chronically woodchuck hepatitis virus (WHV)-infected woodchucks.

Immunogenicity and therapeutic efficacy of a dual-component genetic cancer vaccine cotargeting carcinoembryonic antigen and HER2/neu in preclinical models.

Several cancer vaccine efforts have been directed to simultaneously cotarget multiple tumor antigens, with the intent to achieve broader immune responses and more effective control of cancer growth. Genetic cancer vaccines based on in vivo muscle electro-gene-transfer of plasmid DNA (DNA-EGT) and adenoviral vectors represent promising modalities to elicit powerful immune responses against tumor-associated antigens (TAAs) such as carcinoembryonic antigen (CEA) and human epidermal growth factor receptor-2 (HER2)/neu. Combinations of these modalities of immunization (heterologous prime-boost) can induce superior immune reactions as compared with single-modality vaccines. We have generated a dual component-dual target genetic cancer vaccine consisting of a DNA moiety containing equal amounts of two plasmids, one encoding the extracellular and transmembrane domains of HER2 (ECD.TM) and the other encoding CEA fused to the B subunit of Escherichia coli heat-labile toxin (LTB), and of an adenoviral subtype 6 dicistronic vector carrying the same two tumor antigens gene constructs. The CEA/HER2 vaccine was tested in two different CEA/HER2 double-transgenic mouse models and in NOD/scid-DR1 mice engrafted with the human immune system. The immune response was measured by enzyme-linked immunospot assay, flow cytometry, and ELISA. The CEA/HER2 vaccine was able to break immune tolerance against both antigens. Induction of a T cell and antibody immune response was detected in immune-tolerant mice. Most importantly, the vaccine was able to slow the growth of HER2/neu⁺ and CEA⁺ tumors. A significant T cell response was measured in NOD/scid-DR1 mice engrafted with human cord blood cells. In conclusion, the CEA/HER2 genetic vaccine was immunogenic and able to confer significant therapeutic effects. These data warrant the evaluation of this vaccination strategy in human clinical trials.

A novel minigene scaffold for therapeutic cancer vaccines.

Genetic vaccines are emerging as a powerful modality to induce T-cell responses to target tumor associated antigens (TAA). Viral or plasmid DNA or RNA vectors harbor an expression cassette encoding the antigen of choice delivered in vivo by vaccination. In this context, immunizations with minigenes containing selected, highly antigenic, T-cell epitopes of TAAs may have several advantages relative to full-length proteins. The objective of this study was to identify an optimal scaffold for minigene construction. We generated a number of minigenes containing epitopes from the carcinoembryonic antigen (CEA) model TAA and utilized muscle DNA electro-gene-transfer (DNA-EGT) to vaccinate HLA-A*0201 (HHD) and CEA/HHD double transgenic mice. The components utilized to construct the minigenes included CD8+ T cell epitopes and (or) anchor modified analogs that were selected on the basis of their predicted binding to HLA-*A0201, their uniqueness in the human proteome, and the likelihood of cancer cell natural processing and presentation via MHC-I. Other candidate components comparatively tested included: helper CD4+ T-cell epitopes, flanking regions for optimal epitope processing (including both proteasome-dependent and furin-dependent polypeptide processing mechanisms), and immunoenhancing moieties. Through a series of comparative studies and iterations we have identified an optimal minigene scaffold comprising the following elements: human tissue plasminogen activator (TPA) signal peptide, T-cell epitopes connected by furin sensitive linkers, and the E. Coli enterotoxin B subunit. The selected epitope modified minigenes (EMM) delivered by DNA-EGT were able to break immune tolerance in CEA/HHD mice and induce a strong immune response against all epitopes tested, independently of their relative positions within the scaffold. Furthermore, the optimized EMMs delivered via DNA-EGT were more immunogenic and exerted more powerful antitumor effects in a B16-CEA/HHD metastatic melanoma model than a DNA vector encoding the full-length protein or a mixture of the same peptides injected subcutaneously. Our data may shed light on the optimal design of a universal vehicle for epitope-targeted, genetic cancer vaccines.