DNA-transfected Cells Research Articles

Inhibitory effect of a novel thiazolidinedione derivative on hepatitis B virus entry

The development of novel antivirals to treat hepatitis B virus (HBV) infection is still needed because currently available drugs do not completely eradicate chronic HBV in some patients. Recently, troglitazone and ciglitazone, classified among the compounds including the thiazolidinedione (TZD) moiety, were found to inhibit HBV infection, but these compounds are not clinically available. In this study, we synthesized 11 TZD derivatives, compounds 1–11, and examined the effect of each compound on HBV infection in HepG2 cells expressing NTCP (HepG2/NTCP cells). Among the derivatives, (Z)-5-((4'-(naphthalen-1-yl)-[1,1′-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (compound 6) showed the highest antiviral activity, with an IC50 value of 0.3 μM and a selectivity index (SI) of 85, but compound 6 did not affect HCV infection. Treatment with compound 6 inhibited HBV infection in primary human hepatocytes (PHHs) but did not inhibit viral replication in HepG2.2.15 cells or HBV DNA-transfected Huh7 cells. Moreover, treatment with compound 6 significantly impaired hepatitis delta virus (HDV) infection and inhibited a step in HBV particle internalization but did not inhibit attachment of the preS1 lipopeptide or viral particles to the cell surface. These findings suggest that compound 6 interferes with HBV infection via inhibition of the internalization process.

Intranasal Antifungal Vaccination Using DNA-Transfected Dendritic Cells.

Dendritic cells are the most potent antigen-presenting cells, and are critical for the generation of an antigen-specific immune response and protective immunity. These unique features have been applied to dendritic cell-based immunization in a number of disease conditions. Our published results have demonstrated that the immunity induced by intranasal immunization with DNA-transfected dendritic cells results in reduced fungal burden, and alleviated lung tissue damage in a mouse model of pulmonary fungal infection. In this article, approaches for the preparation and characterization of DNA-transfected dendritic cells and intranasal immunization in mice are described.

Abstract 1253: Improvement of the potency of the dendritic cell-based vaccine immunotherapy through local inhibition of TGF-β in tumor-draining lymph nodes.

Abstract The dendritic cell (DC) -based vaccine immunotherapy has been a promising anti-cancer immunotherapy, but has been unable to elicit anti-tumor immune responses enough to eradicate the tumor in patients with advanced cancer. In order to develop the potent DC-based vaccine immunotherapy, it should be required to focus on the unique environment implicated in the induction of anti-tumor immune responses. Transforming growth factor-β (TGF-β), an immunosuppressive cytokine, is produced in the tumor microenvironment, and flows into tumor-draining lymph nodes (TDLs), primary priming sites for generation of anti-tumor immune responses. In TDLs, TGF-β plays a critical role in suppressing the induction of anti-tumor immune responses. In this study, we focused on TGF-β-mediated immunosuppression in TDLs, and examined whether local inhibition of TGF-β in TDLs could improve the potency of the DC-based vaccine immunotherapy in tumor-bearing mouse model. C57BL/6 mice bearing Lewis lung carcinoma (LLC1) subcutaneously were vaccinated with DCs loading LLC1-derived antigen. In order to inhibit TGF-β in TDLs, those mice were administered the plasmid DNA encoding the extracellular domain of TGF-β type II receptor fused to the human IgG heavy chain(TGFR DNA) near the established tumor intramuscularly. Soluble TGF-β type II receptor secreted from TGFR DNA-transfected cells is expected to function to attenuate TGF-β level in TDLs. After this combination therapy, anti-tumor immune responses elicited in those mice were evaluated. As results, the level of TGF-β in TDLs was decreased by administrations of TGFR DNA near the established tumor. The attenuation of TGF-β level in TDLs by TGFR DNA was able to suppress the proliferative activity of Foxp3+ regulatory T cells in TDLs. Regarding the induction of effector cells in TDLs, tumor antigen-specific CD8+ cells producing interferon-gamma were significantly increased by this combination therapy. Next, we evaluated the association between the local inhibition of TGF-β in TDLs and systemic anti-tumor immune responses. Tumor antigen-specific cytotoxic responses as well as natural killer activities were significantly enhanced through the local inhibition of TGF-β in TDLs, leading to effective suppression of tumor growth. Such potent anti-tumor immune responses were not achieved in mice that had been administered DC-based vaccine alone. Improvement of the induction of anti-tumor immune responses in TDLs by the local inhibition of TGF-β contributed to enhancement of systemic anti-tumor immune responses. In conclusion, inhibition of TGF-β-mediated immunosuppression in TDLs can enhance the potency of DC-based vaccine immunotherapy. This animal model provides for a novel rationale with DC-based vaccine immunotherapy. Citation Format: Koji Teramoto, Yasuhiko Ohshio, Takuya Fujita, Jun Hanaoka, Yataro Daigo. Improvement of the potency of the dendritic cell-based vaccine immunotherapy through local inhibition of TGF-β in tumor-draining lymph nodes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1253. doi:10.1158/1538-7445.AM2013-1253

Autographa californica M nucleopolyhedrovirus open reading frame 109 affects infectious budded virus production and nucleocapsid envelopment in the nucleus of cells

Autographa californica M nucleopolyhedrovirus (AcMNPV) open reading frame 109 (ac109) is conserved in all known baculovirus genomes, suggesting a crucial role in virus replication. Although viruses lacking ac109 have been previously characterized, the phenotypes differ from production of non-infectious virions to lack of virion production. To re-examine ac109 function, we constructed a recombinant AcMNPV bacmid, AcBAC109KO, with a deletion in ac109. We did not detect infectious budded virus after transfection of AcBAC109KO DNA into cells. In the nucleus, nucleocapsids had envelopment defects and polyhedra lacked virions. DNA synthesis and gene expression between AcBAC109KO and a control virus were similar. However, lower levels of non-infectious budded virus were detected from AcBAC109KO DNA-transfected cells compared to the parental virus using Q-PCR to detect viral DNA or by immunoblotting to detect a budded virus protein. Therefore, deletion of ac109 affects envelopment of nucleocapsids in the nucleus and the production of infectious budded virus.

New clinical trial of DNA-based vaccines for patients with non-small cell lung cancer.

e13034 Background: We report a new immunotherapeutic trial for patients with non-small cell lung cancer (NSCLC). The trial to be conducted at the University of Pittsburgh Cancer Institute is a two-stage uncontrolled non-randomized Phase IB study designed to evaluate the safety, immunogenicity and feasibility of a new vaccine, consisting of human allogeneic fibroblasts transfected with DNA derived from the patient’s own tumor. The vaccine is prepared by transfer of genomic DNA-fragments (25 kb) from the patient’s tumor into MRC-5 cells, a human embryonic fibroblast cell line. The rationale is that numerous mutant and dysregulated genes in the tumor specifying an array of patient-specific tumor antigens are expressed in a highly immunogenic form by the DNA-transfected cells. A large body of preclinical studies in tumor-bearing mice supports this approach. An IND has been issued. Cohort 1 is underway without DLT. Methods: We plan to use a two-stage trial design. Initially, 15 early-stage patients with NSCLC will be enrolled. If there is no evidence of toxicity, and >3 of the initial patients show an immunologic response, the second stage of the study will be opened for the accrual of 22 additional patients. A portion of the primary tumor removed at surgery will be obtained to serve as a source of tumor-DNA. Each vaccine will contain 1 X 10E7 DNA-transfected fibroblasts. The vaccine will be lethally irradiated before it is administered. It will be injected intradermally in the Outpatient Clinic. Immunologic responses to the vaccine and to the autologous tumor will be evaluated by IFN-g secretion in ELISPOT assays prior to and after vaccination for the frequency of T-cells responsive to the autologous tumor. Additional assays will include lymphocyte proliferation determinations in response anti CD3 and anti CD28 monoclonal antibodies and the effect of the immunizations on the proportion of myeloid-derived suppressor T cells in the patient’s peripheral blood. The patients will also be evaluated before and after immunization for the capacity of their T cells to respond to activating signals delivered via the T cell receptor (TcR). Results: Vaccines have been prepared. First patient has been enrolled. Conclusions: No data to report in this early trial.

The Autographa californica M Nucleopolyhedrovirus ac79 Gene Encodes an Early Gene Product with Structural Similarities to UvrC and Intron-Encoded Endonucleases That Is Required for Efficient Budded Virus Production

The Autographa californica M nucleopolyhedrovirus (AcMNPV) orf79 (ac79) gene is a conserved gene in baculoviruses and shares homology with genes in ascoviruses, iridoviruses, and several bacteria. Ac79 has a conserved motif and structural similarities to UvrC and intron-encoded endonucleases. Ac79 is produced at early times during infection and concentrates in the nucleus of infected cells at late times, suggesting a cellular compartment-specific function. To investigate its function, an ac79-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration assays showed that budded virus (BV) production was reduced in the ac79-knockout virus compared to control viruses, following either virus infection or the transfection of bacmid DNA. The ac79-knockout virus-infected cells produced plaques smaller than those infected with control ac79-carrying viruses. No obvious differences were observed in viral DNA synthesis, viral protein accumulation, or the formation of occlusion bodies in ac79-knockout and control viral DNA-transfected cells, indicating progression into the late and very late phases of viral infection. However, comparative analyses of the amounts of BV genomic DNA and structural proteins in a given quantity of infectious virions suggested that the ac79-knockout virus produced more noninfectious BV in infected cells than the control virus. The structure of the ac79-knockout BV determined by transmission electron microscopy appeared to be similar to that of the control virus, although aberrant capsid protein-containing tubular structures were observed in the nuclei of ac79-knockout virus-infected cells. Tubular structures were not observed for ac79 viruses with mutations in conserved endonuclease residues. These results indicate that Ac79 is required for efficient BV production.

Pro-survival Effects of 17β-Estradiol on Osteocytes Are Mediated by Nitric Oxide/cGMP via Differential Actions of cGMP-dependent Protein Kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.

Establishment of a DNA-launched infectious clone for a highly pneumovirulent strain of type 2 porcine reproductive and respiratory syndrome virus: Identification and in vitro and in vivo characterization of a large spontaneous deletion in the nsp2 region

A highly pneumovirulent strain of porcine reproductive and respiratory syndrome virus (PRRSV), ATCC VR2385, was isolated from a pig exhibiting typical PRRS in the early 90s. While passaging the virus in monkey kidney cells, we identified a large spontaneous deletion of a 435-bp in the nsp2 gene. To assess the biological significance of this spontaneous deletion, we first determined the full-length genomic sequence of this virus and established a DNA-launched infectious clone of the passage 14 virus containing the 435-bp nsp2 deletion (designated as pIR-VR2385-CA). The full-length viral genome engineered with two ribozyme elements at both ends was placed under the control of the eukaryotic CMV promoter. The infectious virus was successfully rescued from pIR-VR2385-CA DNA-transfected BHK-21 cells. To characterize the biological and pathological significance of this large nsp2 deletion, we subsequently constructed another DNA-launched infectious clone, pIR-VR2385-R, in which we restored the deleted 435-bp nsp2 sequence back to the pIR-VR2385-CA backbone. The growth characteristics of the two rescued viruses (VR2385-CA and VR2385-R) were compared, and the results showed that the VR2385-CA virus with the nsp2 deletion replicated more efficiently in vitro (1.0–1.5 log titer higher) than the VR2385-R virus with the restored nsp2 sequence but the VR2385-CA virus exhibited a significantly reduced serum viral RNA load in vivo. A comparative pathogenicity study in pigs (n=10) revealed that the nsp2 deletion had no effect on virus virulence, and the restored nsp2 sequence in the VR2385-R virus remains stable during virus replication in pigs. The results from this study indicates that the spontaneous nsp2 deletion plays a role for enhanced PRRSV replication in vitro but has no effect on the pathogenicity of the virus.

Autographa californica Multiple Nucleopolyhedrovirus Ac92 (ORF92, P33) Is Required for Budded Virus Production and Multiply Enveloped Occlusion-Derived Virus Formation

The Autographa californica multiple nucleopolyhedrovirus orf92 (p33), ac92, is one of 31 genes carried in all sequenced baculovirus genomes, thus suggesting an essential function. Ac92 has homology to the family of flavin adenine dinucleotide-linked sulfhydryl oxidases and is related to the ERV/ALR family of sulfhydryl oxidases. The role of ac92 during virus replication is unknown. Ac92 was associated with the envelope of both budded and occlusion-derived virus (ODV). To investigate the role of Ac92 during virus replication, an ac92-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration and plaque assays showed no virus spread in ac92-knockout bacmid DNA-transfected insect cells. Deletion of ac92 did not affect viral DNA replication. However, ac92-knockout bacmid DNA-transfected cells lacked multiply enveloped occlusion-derived nucleocapsids; instead, singly enveloped nucleocapsids were detected. To gain insight into the requirement for sulfhydryl oxidation during virus replication, a virus was constructed in which the Ac92 C(155)XXC(158) amino acids, important for sulfhydryl oxidase activity, were mutated to A(155)XXA(158). The mutant virus exhibited a phenotype similar to that of the knockout virus, suggesting that the C-X-X-C motif was essential for sulfhydryl oxidase activity and responsible for the altered ODV phenotype.

Establishment of a simple detection system for blood group ABO-specific transferase activity in DNA-transfected cells

A/B-transferase is a glycosyltransferase that transfers a sugar substrate onto H-antigen resulting in the synthesis of glycoproteins and glycolipids termed A/B-antigens. The ABO blood group (ABO) gene encoding A/B-transferase possesses numerous polymorphisms affecting the specificity and/or activity of the enzyme. The relationship between genotype and phenotype is very complicated, except for those of some critical polymorphisms. In order to establish a system for evaluating the effect of each polymorphism on the transferase function, an A- or B-transferase cDNA expressing vector was introduced into HeLa cells, a cell line that do not possess endogenous A/B-transferase activity. We successfully detected substrate-specific transferase activity in the cells and in the culture medium. Furthermore, in three different assays, each corresponding A- or B-antigen was detected in the transfectants with high sensitivity. Accordingly, the present study demonstrates a possibility that A/B-transferase variants may be characterized by using this method.

Human Cytomegalovirus IE1-72 Protein Interacts with p53 and Inhibits p53-Dependent Transactivation by a Mechanism Different from That of IE2-86 Protein

Infection of host cells with human cytomegalovirus (HCMV) induces cell cycle dysregulation. Two HCMV immediate-early (IE) proteins, IE1-72 and IE2-86, are promiscuous transactivators that have been implicated in the dysregulatory events. Cellular p53 protein is accumulated to high levels in HCMV-infected cells, but the indicative marker of p53 transcriptional activity, p21, is markedly decreased. Both IE1-72 and IE2-86 were able to transactivate the p53 promoter and interact with p53 protein in DNA-transfected or HCMV-infected cells. HCMV UL84, a multiregulatory protein expressed in early periods of HCMV infection, also interacted with p53. HCMV IE1-72 prevented or disrupted p53 binding to p53-specific DNA sequences, while IE2-86 and/or UL84 enhanced p53 binding and induced supershift of this DNA-protein complex. Both HCMV IE1-72 and IE2-86 were able to inhibit p53-dependent transcriptional activation in plasmid-transfected cells. IE1-72, rather than IE2-86, was found to be responsible for p21 downregulation in HCMV-infected HEL cells. DNA transfection analysis using IE1-72 mutants revealed that exon 2/3 and the zinc finger region of IE1-72 are essential for IE1-72's effect on the repression of p53-dependent transcriptional activation. These data suggest that HCMV IE1-72 and/or IE2-86 transactivates the p53 promoter and induces p53 accumulation, but HCMV IE1-72 represses the p53 transactivation activity by a unique binding hindrance mechanism different from that of IE2-86. Thus, various modes of viral IE proteins and p53 interactions might result in multiple outcomes, such as stimulation of cellular DNA synthesis, cell cycle progression and cell cycle arrest, and prevention of program cell death.

A modified epitope identified for generation and monitoring of PSA-specific T cells in patients on early phases of PSA-based immunotherapeutic protocols

Efficacy of vaccination in cancer patients on immunotherapeutic protocols can be difficult to evaluate. The aim of this study was therefore to identify a single natural or modified epitope in prostate-specific antigen (PSA) with the ability to generate high levels of PSA-specific T cells to facilitate monitoring in patients after vaccination against prostate cancer. To the best of our knowledge, this study describes for the first time the peptide specificity of T cells stimulated by endogenously processed PSA antigen. The peptide specificity of HLA-A*0201-restricted CD8 + T cells against human and rhesus PSA was investigated both in vivo after DNA vaccination in HLA-A*0201-transgenic mice and in vitro after repetitive stimulation of human T cells with DNA-transfected human dendritic cells (DCs). One of seven native PSA peptides, psa53–61, was able to activate high levels of PSA-specific CD8 + T cells in HLA-A*0201-transgenic mice after PSA DNA vaccination. Psa53–61 was also the only peptide that induced human T cells to produce IFNγ after stimulation with PSA transfected DCs, however not in all donors. Therefore, plasmids encoding modified epitopes in predicted HLA-A*0201 sequences were constructed. One of these modified PSA plasmids consistently induced IFNγ producing CD8 + T cells to the corresponding modified peptide as well as to the corresponding native peptide, in all murine and human T cell cultures. This study demonstrates a novel concept of introducing a modified epitope within a self-tumor antigen, with the purpose of eliciting a reliable T cell response from the non-tolerized immune repertoire, to facilitate monitoring of vaccine efficacy in cancer patients on immunotherapeutic protocols. The purpose of such a modified epitope is thus not to induce therapeutically relevant T cells but rather to, in case of weak or divergent T cell responses to self antigens/peptides, help answer questions about efficacy of vaccine delivery and about the possibility to induce immune responses in the selected and often immunosuppressed cancer patients.

Cyclosporin a inhibits HTLV‐I tax expression and shows anti‐tumor effects in combination with VP‐16

Adult T cell leukemia (ATL) is one of the most refractory malignant hematological diseases. Our previous studies demonstrated HTLV-1Tax protein involvement in clinical manifestation of the aggressive type of ATL and suggested the potential application of agents to inhibit Tax expression for ATL treatment. In the present study, we first examined Tax involvement in the resistance to VP-16-induced apoptosis using four HTLV-1 infected T cell clones and cTax DNA-transfected cells. Next, we examined whether cyclosporin A reduced expression of Tax and its related transfer factors on Western blot and CAT assay. We further investigated whether cyclosporin A in combination with VP-16 can induce apoptosis in HTLV-1 infected T cells. Tax-producing T cells, K3T and F6T, were resistant to VP-16 induced growth inhibition compared with that of the nonproducing cells, S1T and Su9T01. Experiments using S1T and Tax-expressing cDNA-transfected S1T demonstrated Tax-induced resistance to VP-16 induction of apoptosis by DNA ladder formation. Cyclosporin A reduced Tax expression in K3T by Western blot analysis and on CAT assay, showing maximal reduction of 61% and 60% compared to control culture using LTR CAT transfected Jurkat cells and K3T cells, respectively. Cyclosporin A also reduced the nuclear expression of two Tax-related transfer factors, ATF-1 and ATF-2 on Western blot. Cyclosporin A alone did not show any cytotoxicity by itself, but sensitized cells to VP-16 when combined with VP-16. Cyclosporin A may be a useful anti-ATL agent when combined with other anti-cancer agents possibly related to Tax inhibition.

A549 폐 상피세포에서 합토글로빈에 의한 염증반응 조절

Haptoglobin (Hp) is an acute phase protein, and its plasma level increases consistently in response to inflammation. To investigate the biological role of Hp in lung epithelial cells, the gene expressions of cyclooxygenase-2 (COX-2) and inflammatory cytokines were analyzed using human Hp gene-transfected A549 cells. Western blot analysis showed that COX-2 expression was markedly increased in Hp DNA-transfected cells (stable transfection and transient transfection) compared with that in vehicle DNA-transfected cells. When the Hp-expressing cells were treated with of LPS or 100 U/ml of for 24 hr, the COX-2 expression was synergistically up-regulated. ACP-based PCR data demonstrated the Hp decreased SPARC expression, but increased IL-4 and S100AI expressions. These findings suggest that the Hp acts as a pro-inflammatory mediator in lung inflammation.

407 AN ATTEMPT TO GENERATE TRANSGENIC CHICKENS BY INJECTION OF EGFP OR YFP GENE-TRANSFECTED BLASTODERMAL CELLS

Transgenic chickens have been generated from blastodermal chimeric chickens created by an injection of the foreign DNA-transfected blastodermal cells into recipient embryos. However, there has been no method that allows the efficient foreign DNA transfection into chicken primary cells without using viral vectors. This method using viral vectors has a limitation on the size of DNA that can be transfected, and homologous recombination is not possible with this method. This study was conducted to establish a DNA transfection method using electroporation, which can transfect up to 5 kb DNAs into cells. Embryos at stage X were obtained from freshly laid eggs (200) of Barred Plymouth Rock chickens. Blastodermal cells (105 cells) were then collected from them and transfected with the linearized EGFP or YFP gene by electroporation using a Nucleofector Device (Amaxa, Inc., Gaithersburg, MD, USA). After transfection, the transfected chicken blastodermal cells were injected into White Leghorn recipient embryos to produce chimeric chickens. By 48 h after gene transfection, EGFP or YFP gene expression in the cells was easily observed under a fluorescence microscope. The gene expression rate, however, could not be determined, because the cultured chicken blastodermal cells were not able to separate and it was impossible to load them on the flow cytometer to determine the rate. The blastodermal cells transfected with the EGFP or YFP gene were cultured over night and injected into recipient embryos. The manipulated embryos were cultured ex ovo until they hatched. Dead embryos before hatch were dissected and observed under the fluorescence microscope to determine whether they had fluorescent organs. EGFP or YFP gene expression was detected at several areas: head, somite, inner tissues, and limb bud of embryos, but not germinal tissues. Six female chicks and 10 male chicks were obtained. Five females and 6 males were raised until they were sexually mature. Five female chickens were artificially inseminated with semen of Barred Plymouth Rock males to obtain progeny from donor transfected cells. However, no progeny was obtained. Thus the contribution of donor cells to the germ cells could not be determined, and it was not clear if the expression of EGFP gene or YFP gene was stable. Furthermore, semen could not be collected from six males because of a technical problem, and males were not mated. Although a large number of cells must be used to transfect a foreign gene, the novel gene transfer method in chicken in this study has great significance in the field of transgenic chicken production. Even if the gene expression is transient, this transfer method will enable us not only to integrate more than 5 kb DNA into the avian genome but also to induce homologous recombination in cultured avian cells.

Antigen Expression Kinetics and Immune Responses of Mice Immunized with Noninfectious Simian-Human Immunodeficiency Virus DNA

In a previous report we demonstrated that three injections of an rt-deleted noninfectious genome of the simian-human immunodeficiency virus SHIV(KU2) induced protection against AIDS in macaques (D. K. Singh, Z. Liu, D. Sheffer, G. A. Mackay, M. Smith, S. Dhillon, R. Hegde, F. Jia, I. Adany, and O. Narayan, J. Virol 79:3419-3428, 2005). To make this DNA safer, we deleted two more genes, the integrase gene and vif, along with the 3' long terminal repeat. We also replaced the gag, pro, and nef genes (SIVmac239 origin) with those of human immunodeficiency virus (HIV) type 1 strain SF2. The resultant construct, designated delta4SHIV(KU2) DNA, was used in this study to evaluate gene expression and immunogenicity in BALB/c mice. DNA-transfected human embryonic kidney epithelial cells (HEK 293) produced all of the major viral proteins and released p24 in the supernatant for 12 days. Inoculation of the vaccine DNA into the gastrocnemius muscles resulted in intense mononuclear cell infiltration at the inoculated sites and the production of viral p24 in myocytes, in infiltrating mononuclear cells, and in cells in the spleen and draining lymph nodes between 3 and 10 days postinoculation. Expression of p24 in the muscle cells peaked at day 7 and became undetectable after day 12. The same 12-day period of expression of p24 was observed in mice that were given a second injection 4 weeks after the first. Evaluation of immune responses in BALB/c mice revealed that the DNA induced enzyme-linked immunospot and antigen-specific proliferative cell-mediated immunity responses. The responses were stronger in mice that were coinjected with a second plasmid expressing granulocyte-macrophage colony-stimulating factor. Since new waves of viral antigen production could be induced with each boosting injection of the vaccine DNA, this DNA could be a safe and efficient agent to induce long-term protection against HIV.

272. Induction of Anti-Tumoral Immune Response and IFN-Regulated Factors by Delivery of Control DNA

Delivery of DNA encoding therapeutic genes in vivo has great potential for treating malignancy as well as genetic diseases. Delivery of placebo DNA without a transgene is used as a control in gene therapy studies. It is tacitly assumed by most investigators that the protein expressed from the transfected DNA has phenotypic consequences, but that the consequences are not from the DNA itself. Here, we demonstrate that transfection of control plasmid DNA (that does not express a gene product) into tumor cell lines induces a dramatic (>10-fold) increase in the expression of the interferon (IFN)-regulated genes IRF7, STAT1, CXCL9, MHCI and CD11a (ITGAL) in tumor cell lines. Induction of these genes inhibits tumor development and tumor growth in immunocompetent mice that are immunized with apoptotic tumor cells. The antibody depletion study indicates that the underlying mechanism by which transfection of control DNA induces IFN-regulated genes is the induction of a secreting factor such as IFN-beta. Three lines of evidence indicate that DNA transfection-mediated induction of IFN-regulatory genes is independent of TLR9. The three lines of evidence are: 1) TLR9 is not expressed in either SCCVII or 4T1 cell line, 2) activation of TLR9 downstream signaling molecules is not associated with the induction of gene expression, and 3) the secretion factor (s) obtained from the conditioned medium of DNA-transfected SCCVII tumor cells induces the same type of gene expression in the 4T1 tumor cell line, which is resistant to the DNA transfection-mediated induction of IFN-regulated genes, can be used to determine the real therapeutic gene function.

Induction of IFN-regulated factors and antitumoral surveillance by transfected placebo plasmid DNA

Delivery of DNA encoding therapeutic genes in vivo has great potential for treating malignancy as well as genetic diseases. Delivery of placebo DNA without a transgene is used as a control in gene therapy studies. It is tacitly assumed by most investigators that the protein expressed from the transfected DNA has phenotypic consequences, but that the consequences are not from the DNA itself. Here, we demonstrate that transfection of control plasmid DNA (that does not express a gene product) into tumor cell lines induces a dramatic (>10-fold) increase in the expression of the interferon (IFN)-regulated genes IRF7, STAT1, MIG (approved gene symbol CXCL9), MHCI (MICA), and CD11a (ITGAL) in tumor cell lines. Induction of these genes inhibits tumor development and tumor growth in immunocompetent mice that are immunized with apoptotic tumor cells. The antibody depletion study indicates that the underlying mechanism by which transfection of control DNA induces IFN-regulated genes is the induction of a secreting factor(s) such as IFN-beta. Three lines of evidence indicate that DNA transfection-mediated induction of IFN-regulatory genes is independent of TLR9. The three lines of evidence are: (1) TLR9 is not expressed in either SCCVII or 4T1 cell line, (2) activation of TLR9 downstream signaling molecules is not associated with the induction of gene expression, and (3) the secretion factor(s) obtained from the conditioned medium of DNA-transfected SCCVII tumor cells induces the same type of gene expression in the 4T1 tumor cell line, which is refractory to the gene induction by DNA transfection. Our finding indicates that the 4T1 tumor cell line, which is resistant to the DNA transfection-mediated induction of IFN-regulated genes, can be used to determine the real therapeutic gene function.

Two common PFIC2 mutations are associated with the impaired membrane trafficking of BSEP/ABCB11†

Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by a mutation in the bile salt export pump (BSEP/ABCB11) gene. However, the mechanisms for the deficiency in the function of two mutations (E297G and D482G), which are frequently found in European patients, have not yet been identified. In the present study, we examined the transport activity and cellular localization of these two mutants in human embryonic kidney 293 and Madin-Darby canine kidney II cells, respectively. Introduction of E297G and D482G mutations into the human BSEP gene by site-directed mutagenesis resulted in a significant reduction in the BSEP expression level, which was associated with impaired membrane trafficking. Most of the D482G BSEP and some of the E297G BSEP underwent only core glycosylation and appeared to be predominantly located in the endoplasmic reticulum. The inhibition of proteasome function by MG132 resulted in the cellular accumulation of the core glycosylation form of the two mutants. In contrast, transport studies for taurocholate and glycocholate with membrane vesicles isolated from complementary DNA-transfected cells indicated that both mutations did not significantly affect the transport function of BSEP per se. In conclusion, E297G and D482G mutations result in impaired membrane trafficking, whereas the transport functions of these mutants remain largely unchanged.

Intraocular implantation of DNA-transfected retinal pigment epithelium cells: a new approach for analyzing molecular functions in the newt retinal regeneration

Adult newts can regenerate their entire retinas, even after surgical removal of the neural retina (retinectomy), through transdifferentiation of the retinal pigment epithelium (RPE) cells. To develop a new experimental system for analyzing molecular functions during retinal regeneration of adult newts, we attempted to deliver a foreign gene into RPE cells of retina-less eye-cups in vitro. Here we used pCS2mt-GFP as a reporter construct, and selected Polyfect as a transfection reagent. DNA-transfection appeared to be restricted to the RPE cells of retina-less eye-cups and its efficiency was 0.1–0.2%. We tried to implant RPE-choroid tissue containing DNA-transfected RPE cells into the eye of a host animal. The tissue was placed into the posterior eye-chamber immediately after retinectomy so that the implanted RPE tissue was facing the cornea (i.e., normal orientation). The implant and host RPE regenerated one continuous hybrid neural retina. Ocular sections after 60 days of implantation showed that a small number of cells in the regenerating retina were intensely stained with an anti-GFP antibody. Some of those cells were believed to be retinal cells such as ganglion cells, amacrine cells and photoreceptors. The GFP-positive cells in the hybrid regenerating retina could represent clones derived from a single RPE cell. These results indicate that this experimental system could become useful in the study of adult newt retinal regeneration.