Yuka Okada

Complementation of placental defects and embryonic lethality by trophoblast-specific lentiviral gene transfer

Placental dysfunction underlies many complications during pregnancy, and better understanding of gene function during placentation could have considerable clinical relevance. However, the lack of a facile method for placenta-specific gene manipulation has hampered investigation of placental organogenesis and the treatment of placental dysfunction. We showed previously that transduction of fertilized mouse eggs with lentiviral vectors leads to transgene expression in both the fetus and the placenta. Here we report placenta-specific gene incorporation by lentiviral transduction of mouse blastocysts after removal of the zona pellucida. All of the placentas analyzed, but none of the fetuses, were transgenic. Application of this method substantially rescued mice deficient in Ets2, Mapk14 (also known as p38α) and Mapk1 (also known as Erk2) from embryonic lethality caused by placental defects. Ectopic expression of Mapk11 also complemented Mapk14 deficiency during placentation.

Immunological properties and vaccine efficacy of murine dendritic cells simultaneously expressing melanoma-associated antigen and interleukin-12.

Interleukin (IL)-12 is a key factor for inducing cellular immune responses, which play a central role in the eradication of cancer. In the present study, in order to create a dendritic cell (DC)-based vaccine capable of positively skewing immune response toward a cellular immunity-dominant state, we analyzed immunological characteristics and vaccine efficacy of DCs cotransduced with melanoma-associated antigen (gp100) and IL-12 gene (gp100+IL12/DCs) by using RGD fiber-mutant adenovirus vector (AdRGD), which enables highly efficient gene transduction into DCs. gp100+IL12/DCs could simultaneously express cytoplasmic gp100 and secretory IL-12 at levels comparable to DCs transduced with each gene alone. In comparison with DCs transduced with gp100 alone (gp100/DCs), upregulation of major histocompatibility complex class I, CD40, and CD86 molecules on the cell surface and more potent T-cell-stimulating ability for proliferation and interferon-γ secretion were observed as characteristic changes in gp100+IL12/DCs. In addition, administration of gp100+IL12/DCs, which were prepared by a relatively low dose of AdRGD-IL12, could induce more potent tumor-specific cellular immunity in the murine B16BL6 melanoma model than vaccination with gp100/DCs. However, antitumor effect and B16BL6-specific cytotoxic T-lymphocyte activity in mice vaccinated with gp100+IL12/DCs diminished with increasing AdRGD-IL12 dose during gene transduction, and paralleled the decrease in presentation levels via MHC class I molecules for antigen transduced with another AdRGD. Collectively, our results suggested that optimization of combined vector dose was required for development of a more efficacious DC-based vaccine for cancer immunotherapy, which relied on genetic engineering to simultaneously express tumor-associated antigen and IL-12.

Gene transduction efficiency and maturation status in mouse bone marrow-derived dendritic cells infected with conventional or RGD fiber-mutant adenovirus vectors.

Since dendritic cells (DCs) play a critical role in establishing antigen-specific adaptive immune responses, in the past several years, therapeutic strategies using genetically modified DCs against cancer and infectious diseases have attracted increasing attention. In the present study, we demonstrated that RGD fiber-mutant adenovirus vector (AdRGD) exhibited markedly superior gene transduction efficiency in mouse bone marrow-derived DCs (mBM-DCs) compared to conventional adenovirus vector (Ad). Likewise, this vector exhibited superior major histocompatibility complex class I-restricted presentation of antigen derived from the delivered gene in mBM-DCs. In order to investigate the effect of Ad-infection on the DC-differentiation process (maturation), we used three types of AdRGD and three conventional Ad to transduce mBM-DCs. These vectors carried either no transgene, LacZ gene, or gp100 gene. Infection by any of the Ad vectors enhanced the expression of MHC class II molecules in mBM-DCs. CD80, CD86, and CD40 expression and IL-12 production were more efficient in AdRGD-infected mBM-DCs than in conventional Ad-infected cells. Contrary to our expectations, endocytotic activity of mBM-DCs decreased only slightly upon Ad-infection, whereas antigen uptake by lipopolysaccharide (LPS)-driven mature mBM-DCs was significantly impaired. However, our reverse transcription-polymerase chain reaction analysis revealed that Ad-infection resulted in the upregulation of the chemokine receptor CCR7 and downregulation of CCR6 in mBM-DCs and LPS-stimulated cells. We, therefore, concluded that Ad-infection directly influenced DC-maturation, although the effects were milder than under LPS-stimulation. In addition, this change in the immunologic properties of DCs resulted primarily from an increase in the number of Ad-particles capable of invading the cells rather than from the expression of foreign genes. AdRGD-infection caused greater induction of maturation than conventional Ad-infection, irrespective of the type of transgene inserted.

Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

Calponin 3 (CNN3), an actin binding molecule, negatively regulates trophoblast differentiation and fusion. CNN3 phosphorylation modulates the actin-binding capacity of CNN3 and most probably regulates cytoskeleton remodeling that renders cells capable of undergoing fusion.

Dendritic cells transduced with gp100 gene by RGD fiber-mutant adenovirus vectors are highly efficacious in generating anti-B16bl6 melanoma immunity in mice

Dendritic cells (DCs) are the most potent professional antigen-presenting cells for the initiation of antigen-specific immune responses, and antigen-loaded DCs have been regarded as promising vaccines in cancer immunotherapy. We previously demonstrated that RGD fiber-mutant adenovirus vector (AdRGD) could attain highly efficient gene transduction into human and murine DCs. The aim of the present study is to demonstrate the predominance of ex vivo genetic DC manipulation using AdRGD in improving the efficacy of DC-based immunotherapy targeting gp100, a melanoma-associated antigen (MAA). Vaccination with murine bone marrow-derived DCs transduced with AdRGD encoding gp100 (AdRGD-gp100/mBM-DCs) dramatically improved resistance to B16BL6 melanoma challenge and pulmonary metastasis as compared with immunization with conventional Ad-gp100-transduced mBM-DCs. The improvement in antimelanoma effects upon immunization with AdRGD-gp100/mBM-DCs correlated with enhanced cytotoxic activities of natural killer (NK) cells and B16BL6-specific cytotoxic T lymphocytes (CTLs). Furthermore, in vivo depletion analysis demonstrated that CD8+ CTLs and NK cells were the predominant effector cells responsible for the anti-B16BL6 immunity induced by vaccination with AdRGD-gp100/mBM-DCs, and that helper function of CD4+ T cells was necessary for sufficiently eliciting effector activity. These findings clearly revealed that highly efficient MAA gene transduction to DCs by AdRGD could greatly improve the efficacy of DC-based immunotherapy against melanoma.

Transcriptional targeting of RGD fiber-mutant adenovirus vectors can improve the safety of suicide gene therapy for murine melanoma

Since RGD fiber-mutant adenovirus vector (AdRGD), which contains an αv-integrin tropism, is highly efficient in gene transduction to melanoma, the AdRGD-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system is an attractive approach for melanoma treatment. However, the intratumoral injection of AdRGD causes limited transgene expression in healthy normal tissue, due to unwanted vector spread. Herein, we describe our attempt to overcome this limitation related to the safety of HSVtk/GCV treatment by using AdRGD carrying either melanoma-specific tyrosinase (Tyr) promoter or tumor-specific telomerase reverse transcriptase (TERT) promoter instead of universal cytomegalovirus promoter. Our in vitro study revealed that Tyr promoter-regulated AdRGD exhibited high transgene expression specificity for melanoma cells, and that TERT promoter-regulated AdRGD could induce efficient gene expression in tumor cells, but was relatively quiescent in normal cells. Anti-B16BL6 melanoma effects in mice injected intratumorally with AdRGD-Tyr/HSVtk or AdRGD-TERT/HSVtk, after which GCV was injected intraperitoneally for 10 days, were comparable to those in mice injected with AdRGD-CMV/HSVtk at 10 times less vector dosage. On the other hand, AdRGD-Tyr/HSVtk and AdRGD-TERT/HSVtk did not induce severe adverse effects even when they were intravenously injected into mice at 109 plaque-forming units (PFU), whereas mice injected with AdRGD-CMV/HSVtk at 108 PFU exhibited body weight reduction and serum level increase of biochemical enzymes for hepatotoxicity. These results indicate that AdRGD combined with transcriptional regulation using Tyr or TERT promoter is a potentially useful and safe vector system for suicide gene therapy for melanoma.

Fiber-mutant technique can augment gene transduction efficacy and anti-tumor effects against established murine melanoma by cytokine-gene therapy using adenovirus vectors

Melanoma cells are relatively resistant to adenovirus vector (Ad)-mediated gene transfer due to the low expression of Coxsackie-adenovirus receptor (CAR), which acts as a primitive Ad-receptor. Therefore, extremely high doses of Ad are required for effective gene therapy against melanoma. In the present study, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob could promote gene delivery and anti-tumor effects in the murine B16 BL6 tumor model. B16 BL6 cells (in vitro) and tumors (in vivo) infected with RGD fiber-mutant Ad containing a tumor necrosis factor alpha gene (Ad-RGD-TNFalpha) produced more TNFalpha than those infected with conventional Ad-TNFalpha. In addition, Ad-RGD-TNFalpha required about one-tenth the dosage of Ad-TNFalpha for induction of equal therapeutic effects upon intratumoral injection into established B16 BL6 tumors. Furthermore, the combination of both TNFalpha- and interleukin 12-expressing RGD fiber-mutant Ads exhibited more effective tumor regression than the Ad expressing each alone. These results suggested that the fiber-mutant for altering Ad-tropism is a very potent technology for advancing gene therapy for melanoma.

Tumor Necrosis Factor α-Gene Therapy for an Established Murine Melanoma Using RGB (Arg-Gly-Asp) Fiber-mutant Adenovirus Vectors

Although adenovirus vectors (Ad) provide high‐level transduction efficacy to many cell types, extremely high doses of Ad are required for sufficient gene transduction into several tumors, including melanoma. Here, we demonstrated that the expression of coxsackie‐adenovirus receptor, a primitive Ad‐receptor, was very low in murine and human melanoma cells. We also found that fiber‐mutant Ad containing the Arg‐Gly‐Asp (RGD) sequence in the fiber knob remarkably augmented gene transduction efficacy in melanoma cells by targeting αv‐integrins. In addition, intratumoral injection of RGD fiber‐mutant Ad containing the tumor necrosis factor α gene (AdRGD‐TNFα) revealed dramatic anti‐tumor efficacy through hemolytic necrosis in an established murine B16 BL6 melanoma model. Ad‐RGD‐TNFα required one‐tenth the dosage of Ad‐TNFα to induce an equal therapeutic effect. These results suggest that αv‐integrin‐targeted Ad will be a very powerful tool for the advancement of melanoma gene therapy.

An investigation of adverse effects caused by the injection of high-dose TNFα-expressing adenovirus vector into established murine melanoma

We previously reported that RGD fiber-mutant adenovirus vector carrying human TNFα cDNA (AdRGD-TNFα) could more effectively induce mouse B16 BL6 melanoma regression than conventional Ad-TNFα on intratumoral injection at less than 109 vector particles (VP). Although mice treated with either Ad type at 1010 VP showed remarkable tumor regression due to hemolytic necrosis, severe adverse effects including extreme reduction in body weight were also induced by Ad treatment. Here, we attempted to elucidate the cause of the adverse effects to optimize the application of AdRGD-TNFα. More than 99% of systemically administered Ad accumulated in the liver, and the rate of Ad leakage into systemic circulation from the B16 BL6 tumors injected with AdRGD or conventional Ad at 1010 VP was about 1% of the administered VP. Although the leaked Ad did not directly induce hepatotoxicity or body weight reduction, excessive TNFα produced in the tumors leaked into the blood at high concentrations and caused systemic inflammation, tissue denaturation, and body weight reduction in mice injected intratumorally with AdRGD-TNFα or Ad-TNFα at 1010 VP. Our results demonstrated that an exact AdRGD-TNFα dosage must be determined to prevent TNFα leakage from tumors into systemic circulation, thereby enabling safe application of AdRGD-TNFα to clinical melanoma gene therapy in the future.

Targeted gene modification in mouse ES cells using integrase-defective lentiviral vectors.

Lentiviral vectors efficiently integrate into the host genome of both dividing and nondividing cells, and so they have been used for stable transgene expression in biological and biomedical studies. However, recent studies have highlighted the risk of insertional mutagenesis and subsequent oncogenesis. Here, we used an integrase‐defective lentiviral (IDLV) vector to decrease the chance of random integration and examined the feasibility of lentiviral vector‐mediated gene targeting into murine embryonic stem (ES) cells. After transduction with wild‐type lentiviral vectors, none of the 512 G418 resistant clones were found to be homologous recombinant clones. Although the transduction efficiency was lower with the IDLV vectors (5.9% of wild‐type), successful homologous recombination was observed in nine out of the 941 G418 resistant clones (0.83 ± 1.32%). Pluripotency of the homologous recombinant ES cells was confirmed by the production of chimeric mice and subsequent germ line transmission. Because lentiviral vectors can efficiently transduce a variety of stem cell types, our strategy has potential relevance for secure gene‐manipulation in therapeutic applications. genesis 47:217–223, 2009.