Polycistronic Retroviral Vector Research Articles

Morphologic and Gene Expression Criteria for Identifying Human Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells can be generated from somatic cells by the forced expression of four factors, Oct3/4, Sox2, Klf4, and c-Myc. While a great variety of colonies grow during induction, only a few of them develop into iPS cells. Researchers currently use visual observation to identify iPS cells and select colonies resembling embryonic stem (ES) cells, and there are no established objective criteria. Therefore, we exhaustively analyzed the morphology and gene expression of all the colonies generated from human fibroblasts after transfection with four retroviral vectors encoding individual factors (192 and 203 colonies in two experiments) and with a single polycistronic retroviral vector encoding all four factors (199 and 192 colonies in two experiments). Here we demonstrate that the morphologic features of emerged colonies can be categorized based on six parameters, and all generated colonies that could be passaged were classified into seven subtypes in colonies transfected with four retroviral vectors and six subtypes with a single polycistronic retroviral vector, both including iPS cell colonies. The essential qualifications for iPS cells were: cells with a single nucleolus; nucleus to nucleolus (N/Nls) ratio ∼2.19: cell size ∼43.5 µm2: a nucleus to cytoplasm (N/C) ratio ∼0.87: cell density in a colony ∼5900 cells/mm2: and number of cell layer single. Most importantly, gene expression analysis revealed for the first time that endogenous Sox2 and Cdx2 were expressed specifically in iPS cells, whereas Oct3/4 and Nanog, popularly used markers for identifying iPS cells, are expressed in colonies other than iPS cells, suggesting that Sox2 and Cdx2 are reliable markers for identifying iPS cells. Our findings indicate that morphologic parameters and the expression of endogenous Sox2 and Cdx2 can be used to accurately identify iPS cells.

Correction: Efficient Generation of Fully Reprogrammed Human iPS Cells via Polycistronic Retroviral Vector and a New Cocktail of Chemical Compounds

Correction: Efficient Generation of Fully Reprogrammed Human iPS Cells via Polycistronic Retroviral Vector and a New Cocktail of Chemical Compounds

Efficient generation of fully reprogrammed human iPS cells via polycistronic retroviral vector and a new cocktail of chemical compounds.

Direct reprogramming of human somatic cells into induced pluripotent stem (iPS) cells by defined transcription factors (TFs) provides great potential for regenerative medicine and biomedical research. This procedure has many challenges, including low reprogramming efficiency, many partially reprogrammed colonies, somatic coding mutations in the genome, etc. Here, we describe a simple approach for generating fully reprogrammed human iPS cells by using a single polycistronic retroviral vector expressing four human TFs in a single open reading frame (ORF), combined with a cocktail containing three small molecules (Sodium butyrate, SB431542, and PD0325901). Our results demonstrate that human iPS cells generated by this approach express human ES cells markers and exhibit pluripotency demonstrated by their abilities to differentiate into the three germ layers in vitro and in vivo. Notably, this approach not only provides a much faster reprogramming process but also significantly diminishes partially reprogrammed iPS cell colonies, thus facilitating efficient isolation of desired fully reprogrammed iPS cell colonies.

417. Simultanous Transfer of the p16INK4a and p21Waf1 Tumor Suppressor Genes Plus GFP Mediated by a Novel Polycistronic Retroviral Vector

The disregulated growth of cancer cells is associated with the hyperphosphorylation of the retinoblastoma gene product, Rb. In the majority of tumors, loss of Rb function is associated with the alteration of expression of CDK inhibitors, such as p16INKa and p21Waf1, but not the loss of Rb itself. Therefore, we have focused our efforts on regaining control of Rb function by the introduction of p16INK4a and p21Waf1 in tumor cells, maintaing Rb in the active, growth-inhibiting form.

Improvement of combination chemotherapy tolerance by introduction of polycistronic retroviral vector drug resistance genes MGMT and MDR1 into human umbilical cord blood CD34 + cells

We obtained a full-length cDNA fragment encoding human O 6-methylguanine-DNA-methyltransferase (MGMT) from the liver tissue of a patient with cholelithiasis by RT-PCR and confirmed by DNA sequencing. The polycistronic retrovirus vector G1Na-MGMT-Neo r-IRES-MDR1 was constructed and verified by restriction endonuclease analysis and DNA sequencing. The vector was transfected into packaging cells GP+E86 and PA317 by the LipofectAMINE method. Cord blood CD34 + cells were transfected with the supernatant of retrovirus containing human MGMT and MDR1cDNA under stimulation of hematopoietic growth factors. PCR, RT-PCR, Southern Blot, Western Blot, FACS and MTT analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34 + cells and expressed efficiently. The transgene cord blood CD34 + cells conferred 5.8–6.3-fold stronger resistance to P-glycoprotein effluxed drugs and 5-fold to BCNU than untransduced cells. The polycistronic retrovirus vector mediated transfer of two different types of drug resistance genes into human cord blood CD34 + cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in clinical practice.

Efficient tumor regression induced by genetically engineered tumor cells secreting interleukin-2 and membrane-expressing allogeneic MHC class I antigen.

To analyze the immunotherapeutic potentials of genetically engineered tumor cells secreting IL-2 and a membrane-expressing allogeneic MHC class I molecule Kb in a murine hepatoma model. In order to express both genes in coordination in the target cells, we constructed a polycistronic retroviral vector containing Kb, IL-2, and NeoR genes using two internal ribosome entry sites (IRES). Tumor growth was carried out by implantation of transduced tumor cells into mouse, while anti-tumor effects were demonstrated by the treatment of established tumors. The infiltrated cells were analyzed by immunohistochemistry. The combined effect of IL-2 secretion and alloantigen expression on immunostimulation was demonstrated by the rejection of transduced tumor cells. In the treatment of established tumors, the Kb/IL-2 co-expressing tumor cells induced strong anti-tumor immunity, superior to that induced by the single gene-transduced cells. The increased diversity of infiltrated cell types in tumor sites indicated that both a specific and non-specific immune response had been activated. Our study provides evidence that tumor cells with IL-2 secretion and membrane-expression of allogeneic MHC class I antigen are capable of inducing both strong tumor rejection and immunity.

Interleukin-12 (IL-12) gene therapy of leukemia: immune and anti-leukemic effects of IL-12-transduced hematopoietic progenitor cells.

Recombinant interleukin-12 (rlL-12) is a potent immunomodulatory cytokine that has been shown to exert strong antitumoral and antimetastatic activity against several mouse tumors grown as solid lesions. The therapeutic efficacy of rIL-12 against hematological tumors and the transfer of IL-12 genes into hematopoietic progenitor cells to deliver IL-12 to the bone marrow (BM) to treat residual leukemia has not been studied adequately. We have investigated the retroviral-mediated transduction of hematopoietic progenitor cells with IL-12 genes and the in vivo anti-leukemic activity of transduced cells against the murine myeloid leukemia cell line 32Dp210. We were able to efficiently transduce the IL-3-dependent 32Dc13 myeloid progenitor cell line and primary hematopoietic progenitor cells using an MFG-based polycistronic retroviral vector containing the cDNAs of p35 and p40 murine IL-12 genes. 32Dc13 myeloid progenitor cells expressing IL-12 genes (32DIL-12 cells) have stably secreted biologically active murine IL-12 for >9 months. Mice transplanted with 32DIL-12 cells transiently express the transgene in the BM and spleen, which is associated with a rapid elevation of interferon-gamma (IFN-gamma) in the circulation and with secretion of IFN-gamma by spleen cells in vitro. In addition, spleen and BM cells of mice injected with 32DIL-12 cells readily acquire the capacity to lyse natural killer cell-sensitive YAC-1 target cells and 32Dp210 myeloid leukemia cells. Furthermore, whereas mice challenged with leukemia cells suffered 100% mortality within 14 days, approximately 40% of mice coinjected with 32Dp210 leukemia cells and 32DIL-12 progenitor cells exhibited long-term, leukemia-free survival (>60 days). This study demonstrates that IL-12 can be stably expressed in hematopoietic cells; in addition, when transplanted, transduced cells induce IFN-gamma production and activation of natural killer cells, both of which may be involved in inhibiting the progression of leukemia in vivo.

Expression of swine MHC class II genes in a cynomolgus monkey: Retro virus‐mediated gene therapy in a preclinical transplantation model

Abstract: Immune reactivity against products of the major histocompatibility complex (MHC) is the major barrier to allotransplantation. Conversely, sharing of MHC class II antigens I appears to be of overwhelming importance in permitting the induction of immune tolerance to vascularized organ allografts, as demonstrated previously in miniature swine. Class II antigen has also been shown to be recognized predominantly in human anti‐pig xenoreactions in vitro. To achieve tolerance in the xenogeneic pig‐to‐primate model, we are therefore investigating an approach involving retrovirus‐mediated gene therapy to transfer swine MHC (SLA) class II genes into primate primitive hematopoietic stem cells, so that swine MHC class II antigens may participate in the education of the recipient's newly developing T cell repertoire. We report here the in vitro and in vivo use of a recombinant retrovirus containing a polycistronic retroviral vector which carries swine MHC class II DRA and DRB cDNA sequences to transduce CD34+ bone marrow cells (BMC) from a cynomolgus monkey. Transduction efficiency was assessed by reverse transcriptase‐polymerase chain reaction analysis of the colony‐forming unit progenitor colonies grown in the absence of gentamycin; 55% and 24% of the progenitor colonies were determined to express the retroviral transcript at 1 week and 3 weeks post‐transduction, respectively. These in vitro studies have been extended to the transplantation of retrovirally transduced autologous stem cells into a cynomolgus monkey prepared with a non‐myeloablative conditioning regimen. Prolonged expression of SLA‐DR transcripts in monkey peripheral blood mononuclear cells (PBMC) has been documented over a 56‐week period after transplantation of retrovirus‐transduced bone marrow cells. However, we could not detect any protein expression by FACS analysis on the surface of primate PBMC or bone marrow, using a porcine SLA‐DR‐specific antibody. Engraftment of hematopoietic cells with the transduced genes was further detected in the progenitor colonies grown from the bone marrow cells harvested at 4 weeks and 25 weeks after bone marrow transplantation. Our results thus document that long‐term engraftment of retrovirally transduced hematopoietic cells can be achieved in a primate model using a non‐myeloablative preparative I regimen.

A polycistronic retrovirus vector for expression of swine MHC class II DRaα/β heterodimers

Abstract: A recombinant polycistronic retroviral vector was generated to express multimeric proteins from a single transcript by using internal ribosomal entry sites which allow independent initiation of translation from internal cistrons. In addition, an enhancerless SV40 origin of replication was incorporated into the recombinant plasmids between the retroviral long terminal repeat sequences to facilitate the testing of expression of multiple proteins in a transient COS cell transfection assay.With the ultimate goal of utilizing these vector types for the induction of transplantation tolerance via retrovirus‐mediated gene transfer (LeGuern et al. J Mol Med 1995: 42: 269 [1]; Sachs Transplant Sci 1993: 42: 59 [2]) we have incorporated miniature swine MHC class II DRA and DRB cDNAs which encode for the SLA‐DRα and SLA‐DRβ polypeptide chains, respectively, into the retroviral construct. The pro viral DNA integrated within the producer clone genome, was found to be stable over a 3‐month period and no loss of expression of DR heterodimers was observed on the cell surface of either the retrovirus producer cells or the retrovirally transduced fibroblast cell line. High titers (>1 times 106 CFU/ml) of recombinant particles, devoid of replication competent helper viruses, were obtained. Such vectors represent the first successful attempt to express multimeric proteins at the cell surface of transduced cells.

Effective eradication of established murine tumors with IL-12 gene therapy using a polycistronic retroviral vector.

Our recent studies using IL-12 protein or fibroblasts genetically engineered to secrete IL-12 have demonstrated profound antitumor effects of IL-12 in murine models. The antitumor effects of local, high level IL-12 expression were examined using a retroviral vector, which can express both IL-12 subunits (p35 and p40) and the neomycin phosphotransferase (Neo)-marker gene from a polycistronic message utilizing internal ribosome entry site sequences. All animals intradermally (i.d.) receiving MCA207 murine sarcoma cell line nontransfected or Neo-transfected had progressively growing tumor, whereas all animals injected with MCA207 transfected with IL-12 were tumor free and were subsequently determined to be immune to a rechallenge of nontransfected MCA207 i.d. Similar results were obtained in experiments using the poorly immunogenic MCA102 murine sarcoma cell line. The inoculation of live MCA207-IL-12 tumor cells also caused the regression of contralateral nontransfected MCA207 inoculated either at the same time (80% protection) or up to 3 days before (33% protection) to the therapeutic tumor inoculation. In vivo depletion studies suggest that NK cells and IFN-gamma play important roles in the development of the early phase of the antitumor response, but that T cells (both CD4+ and CD8+) play the major role in the subsequent events, leading to long-term immunity. The potent antitumor effects observed for paracrine gene-delivered administration of IL-12 have thus been confirmed for multiple tumor cell types and in multiple murine strains. We believe that these results support the feasibility of IL-12 gene therapy for the treatment of human cancer.

Induction of anti-tumor immunity by mouse tumor cells transfected with mouse interleukin-12 gene.

Interleukin-12 (IL-12) is a heterodimeric cytokine. In order to transduce both cDNAs for p35 and p40 of IL-12 in the tumor cells, a polycistronic retroviral vector was constructed by inserting the internal ribosome entry site gene of encephalomyocarditis virus between two cDNAs. On the other hand, two cDNAs were sequentially transfected in the tumor cells. Both polycistronic gene transfectants and double transfectants produced biologically active mouse IL-12. IL-12-expressing tumor cells were all rejected in syngeneic mice, and induced cytotoxic T lymphocyte activity. The capacity to induce anti-tumor memory may depend on the amount of IL-12 produced by the transfectants, because the relatively higher IL-12 producer tumor cell line induced the anti-tumor memory in the rejected mice, but the lower producer did not.

CD4 T cells inhibit in vivo the CD8‐mediated immune response against murine colon carcinoma cells transduced with interleukin‐12 genes

Retroviral-mediated cytokine gene transfer into tumor cells is a highly effective way of inducing tumor inhibition and immunity. We analyzed the tumorigenicity of C-26 murine colon carcinoma cells transduced with genes encoding the two subunits of murine interleukin-12 (IL-12) in a polycistronic retroviral vector and selected for resistance to G418 and for IL-12 production (30-80 pg/ml). BALB/c mice injected s.c., i.v. and intrasplenically with C-26/IL-12 cells from three different IL-12-producing clones showed delayed tumor onset as compared with mice injected with control NeoR-transduced or parental tumor cells. Although C-26/IL-12 tumor-bearing mice eventually died of lung metastasis, their survival time was twice as long as that of mice injected with control cells. In experiments with mice selectively depleted of natural killer (NK) cells before tumor cell injection, the time of tumor onset and survival of mice injected with C-26/IL-12 s.c. and i.v., respectively, was reduced. CD8+ T cell depletion had no effect on latency or survival, whereas removal of CD4+ T cells led to C-26/IL-12 tumor regression in about 40% of mice. Histological and immunocytochemical characterization of leukocytes infiltrating C-26/IL-12 tumors showed only slight infiltration with few T cells in non-depleted mice but abundant infiltration by CD8+ T cells and asialo-GM1+ NK cells in tumors of mice depleted of CD4+ T cells. The lack of CD8+ T cell infiltration is not due to a CD4-mediated suppression of their activation because irradiated C-26/IL-12 cells primed for the induction of a strong cytotoxic T lymphocyte response against C-26 parental cells and induced CD8+ effector cells that protected against C-26/IL-12 in a Winn assay. Rather, the results suggest that, although C-26/IL-12 cells injected in vivo stimulate both NK and CD8+ T cells, tumor infiltration by the latter is inhibited by CD4+ T cells.