The role of telomerase and viruses interaction in cancer development, and telomerase-dependent therapeutic approaches.

Human telomerase reverse transcriptase (hTERT) is an enzyme that is critically involved in elongating and maintaining telomeres length to control cell life span and replicative potential. Telomerase activity is continuously expressed in human germ-line cells and most cancer cells, whereas it is suppressed in most somatic cells. In normal cells, by reducing telomerase activity and progressively shortening the telomeres, the cells progress to the senescence or apoptosis process. However, in cancer cells, telomere lengths remain constant due to telomerase's reactivation, and cells continue to proliferate and inhibit apoptosis, and ultimately lead to cancer development and human death due to metastasis. Studies demonstrated that several DNA and RNA oncoviruses could interact with telomerase by integrating their genome sequence within the host cell telomeres specifically. Through the activation of the hTERT promoter and lengthening the telomere, these cells contributes to cancer development. Since oncoviruses can activate telomerase and increase hTERT expression, there are several therapeutic strategies based on targeting the telomerase of cancer cells like telomerase-targeted peptide vaccines, hTERT-targeting dendritic cells (DCs), hTERT-targeting gene therapy, and hTERT-targeting CRISPR/Cas9 system that can overcome tumor-mediated toleration mechanisms and specifically apoptosis in cancer cells. This study reviews available data on the molecular structure of telomerase and the role of oncoviruses and telomerase interaction in cancer development and telomerase-dependent therapeutic approaches to conquest the cancer cells.

Intracellular prodrug gene therapy for cancer mediated by tumor cell suicide gene exosomes.

Recently, we reported about exosomes possessing messenger RNA (mRNA) of suicide gene secreted from mesenchymal stem/stromal cells (MSCs) engineered to express the suicide gene-fused yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT). The yCD::UPRT-MSC exosomes are internalized by tumor cells and intracellularly convert prodrug 5-fluorocytosine (5-FC) to cytotoxic drug 5-fluorouracil (5-FU). Human tumor cells with the potential to metastasize release exosomes involved in the creation of a premetastatic niche at the predicted organs. We found that cancer cells stably transduced with yCD::UPRT gene by retrovirus infection released exosomes acting similarly like yCD::UPRT-MSC exosomes. Different types of tumor cells were transduced with the yCD::UPRT gene. The homogenous cell population of yCD::UPRT-transduced tumor cells expressed the yCD::UPRT suicide gene and secreted continuously exosomes with suicide gene mRNA in their cargo. All tumor cell suicide gene exosomes upon internalization into the recipient tumor cells induced the cell death by intracellular conversion of 5-FC to 5-FU and to 5-FUMP in a dose-dependent manner. Most of tumor cell-derived suicide gene exosomes were tumor tropic, in 5-FC presence they killed tumor cells but did not inhibit the growth of human skin fibroblast as well as DP-MSCs. Tumor cell-derived suicide gene exosomes home to their cells of origin and hold an exciting potential to become innovative specific therapy for tumors and potentially for metastases.

Mesenchymal Stem Cell Exosome-Mediated Prodrug Gene Therapy for Cancer.

Exosomes derived from human mesenchymal stem cells (MSCs) engineered to express the suicide gene yeast cytosine deaminase::uracil phosphoribosyl transferase (yCD::UPRT) represent a new therapeutic approach for tumor-targeted innovative therapy. The yCD::UPRT-MSC-exosomes carry mRNA of the suicide gene in their cargo. Upon internalization by tumor cells, the exosomes inhibit the growth of broad types of cancer cells in vitro, in the presence of a prodrug. Here we describe the method leading to the production and testing of these therapeutic exosomes. The described steps include the preparation of replication-deficient retrovirus possessing the yCD::UPRT suicide gene, and the preparation and selection of MSCs transduced with yCD::UPRT suicide gene. We present procedures to obtain exosomes possessing the ability to induce the death of tumor cells. In addition, we highlight methods for the evaluation of the suicide gene activity of yCD::UPRT-MSC-exosomes.

Influence of Retronectin-Mediated T-Cell Activation on Expansion and Phenotype of CD19-Specific Chimeric Antigen Receptor T Cells.

Enhanced in vivo expansion, long-term persistence of chimeric antigen receptor T (CART) cells, and efficient tumor eradication through these cells are linked to the proportion of less-differentiated cells in the CART cell product. Retronectin is well established as an adjuvant for improved retroviral transduction, while its property to enrich less-differentiated T cells is less known. In order to increase these subsets, this study investigated the effects of retronectin-mediated T-cell activation for CD19-specific CART cell production. Peripheral blood mononuclear cells of healthy donors and untreated chronic lymphocytic leukemia (CLL) patients without or with positive selection for CD3+ T cells were transduced with a CD19.CAR.CD28.CD137zeta third-generation retroviral vector. Activation of peripheral blood mononuclear cells was performed by CD3/CD28, CD3/CD28/retronectin, or CD3/retronectin. Interleukin-7 and -15 were supplemented to all cultures. Retronectin was used in all three activation protocols for retroviral transduction. Expansion was assessed by trypan blue staining. Viability, transduction efficiency, immune phenotype, and cytokine production were longitudinally analyzed by flow cytometry. Cytotoxic capacity of generated CART cells was evaluated using a classical chromium-51 release assay. Retronectin-mediated activation resulted in an enrichment of CD8+ cytotoxic CART cells and less-differentiated naïve-like T cells (CD45RA+CCR7+). Retronectin-activated CART cells showed increased cytotoxic activity. However, activation with retronectin decreased viability, expansion, transduction efficiency, and cytokine production, particularly of CLL patient-derived CART cells. Both retronectin-mediated activation protocols promoted a less-differentiated CART cell phenotype without comprising cytotoxic properties of healthy donor-derived CART cells. However, up-front retronectin resulted in reduced viability and expansion in CLL patients. This effect is probably attributed to the retronectin-mediated activation of B cells with prolonged CLL persistence. Consequently, CART cell expansion and generation failed. In summary, activation with retronectin should be performed with caution and may be limited to patients without a higher percentage of tumor cells in the peripheral blood.

In vivo reprogramming reactive glia into iPSCs to produce new neurons in the cortex following traumatic brain injury.

Traumatic brain injury (TBI) results in a significant amount of cell death in the brain. Unfortunately, the adult mammalian brain possesses little regenerative potential following injury and little can be done to reverse the initial brain damage caused by trauma. Reprogramming adult cells to generate induced pluripotent stem cell (iPSCs) has opened new therapeutic opportunities to generate neurons in a non-neurogenic regions in the cortex. In this study we showed that retroviral mediated expression of four transcription factors, Oct4, Sox2, Klf4, and c-Myc, cooperatively reprogrammed reactive glial cells into iPSCs in the adult neocortex following TBI. These iPSCs further differentiated into a large number of neural stem cells, which further differentiated into neurons and glia in situ, and filled up the tissue cavity induced by TBI. The induced neurons showed a typical neuronal morphology with axon and dendrites, and exhibited action potential. Our results report an innovative technology to transform reactive glia into a large number of functional neurons in their natural environment of neocortex without embryo involvement and without the need to grow cells outside the body and then graft them back to the brain. Thus this technology offers hope for personalized regenerative cell therapies for repairing damaged brain.

Cynomolgus Monkey Induced Pluripotent Stem Cells Generated By Using Allogeneic Genes.

Induced pluripotent stem (iPS) cells that are potentially similar to embryonic stem (ES) cells can be artificially established by introduction into somatic cells of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4, and c-MYC. In cynomolgus monkeys (Macaca fascicularis), iPS cells generated by using these four allogeneic transgenes should be an important resource for various types of biomedical research because the use of xenogeneic transgenes may cause complications. To establish such iPS cells, cynomolgus monkey somatic cells were infected with amphotropic retroviral vectors, which were derived from Plat-A cells, containing cDNA for the cynomolgus monkey genes POU5F1, SOX2, KLF4, and c-MYC. As a result, iPS cells could be established from somatic cells from fetal liver and newborn skin of cynomolgus monkeys, similarly to the case for mouse and human somatic cells.

Lipoprotein lipase is an important modulator of lipid uptake and storage in hypothalamic neurons.

LPL is the rate-limiting enzyme for uptake of TG-derived FFA in peripheral tissues, and the enzyme is expressed in the brain and CNS. We previously created a mouse which lacks neuronal LPL. This animal becomes obese on a standard chow, and we observed reduced lipid uptake in the hypothalamus at 3 months preceding obesity. In our present study, we replicated the animal phenotype in an immortalized mouse hypothalamic cell line (N41) to examine how LPL affects expression of AgRP as well as entry and storage of lipids into neurons. We show that LPL is able to modulate levels of the orexigenic peptide AgRP. LPL also exerts effects on lipid uptake into culture neurons, and that uptake of neutral lipid can be enhanced even by mutant LPL lacking catalytic activity. N41 cells also accumulate neutral lipid in droplets, and this is at least in part regulated by LPL. These data in addition to those published in mice with neuron-specific deletion of LPL suggest that neuronal LPL is an important regulator of lipid homeostasis in neurons and that alterations in LPL levels may have important effects on systemic metabolism and neuronal lipid biology.

Tanshinone IIA enhances bystander cell killing of cancer cells expressing Drosophila melanogaster deoxyribonucleoside kinase in nuclei and mitochondria.

Heterologous expression of the Drosophila melanogaster multi-substrate deoxyribonucleoside kinase (Dm-dNK) increases the sensitivity of cancer cells to several cytotoxic nucleoside analogs. Thus, it may be used as a suicide gene in combined gene/chemotherapy treatment of cancer. To further characterize this potential suicide gene, we constructed two retroviral vectors that enabled the expression of Dm-dNK in cancer cells. One vector harbored the wild­type enzyme that localized to the nucleus. The other vector harbored a mitochondrial localized mutant enzyme that was constructed by deleting the nuclear localization signal and fusing it to a mitochondrial import signal of cytochrome c oxidase. A thymidine kinase-deficient osteosarcoma cell line was transduced with the recombinant viruses. The sensitivity and bystander cell killing in the presence of pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)­2'­deoxyuridine and 1-ß-D-arabinofuranosylthymine were investigated. Tanshinone IIA is a constituent of Danshen; a traditional Chinese medicine used in the treatment of cardiovascular diseases. This study also looked at the influence of Tanshinone IIA on the bystander effect and the underlying mechanisms. We showed that sensitivity of the osteosarcoma cell line to the nucleoside analogs and the efficiency of bystander cell killing were independent of the subcellular localization of Dm-dNK. The enhanced effect of tanshinone IIA on the bystander effect was related to the increased expression of Cx43 and Cx26.

Intravenous administration of retroviral replicating vector, Toca 511, demonstrates therapeutic efficacy in orthotopic immune-competent mouse glioma model.

Toca 511 (vocimagene amiretrorepvec), a nonlytic, amphotropic retroviral replicating vector (RRV), encodes and delivers a functionally optimized yeast cytosine deaminase (CD) gene to tumors. In orthotopic glioma models treated with Toca 511 and 5-fluorocytosine (5-FC) the CD enzyme within infected cells converts 5-FC to 5-fluorouracil (5-FU), resulting in tumor killing. Toca 511, delivered locally either by intratumoral injection or by injection into the resection bed, in combination with subsequent oral extended-release 5-FC (Toca FC), is under clinical investigation in patients with recurrent high-grade glioma (HGG). If feasible, intravenous administration of vectors is less invasive, can easily be repeated if desired, and may be applicable to other tumor types. Here, we present preclinical data that support the development of an intravenous administration protocol. First we show that intravenous administration of Toca 511 in a preclinical model did not lead to widespread or uncontrolled replication of the RVV. No, or low, viral DNA was found in the blood and most of the tissues examined 180 days after Toca 511 administration. We also show that RRV administered intravenously leads to efficient infection and spread of the vector carrying the green fluorescent protein (GFP)-encoding gene (Toca GFP) through tumors in both immune-competent and immune-compromised animal models. However, initial vector localization within the tumor appeared to depend on the mode of administration. Long-term survival was observed in immune-competent mice when Toca 511 was administered intravenously or intracranially in combination with 5-FC treatment, and this combination was well tolerated in the preclinical models. Enhanced survival could also be achieved in animals with preexisting immune response to vector, supporting the potential for repeated administration. On the basis of these and other supporting data, a clinical trial investigating intravenous administration of Toca 511 in patients with recurrent HGG is currently open and enrolling.

Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery.

Designer nucleases, like zinc-finger nucleases (ZFNs), represent valuable tools for targeted genome editing. Here, we took advantage of the gamma-retroviral life cycle and produced vectors to transfer ZFNs in the form of protein, mRNA and episomal DNA. Transfer efficacy and ZFN activity were assessed in quantitative proof-of-concept experiments in a human cell line and in mouse embryonic stem cells. We demonstrate that retrovirus-mediated protein transfer (RPT), retrovirus-mediated mRNA transfer (RMT), and retrovirus-mediated episome transfer (RET) represent powerful methodologies for transient protein delivery or protein expression. Furthermore, we describe complementary strategies to augment ZFN activity after gamma-retroviral transduction, including serial transduction, proteasome inhibition, and hypothermia. Depending on vector dose and target cell type, gene disruption frequencies of up to 15% were achieved with RPT and RMT, and >50% gene knockout after RET. In summary, non-integrating gamma-retroviral vectors represent a versatile tool to transiently deliver ZFNs to human and mouse cells.

Generation of human induced pluripotent stem (Ips) cells in serum- and feeder-free defined culture and TGF-Β1 regulation of pluripotency.

Human Embryonic Stem cells (hESCs) and human induced Pluripotent Stem cells (hiPSCs) are commonly maintained on inactivated mouse embryonic fibroblast as feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture medium containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. In addition, there is no consensus as to the optimal formulation, or the nature of the cytokine requirements of the cells to promote their self-renewal and inhibit their differentiation. In this study, we successfully generated hiPSCs from human dental pulp cells (DPCs) using Yamanaka's factors (Oct3/4, Sox2, Klf4, and c-Myc) with retroviral vectors in serum- and feeder-free defined culture conditions. These hiPSCs retained the property of self-renewal as evaluated by the expression of self-renewal marker genes and proteins, morphology, cell growth rates, and pluripotency evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo. In this study, we found that TGF-ß1 increased the expression levels of pluripotency markers in a dose-dependent manner. However, increasing doses of TGF-ß1 suppressed the growth rate of hiPSCs cultured under the defined conditions. Furthermore, over short time periods the hiPSCs cultured in hESF9 or hESF9T exhibited similar morphology, but hiPSCs maintained in hESF9 could not survive beyond 30 passages. This result clearly confirmed that hiPSCs cultured in hESF9 medium absolutely required TGF-ß1 to maintain pluripotency. This simple serum-free adherent monoculture system will allow us to elucidate the cell responses to growth factors under defined conditions and can eliminate the risk might be brought by undefined pathogens.

Retrocyte Display® technology: generation and screening of a high diversity cellular antibody library.

Over the last nearly three decades in vitro display technologies have played an important role in the discovery and optimization of antibodies and other proteins for therapeutic applications. Here we describe the use of retroviral expression technology for the display of full-length IgG on B lineage cells in vitro with a hallmark of a tight and stable genotype to phenotype coupling. We describe the creation of a high-diversity (>1.0E09 different heavy- and light-chain combinations) cell displayed fully human antibody library from healthy donor-derived heavy- and light-chain gene libraries, and demonstrate the recovery of high affinity target-specific antibodies from this library by staining of cells with a labeled target antigen and their magnetic- and flow cytometry-based cell sorting. The present technology represents a further evolution in the discovery of full-length, fully human antibodies using mammalian display, and is termed Retrocyte Display® (Retroviral B lymphocyte Display).

Trim24-repressed VL30 retrotransposons regulate gene expression by producing noncoding RNA.

Trim24 (Tif1α) and Trim33 (Tif1γ) interact to form a co-repressor complex that suppresses murine hepatocellular carcinoma. Here we show that Trim24 and Trim33 cooperatively repress retinoic acid receptor-dependent activity of VL30-class endogenous retroviruses (ERVs) in liver. In Trim24-knockout hepatocytes, VL30 derepression leads to accumulation of reverse-transcribed VL30 cDNA in the cytoplasm that correlates with activation of the viral-defense interferon responses mimicking the preneoplastic inflammatory state seen in human liver following exogenous viral infection. Furthermore, upon derepression, VL30 long terminal repeats (LTRs) act as promoter and enhancer elements deregulating expression of neighboring genes and generating enhancer RNAs that are required for LTR enhancer activity in hepatocytes in vivo. These data reinforce the role of the TRIM family of proteins in retroviral restriction and antiviral defense and provide an example of an ERV-derived oncogenic regulatory network.

Complementary treatment of siTERT for improving the antitumor effect of TERT-specific I-131 therapy.

Sodium iodide symporter (NIS)-based radionuclide therapy provides an effective means of treating malignant tumors. However, it is sometimes inadequate because of limited effects on radio-resistant tumors, and thus, combination therapies with other therapeutic options have been requested to enhance its efficacy. Human telomerase reverse transcriptase (hTERT) has been reported to be involved in the progression of most cancers and also been viewed as a good candidate for targeting tumor. Application of TERT-specific radionuclide therapies using NIS gene transfer have been reported to treat TERT-positive tumors, but this approach only demonstrated tumor regression rather than eradication. As inhibiting TERT expression by introducing the hTERT-specific shRNA (siTERT) has been suggested as a therapeutic option, we investigated the complementary role of siTERT treatment after the TERT-specific I-131 therapy and its possibility as a novel anticancer therapeutic strategy. Retroviruses containing TERT promoter/NIS for TERT specific Radionuclide therapy and siTERT for TERT targeting antisense therapy were produced. Hep3B cells expressing TERT specific NIS (Hep3B-TERT/NIS) were xenografted into nude mouse and visualized with micro-SPECT/CT for monitoring NIS activity. The levels of hTERT mRNA, protein and its activity were confirmed by RT-PCR, Western blotting and Telomerase repeat amplification protocol assay. Cell proliferation was monitored by MTT assay and induced apoptosis was confirmed by Annexin-V-PI staining. Therapeutic effects of I-131 and/or siTERT were evaluated by clonogenic assay and mouse tumor model. Reduction of hTERT mRNA, protein and TERT activity by siTERT were observed in Hep3B-TERT/NIS cells. The viabilities of the infected cells were significantly decreased to 50% versus siScramble treated controls. The early apoptotic cell population was increased by siTERT. The survival rates of cells treated with siTERT or I-131 alone were 72.4±7.6% and 56.2±5.2%, respectively. However, the survival rate of cells treated with I-131 and siTERT were decreased to 22.1±2.8%. From mouse xenograft model, we also found that the siTERT gene therapy showed synergism to the radioiodine therapy for reducing tumor growth in vivo. Our Results suggested that complementary siTERT gene therapy offers a novel strategy of cancer therapy to improve the therapeutic efficacy of TERT-specific I-131.

Creation and validation of a ligation-independent cloning (LIC) retroviral vector for stable gene transduction in mammalian cells.

BACKGROUND: Cloning vectors capable of retroviral transduction have enabled stable gene overexpression in numerous mitotic cell lines. However, the relatively small number of feasible restriction enzyme sequences in their cloning sites can hinder successful generation of overexpression constructs if these sequences are also present in the target cDNA insert. RESULTS: Utilizing ligation-independent cloning (LIC) technology, we have modified the highly efficient retroviral transduction vector, pBABE, to eliminate reliance on restriction enzymes for cloning. Instead, the modified plasmid, pBLIC, utilizes random 12/13-base overhangs generated by T4 DNA polymerase 3' exonuclease activity. PCR-based introduction of the complementary sequence into any cDNA of interest enables universal cloning into pBLIC. Here we describe creation of the pBLIC plasmid, and demonstrate successful cloning and protein overexpression from three different cDNAs, Bax, catalase, and p53 through transduction into the human prostate cancer cell line, LNCaP or the human lung cancer line, H358. CONCLUSIONS: Our results show that pBLIC vector retains the high transduction efficiency of the original pBABE while eliminating the requirement for checking individual cDNA inserts for internal restriction sites. Thus it comprises an effective retroviral cloning system for laboratory-scale stable gene overexpression or for high-throughput applications such as creation of retroviral cDNA libraries. To our knowledge, pBLIC is the first LIC vector for retroviral transduction-mediated stable gene expression in mammalian cells.

[Establishment of a novel cell model targeted on FGF-21 receptor for screening anti-diabetic drug candidates].

The aim of this project is to establish a fibroblast growth factor-21 (FGF-21) signaling pathway targeted cell model, for screening a class of FGF-21 receptor agonists as anti-diabetic candidates. FGF-21 requires beta klotho transmembrane proteins as co-receptor for the activation of tyrosine kinase FGF receptor (FGFR) signaling, thereby activating a series of intracellular signaling pathways and regulating gene transcription for glucose metabolism. Firstly a recombinant plasmid expressing co-receptor beta klotho and EGFP reporter genes was constructed. After introducing the recombinant plasmid into package cells, the cell culture supernatant was used to infect 3T3-L1 cells, which were then screened for stably expressing beta klotho gene. Administration of FGF-21 increased the expression of GLUT1 and stimulated GLUT1-mediated glucose uptake. This novel cell model can be conveniently used in high-throughput drug screening of FGF-21 or FGF-21 analogues.

Comparative evaluation of preclinical in vivo models for the assessment of replicating retroviral vectors for the treatment of glioblastoma.

Despite impressive improvements in neurosurgical techniques, radiation and chemotherapy during the past few years, little progress has been made in the treatment of malignant gliomas. Recently, the efficacy of suicide gene therapy based on replication-competent retroviral (RCR) vectors as delivery vehicles for the therapeutic gene has been described in the treatment of experimental cancer, including gliomas. In this study, we have thus critically evaluated a panel of human and rodent glioma/glioblastoma cell lines (U-87MG, U-118MG, LN-18, LN-229, 8-MG-BA, 42-MG-BA, A-172, T-98G, UVW, C6, 9L, G-26, GL-261, Tu-2449, Tu-9648) with respect to RCR virus vector spread, sensitivity towards the cytosine deaminase (CD)/5-flurocytosine (5-FC)/5-flurouracil (5-FU) suicide system, and orthotopic growth characteristics in mice to identify suitable preclinical animal models for the development of a glioblastoma gene therapy. Rapid virus spread was observed in eight out of nine human cell lines tested in vitro. As expected, only CD-expressing cells became sensitive to 5-FC, due to their ability to convert the prodrug in its toxic form, 5-FU. All LD(50) values were within the range of concentrations obtained in human body fluids after conventional antifungal 5-FC administration. In addition, a significant bystander effect was observed in all human glioma cell lines tested. Injection of the RCR vector into pre-established orthotopic mouse tumor xenografts revealed substantial infection and virus spread of tumor tissue from most cell types.

In vivo expansion of MDR1-transduced cells accompanied by a post-transplantation chemotherapy regimen with mitomycin C and methotrexate.

BACKGROUND: A recurrent breast cancer patient received high-dose chemotherapy, a transplant of multidrug resistance 1 (MDR1)-transduced cells and four different protocols of post-transplantation chemotherapy. We report the analysis of MDR1-transduced cells in this patient. METHODS: MDR1 transgene levels in the peripheral blood mononuclear cells of the patient were evaluated by polymerase chain reaction (PCR). Retroviral integration sites of the MDR1-transduced cells were identified by linear amplification-mediated (LAM)-PCR. RESULTS: Twelve days after transplantation, approximately 1% of the peripheral blood mononuclear cells were MDR1 transgene-positive. The transgene levels decreased quickly, and were at low levels until day 504. A remarkable increase in MDR1 transgene-positive cells was observed on day 532, during combination chemotherapy with mitomycin C and methotrexate. Using LAM-PCR, 31 MDR1-transduced clones were identified, and eight of these were long-life clones that survived for more than 500 days. Among the 31 clones, ten had a retroviral integration site near genes listed in the Retroviral Tagged Cancer Gene (RTCG) Database. Two long-life clones, N-30 and N-31, had retroviral integration sites within the MDS1-EVI1 locus. Another two long-life clones had integration sites close to PRDM16 or CUEDC1. CONCLUSIONS: These results suggest that MDR1-transduced cells were enriched in vivo by an MDR1 substrate, mitomycin C. The possible activation of EVI1 or other RTCGs by retroviral insertion may have affected the survival and persistence of a proportion of the transduced cells.

Embryonic stem cells use ZFP809 to silence retroviral DNAs.

Embryonic stem cells (ESCs) and other primitive stem cells of mice have been known for more than 30 years to potently block retrovirus replication. Infection of ESCs by the murine leukaemia viruses (MLVs) results in the normal establishment of integrated proviral DNA, but this DNA is then transcriptionally silenced, preventing further viral spread. The repression is largely mediated by trans-acting factors that recognize a conserved sequence element termed the primer binding site, an 18-base pair sequence complementary to the 3' end of a cellular transfer RNA. A specific tRNA is annealed to the primer binding site sequence of the viral genomic RNA, and is used to prime DNA synthesis. This same sequence in the context of the integrated proviral DNA is targeted for silencing in ESCs. We have recently shown that a large protein complex binding to the primer binding site in ESCs contains TRIM28 (refs 8, 9), a well-characterized transcriptional co-repressor. An important question remains as to the identity of the factor that directly recognizes integrated retroviral DNAs and recruits TRIM28 to mediate their specific silencing. Here we identify the zinc finger protein ZFP809 as the recognition molecule that bridges the integrated proviral DNA and TRIM28. We show that expression of ZFP809 is sufficient to render even differentiated cells highly resistant to MLV infection. Furthermore, we demonstrate that ZFP809 is able to potently block transcription from DNA constructs of human T-cell lymphotropic virus-1 (HTLV-1), which use the same primer tRNA. These results identify ZFP809 as a DNA-binding factor that specifically recognizes a large subset of mammalian retroviruses and retroelements, targeting them for transcriptional silencing. We propose that ZFP809 evolved as a stem-cell-specific retroviral restriction factor, and therefore constitutes a new component of the intrinsic immune system of stem cells.

RNA modulators of complex phenotypes in mammalian cells.

RNA-mediated gene silencing, in the form of RNA interference (RNAi) or microRNAs (miRNAs) has provided novel tools for gene discovery and validation in mammalian cells. Here, we report on the construction and application of a random small RNA expression library for use in identifying small interfering RNA (siRNA) effectors that can modify complex cellular phenotypes in mammalian cells. The library is based in a retroviral vector and uses convergent promoters to produce unique small complementary RNAs. Using this library, we identify a range of small RNA-encoding gene inserts that overcome resistance to 5-fluorouracil (5-FU)- or tumour necrosis factor alpha (TNF-alpha)- induced cell death in colorectal cancer cells. We demonstrate the utility of this technology platform by identifying a key RNA effector, in the form of a siRNA, which overcomes cell death induced by the chemotherapeutic 5-FU. The technology described has the potential to identify both functional RNA modulators capable of altering physiological systems and the cellular target genes altered by these modulators.