Retroviral Producer Research Articles

A Functional In Vivo RNAi screen Involving Jumonji C Domain Containing Candidates Unravels Kdm5b As a Negative Modulator of Hematopoietic Stem Cell Self-Renewal

Abstract 385Epigenetic modifications influence chromatin accessibility, impacting on cell fate decisions, such as stem cell self-renewal and differentiation, in both normal and leukemic stem cells (LSC). To investigate the putative role of histone demethylases (HDM) in modulating primary hematopoietic stem cell (HSC) fate, an in vivo functional screen was performed, using an RNAi based strategy, involving 25 members of the Jumonji (JmjC) domain protein family.As a first step, expression profile studies of these gene candidates were undertaken. Transcripts of all these enzymes were detected in isolated HSC populations (frequency 1:2) from fetal liver (n=1) and bone marrow (n=2), except for Hairless. As compared to unsorted bone marrow (BM), stem cells harboured higher expression of Jarid1b (relative-fold enrichment (RQ) of 3.9±1.7), Jmjd2d (RQ3.8±1.9), and Jhdm1b (3.1±1.7). Next, 5shRNA were designed against each of the 25 JmjC containing proteins, and cloned into a retroviral LMP vector encoding GFP to permit tracking of transduced cells in vivo. HSC-enriched CD150+CD48−Lin−cells (∼60 LT-HSC) were infected over 5 days by co-culture with retroviral producer cells in an arrayed 96-well format, with one shRNA per well. Directly after infection, the in vivo reconstituting potential of ¼ of each well was evaluated through duplicate competitive repopulation assays involving the co-transplantation of 1.5 × 105 congenic BM competitor cells into irradiated recipients. The remaining cell fraction served to asses gene transfer by GFP epifluorescence measurements, and RNA isolated from sorted GFP+ cells was used to evaluate gene knockdown levels by Q-RT-PCR analysis. Blood reconstitution was evaluated at an early (4wks) and late time point (16–20wks), tracking the contribution of the donor CD45.1+ transduced (GFP+) cells to recipient hematopoiesis over time. As baseline references, sh-RNA to Luciferase (no effect) and the histone acetyl transferase Myst3 (stem cell loss) were used, as well as Hoxb4 over-expression (stem cell expansion). The primary screen, followed by validation experiments, unravelled one positive (Jhdm1f/Phf8) and two negative (Jarid1b, Hif1an) regulators of HSC activity. The strongest impact was seen with Jarid1b knockdown, and the resulting gain in HSC activity. As a confirmation step, cells were kept in culture for one week, to better contrast an increase in HSC activity, compared to control HSC. After 7 days in vitro, 1/8 equivalents of single well cultures were transplanted into 3 mice, and blood reconstitution levels serially assessed. Cells transduced with sh-RNA against Jarid1b contributed more significantly to host hematopoiesis than sh-RNA Luciferase transduced cells (58±16% vs 26±3% GFP), or Hoxb4 over-expressing cells (37±2% GFP), at comparable gene transfer rates, at the 16 week time point and beyond (3 independent experiments). Long-term HSC frequencies were evaluated from these cultures, and found to be 6–10 fold increased in shJari1d1b-cell cultures. In long-term recipients, differentiation potential of these cells was preserved, as evidenced by CD4+CD8+ thymic cells, B220+ splenic cells and CD11b+ bone marrow cells in the GFP positive contingent. Clonality studies on DNA isolated from these sorted populations confirmed oligoclonality of the stem cell expansion, and HSC pluripotency. There were no cases of leukemic transformation in all of the transplant recipients (n>30). As assessed by Q-RT-PCR, levels of HoxA5, HoxA9, HoxA10 and CxCl5 were increased in day7 sh3Jarib1b-cells (vs ctl), while the levels of the tumor suppressors Cav1, Sash1 and Egr1 were decreased. A more detailed assessment of the HoxA cluster revealed predominant expression of 5’ cluster genes in expanding shJarib1b-cells, from HoxA5 to HoxA11, with a concomitant increase in the level of H3K4 tri-methylation, as assessed by ChIP-CHIP.In conclusion, HDM of the JmjC family can modulate HSC activity, both positively and negatively. These data suggest that the H3K4 demethylase Jarid1b (KDM5b) restrains stem cell self-renewal, acting as a co-repressor, possibly via epigenetic regulation of the HoxA gene cluster, among other target genes. This observation could be further exploited as an HSC expansion strategy. Disclosures:No relevant conflicts of interest to declare.

Poster 11: Exploring the Relationship Between Integrin αvβ6 and αβ1 in Oral Squamous Cell Carcinoma

Poster 11: Exploring the Relationship Between Integrin αvβ6 and αβ1 in Oral Squamous Cell Carcinoma

Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation

BackgroundViral vectors provide a method of stably introducing exogenous DNA into cells that are not easily transfectable allowing for the ectopic expression or silencing of genes for therapeutic or experimental purposes. However, some cell types, in particular bone marrow cells, dendritic cells and neurons are difficult to transduce with viral vectors. Successful transduction of such cells requires preparation of highly concentrated viral stocks, which permit a high virus concentration and multiplicity of infection (MOI) during transduction. Pseudotyping with the vesicular stomatitis virus G (VSV-G) envelope protein is common practice for both lentiviral and retroviral vectors. The VSV-G glycoprotein adds physical stability to retroviral particles, allowing concentration of virus by high-speed ultracentrifugation. Here we describe a method report for concentration of virus from large volumes of culture supernatant by means of successive rounds of ultracentrifugation into the same ultracentrifuge tube.MethodStable retrovirus producer cell lines were generated and large volumes of virus-containing supernatant were produced. We then tested the transduction ability of virus following varying rounds of concentration by ultra-centrifugation. In a second series of experiments lentivirus-containing supernatant was produced by transient transfection of 297T/17 cells and again we tested the transduction ability of virus following multiple rounds of ultra-centrifugation.ResultsWe report being able to centrifuge VSV-G coated retrovirus for as many as four rounds of ultracentrifugation while observing an additive increase in viral titer. Even after four rounds of ultracentrifugation we did not reach a plateau in viral titer relative to viral supernatant concentrated to indicate that we had reached the maximum tolerated centrifugation time, implying that it may be possible to centrifuge VSV-G coated retrovirus even further should it be necessary to achieve yet higher titers for specific applications. We further report that VSV-G coated lentiviral particles may also be concentrated by successive rounds of ultracentrifugation (in this case four rounds) with minimal loss of transduction efficiency.ConclusionThis method of concentrating virus has allowed us to generate virus of sufficient titers to transduce bone marrow cells with both retrovirus and lentivirus, including virus carrying shRNA constructs.

Disruption of a Nuclear NFATc2 Protein Stabilization Loop Confers Breast and Pancreatic Cancer Growth Suppression by Zoledronic Acid

The aminobisphosphonate zoledronic acid has elicited significant attention due to its remarkable anti-tumoral activity, although its detailed mechanism of action remains unclear. Here, we demonstrate the existence of a nuclear GSK-3β-NFATc2 stabilization pathway that promotes breast and pancreatic cancer growth in vitro and in vivo and serves as a bona fide target of zoledronic acid. Specifically, the serine/threonine kinase GSK-3β stabilizes nuclear NFATc2 through phosphorylation of the serine-rich SP2 domain, thus protecting the transcription factor from E3-ubiquitin ligase HDM2-mediated proteolysis. Zoledronic acid disrupts this NFATc2 stabilization pathway through two mechanisms, namely GSK-3β inhibition and induction of HDM2 activity. Upon nuclear accumulation, HDM2 targets unphosphorylated NFATc2 for ubiquitination at acceptor lysine residues Lys-684/Lys-897 and hence labels the factor for subsequent proteasomal degradation. Conversely, mutagenesis-induced constitutive serine phosphorylation (Ser-215, Ser-219, and Ser-223) of the SP2 domain prevents NFATc2 from HDM2-mediated ubiquitination and degradation and consequently rescues cancer cells from growth suppression by zoledronic acid. In conclusion, this study demonstrates a critical role of the GSK-3β-HDM2 signaling loop in the regulation of NFATc2 protein stability and growth promotion and suggests that double targeting of this pathway is responsible, at least to a significant part, for the potent and reliable anti-tumoral effects of zoledronic acid.

Endospanins Regulate a Postinternalization Step of the Leptin Receptor Endocytic Pathway

Endospanin-1 is a negative regulator of the cell surface expression of leptin receptor (OB-R), and endospanin-2 is a homologue of unknown function. We investigated the mechanism for endospanin-1 action in regulating OB-R cell surface expression. Here we show that endospanin-1 and -2 are small integral membrane proteins that localize in endosomes and the trans-Golgi network. Antibody uptake experiments showed that both endospanins are transported to the plasma membrane and then internalized into early endosomes but do not recycle back to the trans-Golgi network. Overexpression of endospanin-1 or endospanin-2 led to a decrease of OB-R cell surface expression, whereas shRNA-mediated depletion of each protein increased OB-R cell surface expression. This increased cell surface expression was not observed with OB-Ra mutants defective in endocytosis or with transferrin and EGF receptors. Endospanin-1 or endospanin-2 depletion did not change the internalization rate of OB-Ra but slowed down its lysosomal degradation. Thus, both endospanins are regulators of postinternalization membrane traffic of the endocytic pathway of OB-R.

Poly(ADP-Ribose) Regulates Stress Responses and MicroRNA Activity in the Cytoplasm

Poly(ADP-ribose) is a major regulatory macromolecule in the nucleus, where it regulates transcription, chromosome structure, and DNA damage repair. Functions in the interphase cytoplasm are less understood. Here, we identify a requirement for poly(ADP-ribose) in the assembly of cytoplasmic stress granules, which accumulate RNA-binding proteins that regulate the translation and stability of mRNAs upon stress. We show that poly(ADP-ribose), six specific poly(ADP-ribose) polymerases, and two poly(ADP-ribose) glycohydrolase isoforms are stress granule components. A subset of stress granule proteins, including microRNA-binding Argonaute family members Ago1-4, are modified by poly(ADP-ribose), and such modification increases upon stress, a condition when both microRNA-mediated translational repression and microRNA-directed mRNA cleavage are relieved. Similar relief of repression is also observed upon overexpression of specific poly(ADP-ribose) polymerases or, conversely, upon knockdown of glycohydrolase. We conclude that poly(ADP-ribose) is a key regulator of posttranscriptional gene expression in the cytoplasm.

RNA helicase Mov10 is a potent inhibitor of HIV or retrovirus infectivity and retrotransposition of endogenous mammalian retroviruses

The objective of this research work is to characterize antiviral and anti‐retrotransposon activity of RNA helicase Mov10. We recently reported that ectopic expression of Mov10 restricted the production of HIV‐1 and other lentiviruses as well as the gammaretrovirus, murine leukemia virus. 293T cells were transfected with retrovirus coding plasmid and Mov10 or control plasmid followed by infectivity test. HIV produced in the presence of high levels of Mov10 is restricted at the pre‐reverse transcription stage in target cells. To determine if Mov10 can also modulate transposition of retroviral elements in human genome, we transfected HeLa cells with retrotransposon coding plasmid that contained neomycin resistance cassette with or without Mov10 expressing plasmid. We then quantitated retrotransposon integration frequency by counting G418‐resistant cellular colonies in the presence or absence of Mov10 transfection. Retrotransposition of LINE‐1, MusD and IAP was significantly reduced by Mov10 in a dose dependent manner. These retroelements also showed sensitivity to restriction by other proteins found within the mRNA processing bodies (P‐bodies) such as Ago‐2 and Rent1. These findings suggest a potentially important role for cytoplasmic RNA processing machinery in retroviral infectivity, production and control of mobile genetic elements.This work was supported by NIH Grant R21 AI078839 to D.U.

Involvement of Autophagy in Oncogenic K-Ras-induced Malignant Cell Transformation

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.

BCL-2 Is a Downstream Target of ATF5 That Mediates the Prosurvival Function of ATF5 in a Cell Type-dependent Manner

ATF5 loss of function has been shown previously to cause apoptotic cell death in glioblastoma and breast cancer cells but not in non-transformed astrocytes and human breast epithelial cells. The mechanism for the cell type-dependent survival function of ATF5 is unknown. We report here that the anti-apoptotic factor BCL-2 is a downstream target of ATF5 that mediates the prosurvival function of ATF5 in C6 glioma cells and MCF-7 breast cancer cells. ATF5 binds to an ATF5-specific regulatory element that is downstream of and adjacent to the negative regulatory element in the BCL-2 P2 promoter, stimulating BCL-2 expression. Highlighting the critical role of BCL-2 in ATF5-dependent cancer cell survival, expression of BCL-2 blocks death of C6 and MCF-7 cells induced by dominant-negative ATF5, and depletion of BCL-2 impairs ATF5-promoted cell survival. Moreover, we found that BCL-2 expression is not regulated by ATF5 in non-transformed rat astrocytes, mouse embryonic fibroblasts, and human breast epithelial cells, where expression of BCL-2 but not ATF5 is required for cell survival. These findings identify BCL-2 as an essential mediator for the cancer-specific cell survival function of ATF5 in glioblastoma and breast cancer cells and provide direct evidence that the cell type-specific function of ATF5 derives from differential regulation of downstream targets by ATF5 in different types of cells.

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoietic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells, reviewed. Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced. Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoietic events but prior to antigenic stimulation to induce plasmacytic differentiation.

Influence of chitosan nanofiber scaffold on porcine endogenous retroviral expression and infectivity in pig hepatocytes

To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes. Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assay kits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells. The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group. And in the in-vitro infection experiment, no HEK293 cell was infected by the supernatant. Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission.

Production of Retroviral Vectors: Review

Retroviral vectors are presently amongst the most widely used vectors in gene therapy clinical trials to target pathologies of different origins, such as cancers, genetic diseases or neurological disorders. This review provides an overview on the evolution of retroviral vector design and production for gene therapy applications, including state of the art developments in flexible producer cells and safe vectors. In addition, production and purification processes will be addressed, with a particular focus on the improvements undertaken to increase vector productivity and to reduce the rapid loss of infectivity, which presently represent the main challenges in retroviral vectors production for gene therapy.

Poly(ADP-ribose) Polymerase-1 (PARP-1) Contributes to the Barrier Function of a Vertebrate Chromatin Insulator

The prototypic chromatin insulator cHS4 has proven effective in reducing silencing chromosomal position effects in a variety of settings. Most of this barrier insulator activity has been mapped to a 250-bp core region, as well as to several proteins that bind this region. However, recent studies from our laboratory demonstrated that an extended 400-bp core region of the cHS4 element is necessary to achieve full barrier insulator activity when used as a single copy in the context of recombinant gammaretroviral and lentiviral vectors. In this study, electrophoretic gel mobility shift assays revealed specific DNA-protein binding activities associated with the distal portion of this extended core region. Affinity purification and tandem mass spectrometry studies led to the identification of one of these proteins as poly(ADP-ribose) polymerase-1 (PARP-1). The identity of this binding activity as PARP-1 was subsequently verified by a variety of biochemical studies in vitro and by chromatin immunoprecipitation studies in vivo. Functional studies with gammaretroviral reporter vectors in cell lines and primary mouse bone marrow progenitor cultures showed that cHS4 barrier activity was abrogated upon mutation of the putative PARP-1-binding site or upon treatment with a PARP inhibitor, respectively. The barrier activity of the cHS4 element was also found to be abrogated in studies using bone marrow from Parp1-null mice. Taken together, this study demonstrates that binding of PARP-1 plays a key functional role in the barrier activity of the extended cHS4 insulator core element.

Susceptibility and production of a feline endogenous retrovirus (RD-114 virus) in various feline cell lines

RD-114 virus is a replication-competent feline endogenous retrovirus that has been classified as a xenotropic virus. In this study, we examined the expression of the receptors for RD-114 virus in feline cell lines by conducting a pseudotype virus infection assay. Six out of eight feline cell lines were susceptible to the RD-114 pseudotype virus and two cell lines (MCC and FER cells) were resistant. The two resistant cell lines and one cell line (CRFK cells) weakly sensitive to the RD-114 pseudotype virus were found to produce replication-competent RD114-like viruses by the LacZ marker rescue assay and the interference assay. These data strongly suggest that RD-114 virus is polytropic and resistance to RD-114 virus in certain cell lines is due to receptor interference but not polymorphism of the RD-114 receptors. In addition, we determined the amino acid sequences of the envelope region of RD-114-like viruses produced from MCC, FER and CRFK cells. The sequences were identical with the authentic RD-114 virus. Because many feline cell lines are used to manufacture live attenuated vaccines for companion animals, attention should be paid to contamination of the RD-114 virus in vaccines.

Lipopolysaccharide stress induces cell-type specific production of murine leukemia virus type-endogenous retroviral virions in primary lymphoid cells

Some murine-endogenous retroviruses, making up ∼10 % of the mouse genome, are induced during the course of experimental sepsis in which lipopolysaccharide (LPS), a pathogenic component of Gram-negative bacteria, often plays a critical role. In this study, we investigated whether LPS stress induces the production of murine leukemia virus type-endogenous retrovirus (MuLV-ERV) virions in primary lymphoid cells. LPS treatment of cells (single-cell suspensions and sorted B- and T-cells) isolated from seven lymphoid organs of C57BL/6J mice resulted in a differential increase in the production of MuLV-ERV virions in most cells examined. Interestingly, among the 34 unique MuLV-ERV U3 sequences cloned from the viral genomic RNAs, the nuclear respiratory factor 1 (transcription factor) element was present only in the 20 U3 sequences that were derived from the LPS-induced MuLV-ERV U3 bands. Using the U3 sequences as a probe, 55 putative MuLV-ERV loci were mapped onto the C57BL/6J mouse genome and 15 of them retained full coding potential. Furthermore, one full-length recombinant MuLV-ERV originating from a locus on chromosome 13 was determined to be responsive to LPS stress. The findings from this study suggest that LPS stress differentially activates MuLV-ERV virion production in lymphoid organs in a cell type- and MuLV-ERV-specific manner. Further investigation is needed to define the role of MuLV-ERVs in the LPS signalling pathway(s) in general, as well as in the pathogenesis of sepsis.

SIN Retroviral Vectors Expressing COL7A1 Under Human Promoters for Ex Vivo Gene Therapy of Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by loss-of-function mutations in COL7A1 encoding type VII collagen which forms key structures (anchoring fibrils) for dermal-epidermal adherence. Patients suffer since birth from skin blistering, and develop severe local and systemic complications resulting in poor prognosis. We lack a specific treatment for RDEB, but ex vivo gene transfer to epidermal stem cells shows a therapeutic potential. To minimize the risk of oncogenic events, we have developed new minimal self-inactivating (SIN) retroviral vectors in which the COL7A1 complementary DNA (cDNA) is under the control of the human elongation factor 1alpha (EF1alpha) or COL7A1 promoters. We show efficient ex vivo genetic correction of primary RDEB keratinocytes and fibroblasts without antibiotic selection, and use either of these genetically corrected cells to generate human skin equivalents (SEs) which were grafted onto immunodeficient mice. We achieved long-term expression of recombinant type VII collagen with restored dermal-epidermal adherence and anchoring fibril formation, demonstrating in vivo functional correction. In few cases, rearranged proviruses were detected, which were probably generated during the retrotranscription process. Despite this observation which should be taken under consideration for clinical application, this preclinical study paves the way for a therapy based on grafting the most severely affected skin areas of patients with fully autologous SEs genetically corrected using a SIN COL7A1 retroviral vector.

CPLA2 Regulates the Expression of Type I Interferons and Intracellular Immunity to Chlamydia trachomatis

Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase RAS and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA(2) are activated and are important for the onset of inflammatory responses. In this study we tested if activation of ERK and cPLA(2) occurred as a result of RAS signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. We provide genetic and pharmacological evidence that during infection RAS, ERK, and, to a lesser extent, cPLA(2) activation are uncoupled, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA(2) signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells, inhibition of ERK and cPLA(2) played a significant protective role against C. trachomatis. We determined that cPLA(2)-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of beta interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Furthermore, the MAPK p38 was primarily responsible for cPLA(2) activation in Chlamydia-infected cells and IRG expression. Overall, these findings define a previously unrecognized role for cPLA(2) in the induction of cell autonomous cellular immunity to Chlamydia and highlight the many non-canonical signaling pathways engaged during infection.

Perturbation of the P-Body Component Mov10 Inhibits HIV-1 Infectivity

Exogenous retroviruses are obligate cellular parasites that co-opt a number of host proteins and functions to enable their replication and spread. Several host factors that restrict HIV and other retroviral infections have also recently been described. Here we demonstrate that Mov10, a protein associated with P-bodies that has a putative RNA-helicase domain, when overexpressed in cells can inhibit the production of infectious retroviruses. Interestingly, reducing the endogenous Mov10 levels in virus-producing cells through siRNA treatment also modestly suppresses HIV infectivity. The actions of Mov10 are not limited to HIV, however, as ectopic expression of Mov10 restricts the production of other lentiviruses as well as the gammaretrovirus, murine leukemia virus. We found that HIV produced in the presence of high levels of Mov10 is restricted at the pre-reverse transcription stage in target cells. Finally, we show that either helicase mutation or truncation of the C-terminal half of Mov10, where a putative RNA-helicase domain is located, maintained most of its HIV inhibition; whereas removing the N-terminal half of Mov10 completely abolished its activity on HIV. Together these results suggest that Mov10 could be required during the lentiviral lifecycle and that its perturbation disrupts generation of infectious viral particles. Because Mov10 is implicated as part of the P-body complex, these findings point to the potential role of cytoplasmic RNA processing machinery in infectious retroviral production.

Histone Demethylases as Modulators of Hematopoietic Cell Fate.

Abstract 393Epigenetic modifications influence cell fate decisions, such as stem cell self-renewal and differentiation, both in normal and leukemic stem cells (LSC). On this premise, an expression profile study of the emerging class of histone demethylating enzymes (HDE) in stem cell enriched populations was designed. Transcripts of all these enzymes (n=27) were detected in isolated HSC populations (frequency 1:2) from fetal liver and bone marrow. Similar results were obtained in HoxA9-Meis1 induced leukemia, with an inverse correlation between LSC content and the HDE Jarid1b expression level. As a functional follow-up, systematic knockdown of known or presumed HDE was undertaken in primary HSC using a RNA interference (RNAi) based approach. Five sh-RNA were designed and generated against every HDE and cloned into a retroviral vector, containing a GFP reporter gene. The screen was set as a loss-of-function assay, where sorted CD150+CD48-Lin- cells were co-cultured on retroviral producers for 5 days, in a 96-well setting, and a fraction of each well was then transplanted into 2 mice (day 0), along with competitor cells. The remaining cell fraction served to asses gene transfer by GFP epifluorescence measurements, and RNA isolated from sorted GFP+ cells was used to evaluate gene knockdown levels by Q-PCR analysis. Blood reconstitution was evaluated at regular intervals, tracking the contribution of the donor CD45.1+ transduced (GFP+) cells to recipient hematopoiesis over time. As baseline references, sh-RNA to luciferase (no effect) and the histone acetyl transferase myst3 (stem cell loss) were used, as well as hoxb4 over-expression (stem cell expansion). In the primary screen, RNAi against jarid1a, lsd1 and an arginine demethylating enzyme, resulted in a progressive decline in blood reconstitution levels from the transduced cells, ranging from a 50% to 80% reduction of the GFP content within the graft. On the other hand, knockdown of Jarid1b conferred an in vivo competitive advantage to the transduced cells. As a validation procedure, a 7 day culture step was introduced to the assay, hence increasing the signal to noise ratio, to better detect an increase in HSC activity, as recently reported. After a week in vitro (day 7), 1/8 equivalents of single well cultures were transplanted into 3 independent mice, and blood reconstitution levels serially assessed. Cells transduced with sh-RNA against jarid1b (3 constructs out of 6) contributed more significantly to host hematopoiesis than sh-RNA luciferase transduced cells (58±16% vs 26±3% GFP), or hoxb4 over-expressing cells (37±2% GFP), at comparable gene transfer rates, at the 14 week time point and beyond (3 independent experiments). In long-term recipients, differentiation potential of these cells was preserved, as evidenced by CD4+CD8+ thymic cells, B220+ splenic cells and CD11b+ bone marrow cells in the GFP positive contingent. Clonality studies on DNA isolated from these sorted populations are ongoing. There were no cases of leukemic transformation in all of the transplant recipients (n>30).In conclusion, this RNAi based study performed on primary hematopoietic cells links histone demethylases to modulations in cell fate. An opposite role seems to emerge among the H3K4me3/me2 HDE JARID1a and JARID1b regarding HSC activity, yet these proteins are highly homologous. Sequence alignments pinpoint to differences within the C-terminal portion, containing a PHD domain, and interestingly, dysregulation of this chromatin-binding PHD finger (NUP98-JARID1a) has been recently shown to play a direct role in leukemogenesis Disclosures:No relevant conflicts of interest to declare.

Rapid High Titer Retroviral Production Using a Concentrated Retrovirus Expansion Device (CRED).

Abstract 2468Poster Board II-445One impediment to the successful development of human therapies using genetically modified hemopoietic cells is the cost and complexity of vector manufacture, which limits the number of studies that can be accomplished within a reasonable time and cost. Retroviral transduction is a commonly used method for such ex vivo genetic modification and usually requires large volumes of retroviral supernatants, the titers of which are low compared with other viral vectors. We now describe the use of a novel culture device, called a Concentrated Retrovirus Expansion Device (CRED), which was developed by Wilson Wolf Manufacturing. This CRED is similar in external appearance and shape to other conventional roller bottles, and thus can be accommodated by standard roller racks. However the presence of an internal dialysis membrane allows the virus particles to become concentrated in the cell compartment, while the nutrient compartment is separated but easily accessible and can retain up to 250 ml of cell culture media without causing virus dilution. This CRED has a surface area of 600cm2 in the cell compartment, which, due to diffusion across the dialysis membrane, requires only 10 ml of media for cell survival, a tenfold lower requirement than for an equivalent area in a traditional cell factory or roller bottle.To test the potency and stability of retroviral vectors produced in the CRED we cultured a clinical grade PG-13 producer cell line that produces the retroviral vector SFG-CAR-CD19/CD28/Z, which encodes a chimeric antigen receptor directed to the CD19 antigen (CD19-CAR). This vector is used in a current clinical protocol to transduce T cells for use in patients with advanced CD19+ B cell malignancies. We compared our standard procedure for retrovirus production (using a T175 flask) and 30 ml of IMDM against the CRED using 10ml of IMDM. Retroviral supernatant was harvested and frozen daily once cell confluence was attained (days 4-5 for the CRED and days 2-3 for the T175). Subsequently, to enumerate viable viral particles, 1×104 HeLa cells were seeded in a 6 well plate and after 24 hrs were transduced with the harvested retroviral supernatant using volumes ranging from 10-300ml in the presence of 1ml of Polybrene (10ug/ul Millipore). Six days later the cultures were evaluated by flow cytometric analysis to assess transduction efficiency, using a monoclonal antibody that recognized the CH2CH3 sequence of the CAR-CD19/CD28/Z transgenic protein. The transduction efficiency of virus harvested from the CRED was at least 4 times greater than that of the cells transduced with supernatant prepared using with the conventional method; 10ul (7.5%±4.5% vs 2.5%±0.28%), 20ul (11.9%±6.3% vs 4.9%±0.1%), 50ul (25.9%±7.4% vs 7.9%±0.9%), 100ul (45.2%±11.5% vs 9.3%±0.6%), 200ul (75.15%±15.6% vs 16.9%±1.5%), 300ul (85.7%±8.6% vs 21.4±1.8%) (n=4 independent experiments).To ensure that the CRED-produced vector was stable we exposed the harvested supernatant to 6 consecutive freeze-thaw cycles, and compared titers before and after each. We initially obtained 86.8% transduction efficiency of HeLa cells that progressively but slowly declined after each cycle (82.4%, then 80.2%, 80%, 68.7%, 60.9% and 55.5%). Hence the CRED can produce large quantities of high titer retrovirus without media change. This concentrated virus is sufficiently stable to resist freeze thaw cycles and occupies 10 fold less storage space than conventional reagents. This combination of features should enable this scalable production technique to facilitate clinical studies of retroviral vectors. [Display omitted] Disclosures:Wilson:Wilson Wolf manufacturing: Equity Ownership.