Murine Ecotropic Receptor Research Articles

Rapid establishment of stable retroviral packaging cells and recombinant susceptible target cell lines employing novel transposon vectors derived from Sleeping Beauty

Viral vector particles derived from murine leukemia virus (MLV) mediate highly efficient stable gene transfer used in gene therapeutic approaches and in the generation of transgenic cell lines. However, the establishment of stable viral packaging cells (VPCs) is a time-consuming challenge. To overcome this limitation, we successfully generated novel Sleeping Beauty-derived transposon vectors entailing envelope and packaging expression cassettes as well as a transfer vector. Upon multiplexed transposition in human cells, VPC bulk populations yielding titers of over 1 × 106 transduction-competent vectors were established within three weeks. In contrast, conventional plasmid-based establishment of VPCs, conducted in parallel, took much longer and yielded significantly lower vector productivity and vector fitness. The generated MLV vectors decorated with the envelope proteins of ecotropic MLV PVC-211mc mediated efficient transduction of Chinese hamster ovary (CHO) cells. Cell susceptibility was further elevated upon recombinant expression of the murine ecotropic receptor mCAT employing a transposon vector.

PO-381 Response project: chromatin regulators as biomarkers and drug targets in colorectal cancer

IntroductionColorectal cancer (CRC) is the third most common cancer worldwide. The majority of patients diagnosed at advanced disease develop primary/acquired resistance. Chromatin-regulatory factors modulate accessibility to DNA and showed some alterations in tumours at variable frequencies.Our aim is to identify which chromatin factors could be novel targets for overcoming resistance to chemotherapy, and if they can be considered as novel biomarkers to predict treatment response. We are using a potent genetic loss-of- function screen that will reveal which chromatin factors lead to sensitisation or resistance in non-treated vs. treated cells with drugs that mimic therapeutic regimes in patients.Material and methodsRESPONSE project is an explorative approach based on a pooled retroviral library containing 7300 short hairpin RNAs (shRNA) targeting 912 chromatin factor genes in an optimised miRNA-backbone that allow maximal knockdown by single copy integration. The experiments were performed using HT-29 CRC cell line, modified with a murine ecotropic receptor (EcoR).Co-infection assays were performed with labelled GFP and mCherry vectors using several viral dilutions to confirm the percentage of virus required for single copy integration. Kill-assays were performed to monitor by flow cytometry the shRNA lethality caused by the knockdown of essential MYC and RPA3 genes compared with neutral shRNA Renilla gene. The half maximal inhibitory concentration (IC50) of 5-Fluoroacil (5-FU), Oxaliplatin (Oxa) and Irinotecan (SN-38) drugs were calculated.FOLFOX (5-FU+Oxa) and FOLFIRI (5-FU+SN-38) like treatments were tested in dose-response three-week experiments to determine the suitable drug concentration for the screening. The experiments were analysed by XTT and DiOC/DAPI staining.Results and discussionsEcoR receptor was correctly expressed in transformed HT-29 cells and functional when infected with GFP and mCherry labelled vectors. Infection rates were 40% and 12%, respectively. The suitable viral dilution ranged from 2.5% to 5%. Kill assays confirmed shRNA inhibition efficiency leading HT-29 EcoR infected cells with MYC and RPA3 shRNA to death. The IC50 of 5-FU, Oxa and SN-38 were 10 µM, 2 µM and 5.5 nM respectively and the FOLFOX and FOLFIRI dilution that allowed 70%–80% of cell survival after a long-term treatment were determined.ConclusionWe have successfully set up the conditions to perform the loss-of-function screening. Currently, we are working on the effect of each relevant shRNA to validate the results of the screening.

The Early-Immediate Gene EGR-1 Is Induced by Transforming Growth Factor-β and Mediates Stimulation of Collagen Gene Expression

Transforming growth factor-beta (TGF-beta) stimulates collagen synthesis and accumulation, and aberrant TGF-beta signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL1A2) transcription by TGF-beta involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription factors that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early growth response factor-1 (EGR-1) as a novel Smad3-inducible gene. Previous studies implicated Egr-1 in cell growth, differentiation, and survival. We found that TGF-beta induced rapid and transient accumulation of Egr-1 protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-beta stimulated the activity of the Egr-1 gene promoter, as well as that of a minimal Egr-1-responsive reporter construct. Furthermore, TGF-beta enhanced endogenous Egr-1 interaction with a consensus Egr-1-binding site element and with GC-rich DNA sequences of the human COL1A2 promoter in vitro and in vivo. Forced expression of Egr-1 by itself caused dose-dependent up-regulation of COL1A2 promoter activity and further enhanced the stimulation induced by TGF-beta. In contrast, the TGF-beta response was abrogated when the Egr-1-binding sites of the COL1A2 promoter were mutated or deleted. Furthermore, Egr-1-deficient embryonic mouse fibroblasts showed attenuated TGF-beta responses despite intact Smad activation, and forced expression of ectopic EGR-1 in these cells could restore COL1A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-1 as a novel intracellular TGF-beta target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-1 in the profibrotic responses elicited by TGF-beta and suggest that Egr-1 may play a new and important role in the pathogenesis of fibrosis.

Adaptation of chimeric retroviruses in vitro and in vivo: isolation of avian retroviral vectors with extended host range.

We have designed and characterized two new replication-competent avian sarcoma/leukosis virus-based retroviral vectors with amphotropic and ecotropic host ranges. The amphotropic vector RCASBP-M2C(797-8), was obtained by passaging the chimeric retroviral vector RCASBP-M2C(4070A) (6) in chicken embryos. The ecotropic vector, RCASBP(Eco), was created by replacing the env-coding region in the retroviral vector RCASBP(A) with the env region from an ecotropic murine leukemia virus. It replicates efficiently in avian DFJ8 cells that express murine ecotropic receptor. For both vectors, permanent cell lines that produce viral stocks with titers of about 5 x 10(6) CFU/ml on mammalian cells can be easily established by passaging transfected avian cells. Some chimeric viruses, for example, RCASBP(Eco), replicate efficiently without modifications. For those chimeric viruses that do require modification, adaptation by passage in vitro or in vivo is a general strategy. This strategy has been used to prepare vectors with altered host range and could potentially be used to develop vectors that would be useful for targeted gene delivery.

Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38- subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV.

Recombinant adeno-associated viral (rAAV) vectors have been evaluated for their ability to transduce primitive hematopoietic cells. Early studies documented rAAV-mediated gene expression during progenitor derived colony formation in vitro, but studies examining genome integration and long-term gene expression in hematopoietic cells have yielded conflicting results. Such studies were performed with crude vector preparations. Using improved methodology, we have generated high titer, biologically active preparations of rAAV free of wild-type AAV (less than 1/107particles) and adenovirus. Transduction of CD34+ cells from umbilical cord blood was evaluated with a bicistronic rAAV vector encoding the green fluorescent protein (GFP) and a trimetrexate resistant variant of dihydrofolate reductase (DHFR). Freshly isolated, quiescent CD34+ cells were resistant to transduction (less than 4%), but transduction increased to 23 +/- 2% after 2 days of cytokine stimulation and was further augmented by addition of tumor necrosis factor alpha (51 +/- 4%) at a multiplicity of infection of 106. rAAV-mediated gene expression was transient in that progenitor derived colony formation was inhibited by trimetrexate. Primitive CD34+ and CD34+, CD38- subsets were sequentially transduced with a rAAV vector encoding the murine ecotropic receptor followed by transduction with an ecotropic retroviral vector encoding GFP and DHFR. Under optimal conditions 41 +/- 7% of CD34+ progenitors and 21 +/- 6% of CD34+, CD38- progenitors became trimetrexate resistant. These results document that highly purified rAAV transduce primitive human hematopoietic cells efficiently but gene expression appears to be transient. Gene Therapy (2000) 7, 183-195.

Adenovirus-mediated expresssion of the murine ecotropic receptor facilitates transduction of human hematopoietic cells with an ecotropic retroviral vector.

One factor limiting the ability to modify human repopulating hematopoietic cells genetically with retroviral vectors is the relatively low expression of the cognate viral receptor. We have tested sequential transduction of human hematopoietic cells with an adenoviral vector encoding the ecotropic retroviral receptor followed by transduction with an ecotropic retroviral vector. Adenoviral transduction of K562 erythroleukemia cells was highly efficiently with >95% of cells expressing the ecotropic receptor at a multiplicity of infection (MOI) of 103with a correspondingly high transduction with a retroviral vector. Ecotropic receptor expression in CD34+ cells following transduction with adenoviral vectors was increased by at least two-fold (from 20 to 48%) by replacing the RSV promoter with the CMV E1a promoter, resulting in a parallel increase in retroviral transduction efficiency. Replacing the head portion of the fiber protein in conventional adenoviral vectors (serotype 5) with the corresponding portion from an adenoviral 3 serotype resulted in ecotropic receptor expression in 60% of CD34+ cells at an MOI of 104 and a retroviral transduction of 60% of hematopoietic clonogenic progenitors. The sequential transduction strategy also resulted in efficient transduction of the primitive CD34+CD38- subset suggesting that it may hold promise for genetic modification of human hematopoietic stem cells.

Effects of infection rate and selection pressure on gene expression from an internal promoter of a double gene retroviral vector.

Many commonly used retroviral vectors express one gene from the viral long terminal repeat (LTR) promoter and another gene from an internal promoter. We have investigated factors affecting the expression of the luciferase reporter gene from the internal cytomegalovirus-derived promoter of the retroviral vector, LNCX, which contains a LTR-driven neo gene as a selectable marker. A subline of human HT1080 cells, expressing the murine ecotropic receptor, was infected with retrovirus generated by transient transfection of BOSC 23 packaging cells. Mass populations of cells infected under conditions resulting in different initial infection rates (IIR) and selected with G418, showed highly variable luciferase activity. Luciferase expression in cell populations with IIR < or = 5% was generally low; many populations with IIR < 1% had marginal or no luciferase activity. The loss of luciferase expression in low-IIR populations was associated with G418 selection. In contrast, cell populations with IIR > or = 6% showed higher luciferase expression, which was strongly correlated with the IIR. Southern hybridization analysis showed that most cells of the low-IIR populations carried one integrated provirus, with a high incidence of structural rearrangements that abolished luciferase activity. In contrast, populations with IIR > or = 6% contained two or more copies of integrated provirus per cell, and their luciferase activity correlated with the provirus copy number. Luciferase expression was relatively stable in the populations with IIR > 1% maintained in the absence of G418. Increasing the selective concentration of G418 or prolonged maintenance of cell populations in the presence of G418 resulted in higher incidence of provirus rearrangements and decreased luciferase expression. These results indicate that the negative effect of selection for the LTR-driven gene on gene expression from an internal promoter depends on the selection stringency and can be obviated by increasing the infection rate.

Retroviral Infection of Syrian Hamster BHK Cells Depends on Age and Susceptibility toward Sialidase

Infection with recombinant retroviruses is a standard method to efficiently manipulate murine or human cells genetically. Due to differences in glycosylation of the hamster homologue to the murine ecotropic receptor and the absence of an amphotropic receptor, murine retroviruses are unable to infect hamster cells. Here we describe an isolated cell clone of the Baby Hamster Kidney cell line, BHK-21, that can be successfully infected with ecotropic murine retroviruses. This finding prompted us to investigate the possibility of changing the infectability of other cell strains by manipulating the receptors of these cell linesin vitro.We found that treatment of other BHK cell clones with sialidase fromC. Perfringensmakes these cell clones also permissive to murine ecotropic retroviruses. Long term cultivation of three tested cell clones increased this susceptibility. Although the resulting infectants show a higher rate of infectability, they still require the manipulation with sialidase for this superinfection to be efficient.

Analysis of the functional and host range-determining regions of the murine ectropic and amphotropic retrovirus envelope proteins.

A series of Moloney murine leukemia virus (Mo-MuLV) envelope gene constructs were analyzed for biological activity. Three classes of recombinant envelopes were examined: insertions, deletions, and chimeras. Insertion (4 to 5 amino acids) and deletion (31 to 62 amino acids) mutants spanned most of the SU (gp70)-coding region and were all biologically inactive. Radioimmunoprecipitation demonstrated that the mutant envelope proteins were incorrectly processed. The Pr80env envelope precursor proteins failed to obtain the proper posttranslational modifications and were not cleaved into SU (gp70) and TM (p15E), suggesting that disruption of Pr80env structure prevents intracellular transport and processing. To analyze the functional domains of the SU portion of the Env protein, we assembled several chimeric constructs. In these constructs, portions of the ecotropic Mo-MuLV envelope gene were replaced with corresponding sequences from the 4070A amphotropic MuLV envelope. Using a retroviral vector pseudotyping assay, 5 of 12 chimeric envelope proteins were shown to be biologically active. Host range was determined by retroviral vector transduction of the appropriate cell, by viral interference studies, and by the productive infection of Chinese hamster ovary cells expressing the murine ecotropic receptor. These results permit assignment of the amino acids responsible for host range determination. Ecotropic host range is determined by the first 88 amino acids of the Mo-MuLV SU, while the amphotropic host range-determining region spans the first 157 amino acids of the 4070A SU.