Academic literature on the topic 'Lenti Virus Vector'

Introduction A novel approach that can cover the therapeutic gap in NHL treatment are the autologous T cells, expressing Chimeric Antigen Receptors (CAR-T cells) against tumor markers. Such clinical-grade products based on Lenti (LV) or Retro- vectors have hit the market. An alternative vector system for CAR gene transfer in T-cells are Foamy Viruses (FV). To evaluate the potential of FV vectors in CAR-T cell development, we synthesized an antiCD19 scFv cDNA and cloned it in both an FV and an LV backbone; both vectors were tested in paired experiments Material and Methods The anti-CD19 CAR was under the control of the EF1a promoter; EGFP expression was under the control of an IRES2 element. The anti-CD19 CAR sequence was deduced from published data. FV vectors were made with a 4-plasmid vector system in 293T cells. 2nd generation LV vectors were purchased from Addgene. Cord blood (CB), healthy donor peripheral blood (PB) and CLL patients' PB was used as a source for CD3+ cells using immunomagnetic enrichment. Informed consent has been obtained in all cases of human sample use. T cells were activated by antiCD3/CD28 beads and transduced with antiCD19 LV or FV vectors. Transduction efficiency was assayed by flow cytometry (FCM) using a PE-conjugated anti-mouse Fab antibody. FV and LV CAR-T cells were expanded with Rapid Expansion Protocol (REP) and their cytotoxicity assays was evaluated against the CD19+ cell lines Raji and Daudi. The CLL patient derived CAR-Ts were evaluated against autologous B cells. Cytotoxicity was evaluated with an FCM protocol using CFSE-stained target cells vs unstained effector CARTs in different ratios. At the end of the incubation cells were stained with 7AAD to discriminate against live/dead cells. CAR-T cell activation was also assayed by INF-γ ELISA, following cocultures with target cells at a ratio of 1:1 for 24h. Results Vector titers: LV vector titers were between 3-5x10^5 TU/ml for both LV vectors (with or without EGFP cassette). FV vector titers were between 2-4x10^5 TU/ml regardless of the presence of the EGFP cassette. Tx efficiency: FV can mediate efficient gene transfer on T cells in the presence of heparin at an effective dose of 20-40 U/ml using a spinoculation technique. Transduction efficiency ranged from 40-65% at MOI=3-5, and was comparable to the transduction efficiency of LV vectors at a much higher MOI (10 to 30). Cytotoxicity data on lines: Following REP, the cell population consisted mostly (close to 96% purity) of CAR-T cells regardless of the vector used or of the T cell source. Effector cells were cocultured with the CD19+ cell lines, Daudi and Raji at varying ratios. With cord blood derived FV-CAR-T cells, at 4h post coculture we observed a 39.4% cell lysis at a ratio of 10:1 effector to target (n=1). Similar results were obtained for LV vectors. Peripheral blood derived CAR-T cells at THE same ratio (10:1), demonstrated 83.9% and 93.1% cell lysis for FV-CART and LV-CART cells respectively (n=2). Cytotoxicity data on CLL cells: T-cells from peripheral blood of CLL patients were used to generate LV- and FV-CAR-T cells. At the ratio of 10:1, we observed 73.1% and 69,8% cytotoxicity for FV-CAR-Ts and 70.1% and 70.7% with LV-CAR-Ts, in 2 independent paired experiments. IFN as activation marker: In two paired activation experiments, CB-derived FV-CAR-T cells secrete 560 and 437pg/ml of IFN-γ; similarly, LV-CAR-Ts secrete 534 and 554pg/ml IFN-γ. Untransduced control cells, produced 68pg/ml and 12pg/ml for FV-CAR-T and LV-CAR-T experimental arm respectively. Conclusion In the current work, we developed and tested FV vectors for anti- CD19 CAR-T cell production. We proved that FV viral vectors are capable of mediating efficient gene transfer to human T cells. We developed a method to efficiently transfer FV vectors into T-cells, using a clinically relevant protocol with heparin. The FV-derived CAR T cells demonstrate the same cytotoxic properties in vitro as their LV-derived counterpart and the same activation levels in the presence of CD19 expressing target cells as measured by IFN-γ secretion. FV CARTs derived from PB of CLL patients were capable of mediating comparable cytotoxicity levels as their LV-derived counterparts. Overall, we provide a proof of concept that FVs could be a safe and efficient alternative to LV derived vectors for CAR-T cells. Disclosures No relevant conflicts of interest to declare.

Abstract Chronic granulomatous disease (CGD) is an inherited immune deficiency due to mutations in the genes for the NADPH subunits (the genes for p47phox, p22phox, p67phox, p40phox autosomal chronic granulomatous disease), or gp91phox (X-linked chronic granulomatous disease). This results in a failure to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs), the major defect in chronic granulomatous disease causing recurrent infections and granulomatous complications. Hematopoietic stem cell transplantation (HSCT) with a suitable donor is potentially curative. However, in the absence of HLA-matched donor, gene therapy using autologous gene-corrected HSC offers potential for significant clinical benefit. To date, despite myeloid conditioning, gene therapy for CGD patients using gamma-retroviral vectors have achieved either minimal long-term gene marking and engraftment, or has been associated with insertional mutagenesis. In contrast, lentivector-mediated gene therapy has successfully treated patients with Wiskott-Aldrich syndrome and Metachromatic Leukodystrophy without any dysregulated clonal expansion. We used a lentivector construct which incorporates an MND internal promoter, a modified self-inactivating MoMuLV LTR U3 region with myeloproliferative sarcoma virus enhancer, and a 650bp single chicken b-globin insulator encoding codon-optimized p47phox gene. Mutations in p47phox accounts for the majority of AR-CGD. The production of large-scale, consistently-high-titer lentivector using a transient 4-plasmid transfection system however, is labor- and cost-prohibitive. To address this, we applied concatemeric array transfection of pCL20cW650 MND-p47-OPT into a stable packaging cell line (GPRTG) for HIV-based lentiviral vectors to create a stable producer of VSV-G pseudotyped pCL20cW650 MND-p47OP. The concatemer array of HIV lentiviral vector construct and bleomycin selectable gene cassette showed 10 copies of lentiviral vector in a stable producer line, capable of producing vector at 10^7 IU/ml. Hematopoietic CD34+ stem cells from p47phox- CGD were transduced with pCL20cW650 MND-p47-OPT vector (MOI 10) with 2 overnight transductions following 24 hours pre-activation with SCF, FLT-3L and TPO (100ng/ml). Following three weeks in vitro culture, non-transduced or transduced p47 CGD HSC versus normal HSC were 0%, 42% and 20% p47phox positive, respectively. To determine functional correction, PMA stimulated oxidant production was measured using the dihydrorhodamine assay, confirmation similar levels of oxidant generation in transduced patient cells compared with normal controls. More than 90% of CFU were vector positive, indicating a high level of gene marking. Transduced and control naïve p47phox-patient CD34+ HSC were transplanted into 20 immunodeficient Nodscid-gc deficient (NSG) mice, and at 13 weeks post-transplant the CD13+ human neutrophils arising in mouse bone marrow were assessed for p47phox expression. Over 40% CD13+ neutrophils expressed p47phox protein from NSG mice transplanted with transduced p47-patient CD34 HSC, compared with 74% or 0% in mice transplanted with normal CD34 or p47 patient naive CD34 cells respectively. Detailed histopathology of each transplanted mice confirmed the absence of vector insertion-related myeloid tumors, and deep sequencing of bone marrow CD45+ human cells from each mouse also demonstrated polyclonal distribution of vector integration sites. In conclusion, we provide preclinical data demonstrating the efficacy and safety of high titer VSVg-pseudotyped lentivector (CL20cW650 MND-p47-OPT) generated by our stable GPTRG p47 lenti-producer for correction of p47phox-deficient human CD34 HSC. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 3845 Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors that are critical regulators of vertebrate development. CTGF is a secreted protein that promotes extracellular matrix production, chemotaxis, cell proliferation and integrin expression. Since CTGF is highly expressed in acute lymphoblastic leukemia (ALL) and CTGF expression levels are related to ALL patient survival (Olga ST et al., Blood, 2007), we hypothesized that CTGF plays a role in regulating stromal cell proliferation and leukemia-stroma interaction. Our first goal was to characterize multipotent mesenchymal stromal cells (MSCs) from CTGF-/- mice, which die soon after birth from respiratory failure due to abnormal skeletal growth. We first attempted to isolate MSCs from BALB/C wild type and CTGF-/- newborn mice (Lvkovic S et al., Development, 2003). While we had no difficulty isolating MSCs from wild type mice, we failed to generate MSCs from CTGF-/- newborns (bone marrow, liver, spleen and thymus). As an alternative approach, we suppressed CTGF in bone marrow derived human MSCs with lenti-virus delivered CTGF shRNA (CTGF-KD-MSCs) and achieved knockdown of ≂f65% compared to vector control cells as determined by RT-PCR. Shortly after transduction (4 days) we started to observe major changes in the phenotype of CTGF-KD-MSCs, which grew 6–7 fold slower compared to vector control MSCs. CTGF-KD-MSCs displayed a significant decrease in the number of cells in S phase (from 14.7% ± 0.8% to 3.5%± 0.4%) and a concomitant increase in the number of G0/1 cells (from 68.8%±1.8% to 82.4%± 1.3%) suggesting a G0 or G1 block. To investigate if CTGF affects expression of MSC surface proteins, we analyzed standard MSC markers including CD105, CD90, CD73, CD44, CD140b, CD166 and CD45 (as a negative marker). Surprisingly, no differences in the expression of these markers in CTGF-KD-MSCs compared to vector control MSCs were observed. To determine if CTGF might regulate gene expression in MSCs, we performed a microarray analysis using Illumina arrays. By gene set analysis methods, we observed significant down regulation (p < 10-9 by the hypergeometric distribution test) in CTGF-KD-MSCs of genes involved in cell cycle progression, most notably in the Gene Ontology lists for ribosomal biogenesis and mitosis. As MSCs are known to differentiate into mesodermal cell lineages, we next tested the differentiation potential of CTGF-KD-MSCs as compared to the vector control MSCs. Following standard differentiation protocols, both CTGF-KD- and vector control-MSCs differentiated into osteoblasts and chondrocytes with no differences in their ability to differentiate into these lineages; whereas, CTGF-KD-MSCs showed 6–7 fold increase in their adipocyte differentiation potential compared to vector control MSCs. Another property of MSCs is to self-renew and generate colony forming units-fibroblast like (CFU-F). Fifty or 100 cells of either CTGF-KD-MSCs or vector control MSCs were seeded in alpha-MEM with 10% serum. After 2 weeks of culture in alpha-MEM with 10% serum, the resulting fibroblast-like colonies were counted. Vector control MSCs generated ≂f30-fold higher CFU-F compared to CTGF-KD-MSCs indicating that CTGF-KD-MSCs lack the ability to self renew. Next we determined if suppression of CTGF might impede leukemia cell migration to MSCs. Using standard trans-well assays, we examined human pre-B acute lymphoblastic leukemia (ALL) derived REH cells and found that they migrated 45% ± 4% less to CTGF-KD-MSCs compared to control MSCs. These findings suggest that CTGF plays a major role in stromal cell proliferation, self renewal and adipocyte differentiation of MSCs. In addition, CTGF is involved in leukemia cell migration towards MSCs and inhibition of CTGF impairs the migration of leukemic cells towards stromal cells and thereby provides opportunities to prevent homing of leukemic cells and sensitize them to chemotherapy. Disclosures: Andreeff: MyeloRx LLC: Consultancy, participant in research under an NCI SBIR Contract to MyeloRx LLC.

Abstract Background: Decitabine, reverts hypermethylation of p15INK4B gene in vitro, was used to improves cytopenias and blast excess in over 50% of patients with high-risk myelodys plastic syndrome (MDS). In this study, over-expression of Heme Oxygenase-1(HO-1) was found in MDS cells line SKM-1 cells, and it was closely related to resistance to apoptosis induced by decitabine. Objective: we aimed to further investigated what role of HO-1 exactly played in apoptosis induced by low-does of decitabine in MDS. Method: CCK-8 kits was used to determine the proliferation inhibition of SKM-1 cells. Flow cytometry was used for analyzing cell proliferation rate and apoptosis. The methylation status and expression of P15INK4B in mRNA and protein levels were measured by methylation-specifc polymerase chain reaction (PCR [MSP]). Apoptosis relative factors expression were detected by real-time transcription and Western blot. Result: Up-regulation of HO-1 by transfected it into SKM-1 cells via lenti-virus vector promoted proliferation and protected cells against apoptosis. In contrast, down-regulation of HO-1 enhanced decitabine-induced apoptosis but reduced accumulation of S phase in cell cycle. To explore the mechanism, we detected cell cycle relative protein expression after SKM-1 cells were treated by decitabine in each group. As a result, over-expression of p15 INK4B and CDK4 were observed in SKM-1 cells which HO-1 was inhibited. And p15 INK4B and CDK4 expression-dependent apoptosis was related to caspase3 pathway. Even though HO-1 was silenced, but apoptotic rate never increased as caspase3 pathway was blocked. Conclusion: As we known that p15 INK4B is a keypoint to regulate S phase of cell cycle, in our study, more obvious demethylation of p15 INK4B was seen in group of SKM-1 cells in which HO-1 was down-regulated. It’s equally in patients’ mononuclear cells who suffered from MDS. The worse the prognosis of MDS was judged, the more the mRNA level of HO-1 expressed. In conclusion, over-expression of HO-1 indicated resistance to demethylation of p15 INK4B induced by decitabine. Disclosures No relevant conflicts of interest to declare.