Bicistronic Lentiviral Vector Research Articles

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency.

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has proved highly valuable for research and clinical applications. Interestingly, iPSC reprogramming of cancer cells, such as pancreatic ductal adenocarcinoma (PDAC), has been shown to revert the invasive PDAC phenotype and override the cancer epigenome. The differentiation of PDAC-derived iPSCs can recapitulate PDAC progression from its early pancreatic intraepithelial neoplasia (PanIN) precursor, revealing the molecular and cellular changes that occur early during PDAC progression. Therefore, PDAC-derived iPSCs can be used to model the earliest stages of PDAC for the discovery of early-detection diagnostic markers. This is particularly important for PDAC patients, who are typically diagnosed at the late metastatic stages due to a lack of reliable biomarkers for the earlier PanIN stages. However, reprogramming cancer cell lines, including PDAC, into pluripotency remains challenging, labor-intensive, and highly variable between different lines. Here, we describe a more consistent protocol for generating iPSCs from various human PDAC cell lines using bicistronic lentiviral vectors. The resulting iPSC lines are stable, showing no dependence on the exogenous expression of reprogramming factors or inducible drugs. Overall, this protocol facilitates the generation of a wide range of PDAC-derived iPSCs, which is essential for discovering early biomarkers that are more specific and representative of PDAC cases.

Abstract B112: SNAP23-dependent SNARE complex is a novel molecular target in AL Amyloidosis and Multiple Myeloma by blocking free light chain secretion and triggering a terminal unfolded protein response

Abstract The goal of this study was to investigate whether SNARE proteins are necessary for immunoglobulin (Ig) free light chain (FLC) secretion, thus representing a yet unexplored therapeutic target in AL amyloidosis (AL) and Multiple myeloma (MM). Both these disorders are incurable, and in the former, pathogenesis is invariably related to deposition of Ig FLC fibrils in target organs. The molecular mechanisms underlying FLC folding/secretion have not been studied, and no therapies directly target this intrinsic vulnerability. We hypothesize that FLC secretion is at least in part mediated by SNARE proteins, and their inhibition would block FLC exocytosis, causing retention of FLC-loaded vesicles, leading to activation of a terminal unfolded protein response (UPR) and apoptosis. We therefore hypothesized that novel, effective targets for MM and AL therapy could be identified within SNAREs. As a tool to study SNARE function, we stably expressed 7 distinct, tetracycline-inducible botulinum neurotoxin (BoNT) serotypes (BoNT/A-F) in FLC-secreting AL (ALMC1, ALMC2) and MM (U266, KMS11) cell lines. To monitor BoNT expression, a bicistronic lentiviral vector with a P2A-GFP was used. Upon doxycycline administration, we performed growth competition assay in polyclonally transduced cells and assessed kinetics of GFP expression over time and apoptosis via flow cytometry in monoclones. Western blot (WB) was used to assess cleavage of SNAREs upon expression of each BoNT. We used WB and ELISA to assess FLC secretion on a per-cell base upon BoNT expression. We used TurboID proximity labeling followed by mass spectrometry to identify cytotoxic BoNT interacting partners and CRISPR-Cas9 gene editing to validate putative BoNT targets. By querying the IFM170 gene expression database of newly diagnosed MM patients, we identified VAMP2, VAMP3, SYNTAXIN4, and SNAP23 as the top expressed SNARE and confirmed their high expression in a panel of AL and MM cell lines. Expression of all BoNT serotypes, except BoNT B, led to rapid apoptosis of AL and MM cells, with BoNT F standing out for extent and rapidity of cytotoxicity. Concurrent cleavage of SNAP23, VAMP3, and SYNTAXIN4 correlated with cytotoxicity of BoNT in AL and MM cell lines. Mechanistically, we showed that cytotoxic BoNTs, but not BoNT B, blocked FLC secretion and triggered the PERK-mediated terminal UPR with induction of CHOP and GADD34 followed by massive apoptosis. We identified SNAP23 as an interacting partner of cytotoxic BoNT F, but not BoNT B. Single SNAP23 KO did not completely recapitulate cytotoxic BoNT phenotype, as it was only partially cytotoxic. Based on our preliminary data, we hypothesize SYNTAXIN4 and VAMP3 to be synthetic lethal with SNAP23. Our data provide proof of concept that blocking FLC secretion in AL and MM cells is feasible and results in rapid cell apoptosis by triggering a terminal UPR, thus representing a promising, novel therapeutic approach. Using BoNT as a biological tool, we identified SNAP23 as a critical component of FLC exocytosis, paving the way for the design of novel therapeutics. Citation Format: Emre Karayol, Maria Moscvin, Tianzeng Chen, Peter G. Czarnecki, Annamaria Gulla, Kenneth C. Anderson, Giada Bianchi. SNAP23-dependent SNARE complex is a novel molecular target in AL Amyloidosis and Multiple Myeloma by blocking free light chain secretion and triggering a terminal unfolded protein response [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B112.

A SNAP23-Dependent SNARE Complex Mediates Free Light Chain Secretion in AL Amyloidosis and Multiple Myeloma and Its Blockade Triggers a Terminal Unfolded Protein Response and Massive Apoptosis

Background AL amyloidosis (AL) is an incurable plasma cell (PC) disorder with only one FDA-approved therapy. Deposition of immunoglobulin free light chain (FLC) fibrils in target organs is the sole pathogenic mechanism in AL. However, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, the specific targets of botulinum neurotoxin (BoNT), mediate the docking and fusion of secretory vesicles. We hypothesized that targeting specific sets of SNAREs via BoNT will block FLC exocytosis, causing retention of FLC-loaded vesicles and triggering a terminal unfolded protein response (UPR) and apoptosis in AL cells. We postulate that SNAREs are potential novel targets in AL and other plasma cell disorders. Materials and Methods We stably expressed 7 distinct, inducible BoNT serotypes (BoNT/A-F) in FLC-secreting AL (ALMC1 and ALMC2) and multiple myeloma (MM, KMS11) cell lines via tetracycline-inducible (Tet-On) bicistronic lentiviral vectors with a P2A-GFP which allowed us to use GFP as a biomarker of BoNT expression. We established monoclones of successfully transduced cells for each BoNT. We performed flow cytometry to assess the kinetics of GFP induction and assess for apoptosis via AnnexinV/DAPI staining. Western blot (WB) was used to assess the cleavage of SNAREs upon expression of each BoNT serotype. We used WB and ELISA to assess FLC secretion per cell upon expression of each BoNT serotype. We used TurboID proximity labeling followed by mass spectrometry to identify cytotoxic BoNT interacting partners and used co-IP for confirmation. We used RNP-based CRISPR-Cas9 gene editing to knock out (KO) putative BoNT targets. Results By querying the IFM170 gene expression database of newly diagnosed MM patients, we identified VAMP2, VAMP3, SNAP23, and SYNTAXIN4 as the top expressed SNAREs. Their high expression was retained in a large panel of AL and MM cell lines. Expression of all BoNT serotypes, except for BoNT B, led to rapid apoptosis of AL and MM cells, with BoNT F standing out for the extent and rapidity of cytotoxicity. Dual cleavage of SNAP23 and VAMP3 correlated with cytotoxicity of BoNT in AL and MM cell lines. Mechanistically, we showed that cytotoxic BoNTs, but not non-toxic BoNT B, led to decreased FLC secretion and increased FLC retention and triggered the PERK pathway of the UPR with increased expression of CHOP and GADD34, preceding the onset of apoptosis. We identified SNAP23 as an interacting partner of highly cytotoxic BoNT F, but not non-toxic BoNT B. KO experiment showed single SNAP23 KO to be partially toxic for AL and MM cell lines but did not recapitulate the effects of cytotoxic BoNTs entirely. Based on our preliminary data, we hypothesize VAMP3 and SYNTAXIN4 to be synthetic lethal with SNAP23. Conclusions Our data provide proof of concept that blocking FLC secretion in AL and MM cells is feasible and results in rapid cell apoptosis by triggering a terminal UPR, thus representing a promising, novel therapeutic approach. By using BoNT as a biological tool, we identified SNAP23 as a critical component of FLC exocytosisin AL cell lines, thus representing a novel, promising molecular target in AL amyloidosis and multiple myeloma.

Preliminary Results of Nathali-01: A First-in-Human Phase I/IIa Study of UCART20x22, a Dual Allogeneic CAR-T Cell Product Targeting CD20 and CD22, in Relapsed or Refractory (R/R) Non-Hodgkin Lymphoma (NHL)

Background: UCART20x22 is a dual targeted allogeneic CAR T-cell product candidate generated from healthy donor T-cells. Donor-derived T-cells are transduced with a bicistronic lentiviral vector to express CARs targeting CD20 and CD22. TALEN ® gene editing technology is used to inactivate the TRAC and CD52 genes to minimize graft-versus-host disease (GvHD) and allow for the use of alemtuzumab (CLLS52, an anti-CD52 monoclonal antibody) in the lymphodepletion (LD) regimen, respectively. UCART20x22 previously demonstrated robust and sustained activity in vitro and in vivo (Aranda-Orgilles B, et al. Cancer Immunol Res 2023). Here we present the initial preliminary results from the NatHaLi-01 trial (NCT05607420), the first-in-human Phase 1/2a dose-finding and expansion study evaluating UCART20x22 in R/R B-cell NHL. Methods: The primary endpoints are safety, tolerability, and determining the recommended phase 2 dose of UCART20x22. Additional endpoints include anti-lymphoma activity and cellular kinetics of UCART20x22. Key eligibility criteria are age 18-80y, lymphoma expression of CD20 and/or CD22 by local assessment, and ≥ 2 prior treatment regimens including autologous CD19 CAR T-cell therapy (CAR19), if eligible. After LD with FCA (fludarabine 30 mg/m 2 × 3d, cyclophosphamide 0.5g/m 2 × 3d, CLLS52 12 mg on D1, 24 mg on D2, D3), a single infusion of UCART20x22 is administered at a flat dose level ([DL1] 50 x 10 6 cells; [DL2] 150 x 10 6 cells; and [DL3] 450 x 10 6 cells). Results: As of 01 July 2023, 3 pts were enrolled and treated at DL1. Pt 1 is a 76yo female with double-expressor diffuse large B-cell lymphoma (DLBCL) relapsed after R-CHOP, radiation therapy, and polatuzumab vedotin with bendamustine/rituximab (BR) and was considered ineligible for CAR19 due to recent high dose bendamustine. Pt 2 is a 65yo female with R/R triple-hit lymphoma transformed from follicular lymphoma who was previously treated with radiation therapy, BR, dose-adjusted R-EPOCH, and two separate CAR19 treatments. Pt 3 is an 18yo female with R/R DLBCL transformed from marginal zone lymphoma who previously received chemoimmunotherapy, venetoclax, ibrutinib, BR, CAR19, obinutuzumab, glofitamab, tafasitamab, lenalidomide, and an experimental epigenetic modifier. Cytokine release syndrome (CRS) occurred in all patients. Pt 1 had G1 CRS and received tocilizumab (toci) x3 and dexamethasone (dex) x1. Pt 2 had G2 CRS and received toci and dex x1. Pt 3 had G1 CRS and received toci x1. All pts' CRS resolved with treatment. No immune effector cell associated neurotoxicity (ICANS) or GvHD was observed. There were no UCART20x22 dose limiting toxicities (DLTs), and there was 1 CLLS52 SAE and 1 DLT. The SAE was G3 CMV reactivation and was managed with IV antivirals. The DLT was in Pt 2, who entered the study with G1/2 cytopenias and a hypocellular bone marrow and had prolonged bone marrow aplasia after treatment thought to be due to cumulative chemotherapy exposure. This pt proceeded to allogeneic hematopoietic stem cell transplant (HSCT) from a matched donor while in complete remission from UCART20x22 treatment. Responses were assessed per Lugano criteria beginning on D28. At D28, all 3 pts responded with 2 CRs and 1 PR. Host lymphocytes remained suppressed through D28 for all pts. UCART20x22 expansion was observed by flow cytometry in 3/3 pts and was initially detected on D7 followed by peaks between D9-14. Peak expansion ranged from 16-682 cells/μL and was predominantly CD8 T-cells. Ferritin levels and increases in serum cytokines, including IFNγ, TNFα, IL-2, and IL-6, correlated closely with UCART20x22 expansion. Summary: Treatment with UCART20x22 at DL1 has been safe and well-tolerated, with no UCART20x22 DLTs or ICANS, and CRS was <G3 in all pts. There was 1 DLT of prolonged bone marrow aplasia in a pt with impaired hematopoietic reserve at study entry. UCART20x22 was active at DL1, with all 3 pts responding, including 2 CRs in pts who had previously received CAR19. UCART20x22 expansion was observed in all pts. These initial data support the continued study of UCART20x22 in R/R NHL, and updated results will be presented.

Development of an Off-the-Shelf Chimeric Antigen Receptor (CAR)-T Cell Therapy for T-Cell Acute Lymphoblastic Leukemia (T-ALL) without Gene Editing

CAR-T cell therapy has revolutionized the treatment of B-lineage ALL and other B-cell malignancies, but the development of cell therapies for T-cell malignancies has lagged behind. For T-ALL, CD7 is an excellent CAR target because it is consistently and highly expressed in leukemic cells and is retained during chemotherapy and at relapse. However, CD7 is also expressed in normal T-cells and the expression of anti-CD7 CARs triggers T-cell fratricide, resulting in a marked decrease in viable and/or effective CAR-T cells. We developed an anti-CD7 CAR-T cell product (PCART7) by utilizing a CD7 protein expression blocker (PEBL) to downregulate CD7 surface expression and prevent fratricide. PEBLs consist of a target binding domain fused to an intracellular retention domain that enables relocalization of surface proteins intracellularly (Png et al. Blood Adv 2017; Kamiya et al. Blood Adv 2018; Kamiya et al., JCI 2019). Autologous PCART7 cells lacking surface CD7 and expressing anti-CD7 CARs can be effectively produced under cGMP conditions and the technology is being tested in clinical trials for patients with relapsed/refractory T-ALL or T-cell lymphoblastic lymphoma. Generation of autologous CAR-T cells, however, may be hampered by the presence of a high percentage of leukemic cells in peripheral blood and/or low T cell numbers. An allogeneic anti-CD7 CAR-T cell product available "off-the shelf" would represent a useful tool to reduce leukemic burden and provide a bridge to hematopoietic cell transplant or to autologous CAR-T cell therapy. Because allogeneic T-cells can be activated upon recognition of host tissue antigens by their T-cell receptor (TCR)/CD3 complex, their infusion carries a risk of causing graft-versus-host disease (GvHD). We optimized a CD3 PEBL that can effectively downregulate surface TCR/CD3. We then developed a lentiviral-based process that allows simultaneous expression of CD3 and CD7 PEBLs together with anti-CD7 CAR. The PEBLs retained surface CD7 and TCR/CD3 intracellularly, thereby minimizing fratricide and abrogating TCR-mediated antigen recognition. PCART7 cells lacking TCR/CD3 were manufactured from healthy donor T-cells by using a double transduction with a bicistronic lentiviral vector delivering CD7 PEBL and anti-CD7 CAR (PCART7 LVV), and a second lentiviral vector delivering CD3 PEBL. Robust expansion of PCART7-CD3 PEBL cells was observed and more than 90% of the cells were of the desired CAR+CD7-CD3- phenotype. Following depletion of residual CD3/TCRαβ+ cells, CD3- cell purity was >99% (Fig. 1A). These results were reproduced across multiple donors. The efficient downregulation of surface TCR/CD3 by CD3 PEBL was similar to that achieved by CRISPR knockout of the TCR alpha chain locus (TRAC) and completely abrogated activation in response to TCR stimulation. In vitro short- and long-term assays confirmed strong cytotoxic potency against CD7+ leukemic cells but not CD7- target cells. Importantly, PCART7 cells were equally cytotoxic regardless of whether they were co-transduced with CD3 PEBL. In a xenograft model using the CCRF-CEM T-ALL cell line, PCART7-CD3 PEBL cells, like PCART7 cells, effectively suppressed tumor growth and improved survival (Fig. 1B). This study demonstrates the feasibility of using two PEBLs simultaneously for intracellular protein retention without perturbing critical CAR-T cell functions. The use of PEBL technology to generate allogeneic anti-CD7 CAR-T cells fits seamlessly in cGMP manufacturing protocols and avoids the risk of genotoxicity arising from the use of gene-editing techniques. Because the resulting allogeneic anti-CD7 CAR-T cells had high potency and were devoid of alloreactivity potential, they represent an attractive new tool for off-the-shelf CAR-T cell therapy of T-ALL and other CD7+ malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 569: Mesothelin CAR T cells secreting FAP specific T cell engaging molecule (TEAM) target pancreatic cancer and its tumor microenvironment (TME)

Abstract Targeting solid tumors with CAR T cells has proven to be more difficult due to their heterogenous target antigen expression, antigen escape, and due to their hostile tumor microenvironment (TME). Mesothelin represents a promising surface tumor antigen, since it has been associated with tumor invasion and is highly expressed on various cancer types, including pancreatic adenocarcinoma. As clinical trials of mesothelin targeting CAR have yet to show efficacy, we hypothesized that tumor stromal cells such as cancer-associated fibroblasts (CAFs) may play a role in this resistance. To provide a more favorable TME for CAR T cells, we generated a bicistronic lentiviral vector encoding a mesothelin CAR along with a secreted T cell engaging molecule (TEAM) that targets fibroblast activation protein (FAP), which is expressed by CAFs. We termed our constructs CARTEAM, and in this case, mesoFAP. We have assessed the activation and proliferative capacity of these CARTEAM through in vitro assays. We showed that TEAMs secreted by CAR T cells bind their appropriate target antigen by adding supernatant from CARTEAM to target cells expressing FAP. In a co-culture assay using a transwell system we demonstrated the cytotoxic effect of secreted TEAM interacting and recruiting bystander T cells against CAFs. In a real-time cell analysis (RTCA) co-culture assay with a pancreatic cancer cell line (AsPC1) and FAP expressing CAFs, we showed cell death of AsPC1 upon CAR recognition and cell death of CAFs through TEAM-mediated recruitment of bystander T cells and CART cells. We show in these co-culture systems, mimicking tumor and TME, that our CARTEAM construct is superior in the elimination of both cancer cell line and CAF, in comparison to control constructs, including mesothelin targeting CAR T cells (meso CAR) and meso CAR T cells secreting an unspecific CD19 TEAM (mesoCD19). We also used acoustic force microscopy to evaluate the additive effect of the TEAM molecule secreted to binding to tumor cells by mesothelin CAR T cells. In vivo experiments of subcutaneously injected tumor cells admixed with CAFs, show superior tumor control when treated with CARTEAM in comparison to control constructs. Based on these data, we demonstrate both the effective in vitro elimination of CAFs and pancreatic cancer cells through the application of CARTEAM and control of pancreatic tumor growth in vivo. Our studies provide a deeper insight into a dual targeting strategy using a novel CAR T cell secreting a TEAM against pancreatic cancer and its tumor microenvironment. Citation Format: Marc Wehrli, Adam Kuo, Rebecca Larson, Irene Scarfò, Amanda Bouffard, Korneel Grauwet, Mark Leick, Andrea Schmidts, Stefanie Bailey, Tamina Kienka, Michael Kann, Sonika Vatsa, Harrison Silva, Kathleen Gallagher, Max Jan, Bryan Choi, David Ting, Marcela Maus. Mesothelin CAR T cells secreting FAP specific T cell engaging molecule (TEAM) target pancreatic cancer and its tumor microenvironment (TME) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 569.

Functionality of a bicistronic construction containing HEXA and HEXB genes encoding β-hexosaminidase A for cell-mediated therapy of GM2 gangliosidoses.

Tay-Sachs disease and Sandhoff disease are severe hereditary neurodegenerative disorders caused by a deficiency of β-hexosaminidase A (HexA) enzyme, which results in the accumulation of GM2 gangliosides in the nervous system cells. In this work, we analyzed the efficacy and safety of cell-mediated gene therapy for Sandhoff disease and Sandhoff disease using a bicistronic lentiviral vector encoding cDNA of HexA α- and β-subunit genes separated by the nucleotide sequence of a P2A peptide (HEXA-HEXB). The functionality of the bicistronic construct containing the HEXA-HEXB genetic cassette was analyzed in a culture of HEK293T cells and human umbilical cord blood mononuclear cells (hUCBMCs). Our results showed that the enzymatic activity of HexA in the conditioned medium harvested from genetically modified HEK293T-HEXA-HEXB and hUCBMCs-HEXA-HEXB was increased by 23 and 8 times, respectively, compared with the conditioned medium of native cells. Western blot analysis showed that hUCBMCs-HEXA-HEXB secreted both completely separated HEXA and HEXB proteins, and an uncleaved protein containing HEXA + HEXB linked by the P2A peptide. Intravenous injection of genetically modified hUCBMCs-HEXA-HEXB to laboratory Wistar rats was carried out, and the HexA enzymatic activity in the blood plasma of experimental animals, as well as the number of live cells of immune system organs (spleen, thymus, bone marrow, lymph nodes) were determined. A significant increase in the enzymatic activity of HexA in the blood plasma of laboratory rats on days 6 and 9 (by 2.5 and 3 times, respectively) after the administration of hUCBMCs-HEXA-HEXB was shown. At the same time, the number of live cells in the studied organs remained unchanged. Thus, the functionality of the bicistronic genetic construct encoding cDNA of the HEXA and HEXB genes separated by the nucleotide sequence of the P2A peptide was shown in vitro and in vivo. We hypothesize that due to the natural ability of hUCBMCs to overcome biological barriers, such a strategy can restore the activity of the missing enzyme in the central nervous system of patients with GM2 gangliosidoses. Based on the obtained data, it can be concluded that intravenous administration of hUCBMCs with HexA overexpression is a promising method of the therapy for GM2 gangliosidoses. The animal protocol was approved by the Animal Ethics Committee of the Kazan Federal University (No. 23) on June 30, 2020.

Systematic improvements in lentiviral transduction of primary human natural killer cells undergoing ex vivo expansion

Transduction of primary human natural killer (NK) cells with lentiviral vectors has historically been challenging. We sought to evaluate multiple parameters to optimize lentiviral transduction of human peripheral blood NK cells being expanded to large numbers using a good manufacturing practice (GMP)-compliant protocol that utilizes irradiated lymphoblastoid (LCL) feeder cells. Although prestimulation of NK cells with interleukin (IL)-2 for 2 or more days facilitated transduction with vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped lentivirus, there was a subsequent impairment in the capacity of transduced NK cells to proliferate when stimulated with LCL feeder cells. In contrast, incubation of human NK cells with LCL feeder cells plus IL-2 before transduction in the presence of the TBK1 inhibitor BX795 resulted in efficient lentiviral integration (mean of 23% transgene+ NK cells) and successful subsequent proliferation of the transduced cells. Investigation of multiple internal promoter sequences within the same lentiviral vector revealed differences in percentage and level of transgene expression per NK cell. Bicistronic lentiviral vectors encoding both GFP and proteins suitable for the isolation of transduced cells with magnetic beads led to efficient transgene expression in NK cells. The optimized approaches described herein provide a template for protocols that generate large numbers of fully functional and highly purified lentivirus-transduced NK cells for clinical trials.

Novel bicistronic lentiviral vectors correct β-Hexosaminidase deficiency in neural and hematopoietic stem cells and progeny: implications for in vivo and ex vivo gene therapy of GM2 gangliosidosis

The favorable outcome of in vivo and ex vivo gene therapy approaches in several Lysosomal Storage Diseases suggests that these treatment strategies might equally benefit GM2 gangliosidosis. Tay-Sachs and Sandhoff disease (the main forms of GM2 gangliosidosis) result from mutations in either the HEXA or HEXB genes encoding, respectively, the α- or β-subunits of the lysosomal β-Hexosaminidase enzyme. In physiological conditions, α- and β-subunits combine to generate β-Hexosaminidase A (HexA, αβ) and β-Hexosaminidase B (HexB, ββ). A major impairment to establishing in vivo or ex vivo gene therapy for GM2 gangliosidosis is the need to synthesize the α- and β-subunits at high levels and with the correct stoichiometric ratio, and to safely deliver the therapeutic products to all affected tissues/organs.Here, we report the generation and in vitro validation of novel bicistronic lentiviral vectors (LVs) encoding for both the murine and human codon optimized Hexa and Hexb genes. We show that these LVs drive the safe and coordinate expression of the α- and β-subunits, leading to supranormal levels of β-Hexosaminidase activity with prevalent formation of a functional HexA in SD murine neurons and glia, murine bone marrow-derived hematopoietic stem/progenitor cells (HSPCs), and human SD fibroblasts. The restoration/overexpression of β-Hexosaminidase leads to the reduction of intracellular GM2 ganglioside storage in transduced and in cross-corrected SD murine neural progeny, indicating that the transgenic enzyme is secreted and functional. Importantly, bicistronic LVs safely and efficiently transduce human neurons/glia and CD34+ HSPCs, which are target and effector cells, respectively, in prospective in vivo and ex vivo GT approaches.We anticipate that these bicistronic LVs may overcome the current requirement of two vectors co-delivering the α- or β-subunits genes. Careful assessment of the safety and therapeutic potential of these bicistronic LVs in the SD murine model will pave the way to the clinical development of LV-based gene therapy for GM2 gangliosidosis.

P5725Identification of novel cardiomyogenic factors by transcriptome analysis of conditionally immortalized atrial myocytes

Abstract Background Cardiac development involves the properly timed expression of cardiomyogenic differentiation factors (CDFs). CDFs have mainly been discovered using animal models and, more recently, pluripotent stem cell-derived cardiomyocytes (PSC-CMCs). These models are, however, laborious, time-consuming and costly. Also, cardiomyogenic differentiation of CMCs is heterogeneous and yields phenotypically immature CMCs. Recently, our research group generated a monoclonal line of conditionally immortalized atrial myocytes, called iAM-1. After removal of the proliferation stimulus these cells spontaneously and synchronously differentiate into mature atrial myocytes, making them ideally suited for transcriptome analysis and the discovery of novels factors involved in cardiomyogenic differentiation. Methods Whole transcriptome analysis of iAM-1 cells was performed at 9 different time points during cardiomyogenic differentiation and subsequent dedifferentiation by RNA sequencing. Six genes upregulated during cardiomyogenic differentiation were selected for knockdown studies in differentiating iAM-1 cells. Each of these genes was targeted by a bicistronic lentiviral vector (LV) driving expression of a specific short hairpin RNA (shRNA) and of enhanced green fluorescent protein (eGFP). Knockdown effects during cardiomyogenic differentiation were studied by immunocytology. The LVs were also used in primary neonatal atrial and ventricular rat cardiomyocytes to study the role of the selected genes in cardiomyocyte homeostasis. Results Whole transcriptome analysis of differentiating iAM-1 cells identified the dynamic expression levels of ± 13.000 genes, including the expected profile for genes known to play a role in atrial myocyte differentiation, like Nkx2–5, Tbx3, Tbx5 and Nppa. Six genes with an unknown role in cardiomyocyte differentiation and homeostasis were selected based on significant upregulation during iAM-1 differentiation, substantial mRNA levels and selective expression in cardiac tissue. Inhibiting gene expression by lentiviral RNA interference resulted for Nkx2–5 as well as for 3 out of 6 target genes in disturbed iAM-1 differentiation, as evinced by loss of sarcomeric cross-striations. Similar effects were observed in shRNA-expressing (i.e. eGFP-positive) primary atrial and ventricular neonatal rat myocytes. Taken together, these results highlight the importance of these novel genes during cardiomyogenic differentiation and homeostasis in atrial as well as ventricular myocytes. Conclusions Transcriptome analysis of cardiomyogenic differentiation in conditionally immortalized atrial myocytes combined with genetic knockdown experiments led to the identification of several novel factors involved in the differentiation and homeostasis of atrial and ventricular myocytes. These results highlight the suitability of iAM-1 as model for fundamental research of cardiomyogenic differentiation. Acknowledgement/Funding ZonMW

T-Box20 inhibits osteogenic differentiation in adipose-derived human mesenchymal stem cells: the role of T-Box20 on osteogenesis

BackgroundSkeletal development and its cellular function are regulated by various transcription factors. The T-box (Tbx) family of transcription factors have critical roles in cellular differentiation as well as heart and limbs organogenesis. These factors possess activator and/or repressor domains to modify the expression of target genes. Despite the obvious effects of Tbx20 on heart development, its impact on bone development is still unknown.MethodsTo investigate the consequence by forced Tbx20 expression in the osteogenic differentiation of human mesenchymal stem cells derived from adipose tissue (Ad-MSCs), these cells were transduced with a bicistronic lentiviral vector encoding Tbx20 and an enhanced green fluorescent protein.ResultsTbx20 gene delivery system suppressed the osteogenic differentiation of Ad-MSCs, as indicated by reduction in alkaline phosphatase activity and Alizarin Red S staining. Consistently, reverse transcription-polymerase chain reaction analyses showed that Tbx20 gain-of-function reduced the expression levels of osteoblast marker genes in osteo-inductive Ad-MSCs cultures. Accordingly, Tbx20 negatively affected osteogenesis through modulating expression of key factors involved in this process.ConclusionThe present study suggests that Tbx20 could inhibit osteogenic differentiation in adipose-derived human mesenchymal stem cells.

Overexpression of MicroRNA-148b-3p stimulates osteogenesis of human bone marrow-derived mesenchymal stem cells: the role of MicroRNA-148b-3p in osteogenesis

BackgroundMesenchymal stem cells (MSCs) are attractive choices in regenerative medicine and can be genetically modified to obtain better results in therapeutics. Bone development and metabolism are controlled by various factors including microRNAs (miRs) interference, which are small non-coding endogenous RNAs.MethodsIn the current study, the effects of forced miR-148b expression was evaluated on osteogenic activity. Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transduced with bicistronic lentiviral vector encoding hsa-miR-148b-3p or -5p and the enhanced green fluorescent protein. Fourteen days post-transduction, immunostaining as well as Western blotting were used to analyze osteogenesis.ResultsOverexpression of miR-148b-3p increased the osteogenic differentiation of human BM-MSCs as demonstrated by anenhancement of mineralized nodular formation and an increase in the levels of osteoblastic differentiation biomarkers, alkaline phosphatase and collagen type I.ConclusionsSince lentivirally overexpressed miR-148b-3p increased osteogenic differentiation capability of BM-MSCs, this miR could be applied as a therapeutic modulator to optimize bone function.

Production of breast cancer cell lines expressing viral gene VIF

Introduction: Virion Infectivity factor (Vif) is a protein required for lentiviruses successful infection of human target cells, through the degradation of host restriction factors, APOBEC3 [1]. In the last years, the activity of APOBEC3 deaminases has also been associated with mutation patterns found in cancer cells, including breast cancer cells [2]. We hypothesize that Vif protein can be used to inhibit APOBEC3 avoiding breast cancer progression and resistance to chemotherapy. This hypothesis should be tested in breast cancer cells expressing Vif proteins from HIV-1 (Vif1), and HIV-2 (Vif2) treated with chemotherapy agents. The aim of this work was the production of breast cell lines expressing constitutively Vif1 and Vif 2 proteins, using lentiviruses as a gene-delivering system. Materials and methods: Vif1 and Vif2 delivering lentivirus vectors were obtained using the fourth generation lentiviral packaging systems (Clontech). Briefly, Vif 1 and Vif 2 genes were cloned in a bicistronic lentiviral expression vector (pLVX-IRES-zsGreen1) which allowed the co-expression of Vif and the reporter gene (ZsGreen) from a single mRNA transcript. VSV-G pseudotyped lentiviruses were produced by transfection of packaging cells, Lenti-X 293T cells (Clontech). Viruses were then used to transduce MCF10A and HCC1806 breast cells. Transduced cells stably expressing fluorescent marker were isolated by Fluorescence Activated Cell Sorting (FACS-AriaIII). The isolated cells were expanded and characterized. RNA was extracted from the cell lines MCF10A-Vif and HCC1806-Vif, and cDNA synthesis was performed. Expression of Vif was confirmed by PCR amplification of cDNA and APOBEC3G, 3B and 3C expression levels were also confirmed by qRT-PCR (TaqMan Technology). Results: We obtained high-titer of Vif delivering lentiviruses used to transduce the breast cells. FACS allowed us to obtain sorting populations with highly purified transduced cells (sorting populations with 97 to 99% purity). Characterization of the new cell populations showed that lentiviral constructs integrated either Vif1 or Vif2 genes into genomic DNA. Moreover, Vif genes are expressed in these populations with no significant effect on the mRNA levels of APOBEC3G, 3C, and 3B (∼1.5 to 2 fold increases). Discussion and conclusions: The genomic DNA of MCF10A (normal breast cell line) and HCC1806 (breast cancer cell line) were modified with Vif gene (and ZsGreen gene) integration generating new stable breast cell lines. In the near future, we will be able to confirm or not that inhibition of APOBEC3 by Vif sensitize to chemotherapy the breast cancer cells, and hopefully decrease recurrent relapses. We believe that the use of Vif to inhibit APOBEC3 is a promising therapeutic tool model with application in breast cancer disease.

Cardiomyogenic differentiation of human adipose-derived mesenchymal stem cells transduced with Tbx20-encoding lentiviral vectors.

Ischemic heart disease often results in myocardial infarction and is the leading cause of mortality and morbidity worldwide. Improvement in the function of infarcted myocardium is a main purpose of cardiac regenerative medicine. One possible way to reach this goal is via stem cell therapy. Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types but display limited cardiomyogenic differentiation potential. Members of the T-box family of transcription factors including Tbx20 play important roles in heart development and cardiomyocyte homeostasis. Therefore, in the current study, we investigated the potential of Tbx20 to enhance the cardiomyogenic differentiation of human adipose-derived MSCs (ADMSCs). Human ADMSCs were transduced with a bicistronic lentiviral vector encoding Tbx20 (murine) and the enhanced green fluorescent protein (eGFP) and analyzed 7 and 14 days post transduction. Transduction of human ADMSCs with this lentiviral vector increased the expression of the cardiomyogenic differentiation markers ACTN1, TNNI3, ACTC1, NKX2.5, TBX20 (human), and GATA4 as revealed by RT-qPCR. Consistently, immunocytological results showed elevated expression of α-actinin and cardiac troponin I in these cells in comparison to the cells transduced with control lentiviral particles coding for eGFP alone. Accordingly, forced expression of Tbx20 exerts cardiomyogenic effects on human ADMSCs by increasing the expression of cardiomyogenic differentiation markers at the RNA and protein level.

Expression of Duplex shRNAs through a Lentiviral Vector against Cellular and Viral Genes Inflicts Sustained Inhibition of Hepatitis C Virus Replication

Background: The RNAi-based transient therapeutic approach has been well explored for its potential against the hepatitis V virus (HCV). However, to achieve a sustained virological response, a consistent presence of siRNA is needed and it can be achieved by constitutively expressing shRNAs. In this context, the lentiviral vector has emerged as an attractive tool for shRNA delivery against HCV. Methods: We monitored HCV inhibition after single and multiple rounds of siRNA treatments against La autoantigen and HCV-NS5B in Huh-7.5 cells infected with the FL-J6/JFH chimeric HCV strain. A bicistronic self-inactivating third-generation lentiviral vector expressing shRNA under U6 and H1 promoters was constructed. To ascertain the long-term HCV inhibition, cells were transduced with lentiviral vectors and HCV inhibition was monitored by RT-PCR and Western blotting at regular intervals. Results: We observed transient antiviral activity after a single round of siRNA treatment, and consecutive rounds of treatments with siRNA demonstrated a sustained HCV inhibition. Delivery of duplex shRNA expressing lentiviral vectors provided constant expression of shRNA leading to synergistic and sustained HCV inhibition. Conclusion: A lentiviral vector-based delivery system is a “single-shot” therapeutic strategy. It can express duplex shRNA for long-term synergistic inhibition of HCV and qualify as a promising therapeutic approach for sustained inhibition of HCV replication.

Selection of an optimal promoter for gene transfer in normal B cells.

Gene transfer into normal quiescent human B cells is a challenging procedure. The present study aimed to investigate whether it is possible to increase the levels of transgene expression by using various types of promoters to drive the expression of selected genes-of-interest. To produce lentiviral particles, the present study used the 2nd generation psPAX2 packaging vector and the vesicular stomatitis virus -expressing envelope vector pMD2.G. Subsequently, lentiviral vectors were generated containing various promoters, including cytomegalovirus (CMV), elongation factor-1 alpha (EF1α) and spleen focus-forming virus (SFFV). The present study was unable to induce satisfactory transduction efficiency in quiescent normal B cells; however, infection of normal B cells with Epstein-Barr virus resulted in increased susceptibility to lentiviral transduction. In addition, the SFFV promoter resulted in a higher level of transgene expression compared with CMV or EF1α promoters. As a proof-of concept that this approach allows for stable gene expression in normal B cells, the present study used bicistronic lentiviral vectors with genes encoding fluorescent reporter proteins, as well as X-box binding protein-1 and binding immunoglobulin protein.

Lentiviral expression system for the purification of secreted proteins from human cell cultures.

BackgroundRecombinant proteins of therapeutic use are ideally produced in human cells to ensure appropriate co- and post-translational modifications. However, purification of secreted proteins from the culture media is impeded by low expression from transfected cell lines and the presence of serum proteins. Here we describe a simple and cost-effective approach based on lentiviral vector-mediated gene delivery and expression of a secreted His-tagged protein from human embryonic kidney 293 T cells and direct affinity chromatography purification from the cell culture media.ResultsUsing a protein-based HIV entry inhibitor, soluble CD4 (sCD4), we demonstrated that 293 T cells transduced with a lentiviral vector mediated over 10-fold higher secretion of sCD4 in comparison to 293 T cells transfected with the corresponding plasmid. Secretion of sCD4 increased with the dose of the lentiviral vector up to a multiplicity of infection of 50. Exchanging the native signal peptide of sCD4 with the signal peptide of human alpha-1 antitrypsin increased expression by 50 %. There was no difference in expression from a monocistronic or bicistronic lentiviral vector. Reduction of the serum concentration in the culture media had no significant effect on the secretion of sCD4. Small-scale purification from 50 ml of culture media with reduced serum content yielded up to 1 mg of pure sCD4. Purified sCD4 bound to recombinant HIV envelope glycoprotein 120 (Env gp120) and inhibited HIV entry at concentrations comparable to published results.ConclusionThe procedure outlined in this study can be performed without the need for specialized reagents or equipment and could easily be adapted by any laboratory. Furthermore, the method could be used to produce sCD4 fusion proteins or other His-tagged proteins.

Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model.

Advances in the treatment of metastatic clear cell renal cell carcinoma (ccRCC) have led to improved progression-free survival of many patients; however the therapies are toxic, rarely achieve durable long-term complete responses and are not curative. Herein we used a single bicistronic lentiviral vector to develop a new combination immunotherapy that consists of human anti-carbonic anhydrase IX (CAIX)-targeted chimeric antigen receptor (CAR) T cells engineered to secrete human anti-programmed death ligand 1 (PD-L1) antibodies at the tumor site. The local antibody delivery led to marked immune checkpoint blockade. Tumor growth diminished 5 times and tumor weight reduced 50–80% when compared with the anti-CAIX CAR T cells alone in a humanized mice model of ccRCC. The expression of PD-L1 and Ki67 in the tumors decreased and an increase in granzyme B levels was found in CAR T cells. The anti-PD-L1 IgG1 isotype, which is capable of mediating ADCC, was also able to recruit human NK cells to the tumor site in vivo. These armed second-generation CAR T cells empowered to secrete human anti-PD-L1 antibodies in the ccRCC milieu to combat T cell exhaustion is an innovation in this field that should provide renewed potential for CAR T cell immunotherapy of solid tumors where limited efficacy is currently seen.

Selective suppression of tumor cells by a tumor-specific bicistronic lentiviral vector

Lentiviral gene delivery is now considered as a major candidate in the future of cancer therapy. To avoid common side-effects associated with methods of cancer therapy, the survivin promoter shows great promise due to its high expression level in multiple cancers. In this research, using a 2A peptide, Noxa was coexpressed with hemagglutinin neuraminidase (HN) driven by the survivin promoter (Surp). Coding genes of Noxa and HN were connected to the upstream and downstream of a 2A peptide using SOE PCR. After cloning of Noxa-2A-HN and Surp into the lentiviral vector, the final construct was transfected to SK-BR3 tumor cells and human embryonic kidney 293T cells. Quantitative real-time PCR implied efficient transfection and integration of the lentiviral vector in target cells. Western blotting analysis was performed to verify the expression of Noxa and HN and we determined cell viability by MTT calorimetric assay after transfection. Our results showed that the expression of Noxa and HN, though not utterly specific, was relatively higher in tumor cells and led to the selective suppression of tumor cells, which eventually resulted in a cell survival reduction of 76%. In conclusion, the novel pLN2AH-Surp lentiviral vector proved the potential for further in vivo and clinical experiments.

The stimulatory effects of eNOS/F92A-Cav1 on NO production and angiogenesis in BMSCs.

Nitric oxide (NO) is generated in endothelial cells by endothelial nitric oxide synthase (eNOS). Caveolin-1 (Cav1) inhibits eNOS function and NO production. Modifying Cav1 scaffold domain, in particular Phenylalanine at position 92 (F92) is critical for the inhibitory actions of Cav1 toward eNOS. The aims of this study were to investigate the effect of enhanced NO production in term of in vitro angiogenesis on rat bone marrow derived mesenchymal stem cells (BMSCs) transduced with a novel bicistronic lentiviral vector co-expressing eNOS and mutant Cav1 (F92A). A bicistronic eNOS/F92-Cav1 lentiviral vector was constructed, and used to transduce rat BMSCs. The expression of eNOS and VEGF protein were confirmed by western-blot. NO production was detected by the greiss assay and in vitro angiogenesis was assessed by matrigel assisted capillary tube formation. The cell viability was evaluated using a Cell Counting Kit (CCK)-8. The bicistronic eNOS/F92A-Cav1 lentiviral vector increased eNOS and VEGF protein expression, NO production compared to controls. In vitro capillary formation was increased in eNOS-F92A transduced cells and cell viability was not affected by transduction. Transduction of rat BMSCs with an eNOS-F92A-Cav1 lentiviral vector can increase NO production by enhancing eNOS protein expression. The increased NO production did not reduce cell viability. This study demonstrates that genetic modification of BMSCs to enhance NO producton could be applied in stem cell based therapeutic approaches to treat diseases such as pulmonary arterial hypertension (PAH) which is characterized by decreased endothelial NO release.