Lentiviral Transduction Efficiency Research Articles

Abstract 5255: A B7-H3-CAR-modified Vδ1 T cells showed potentanti-solid tumor potential

Abstract Background: CAR-αβT cells have made limited advancement in treating solid tumors. Vδ1 T cells, a subtype of γδT cells, are rare in human peripheral blood, but they are the main tissue-resident immune cells found in epithelial and mucosal tissues. This suggests that Vδ1 T cells might be a potential modality for treating solid tumors. To explore this possibility, we hypothesized that Vδ1 T cells modified with B7-H3 CAR (B7-H3-CAR-Vδ1 T) could have great potential of anti-solid tumor activity, making them a promising candidate for further development as a universal cellular therapy product. Methods: A proprietary manufacturing procedure to produce Vδ1 T cells from the leukopak of healthy donors has been established under GMP compliance. The immune checkpoint molecule B7-H3 was selected as the target. The allogeneic B7-H3-CAR-Vδ1 T cells carrying IL-2 (UTAA06), which displayed the most prolonged anti-tumor activity, were chosen for comprehensive functional validation. Single-cell sequencing and mass spectrometry flow cytometry were utilized to characterize the biology of UTAA06. In vivo effectiveness of UTAA06 was confirmed in animal models. Results: A clinical-grade manufacturing process for Vδ1 T cells is developed. This process results in a purity of over 95% of Vδ1 T cells, with an expansion of more than 20,000-fold, and a CAR lentiviral transduction efficiency of over 50%.žSingle-cell sequencing and mass cytometry analysis both demonstrated that the Vδ1 T cells produced by our approach exhibited high expression levels of natural cytotoxic receptors (including NKG2D, DNAM1, and NKp series receptors), chemokine receptors (such as CXCR3 and CXCR6), anti-tumor related molecules (such as CD27 and CD137), and low expression levels of inhibitory molecules (such as PD1 and LAG3).ž UTAA06 cells did not produce IL17A, which is a signal protein with pro-tumor activity. Meanwhile, UTAA06 demonstrated significant and specific inhibitory effects on the growth of various solid tumor cell lines.žUTAA06 cells were found to significantly suppress tumor cell growth in solid tumor mouse models. The complete remission (CR) for each tumor model was 83.3% of neuroblastoma (SK-N-AS, N=6), 66.7% of pancreatic cancer (SW1990, N=6), 100% of lung cancer (A549, N=6), and 70% of colorectal cancer (HCT-15, N=10) respectively. In addition, a potent capacity of UTAA06 to penetrate tumor tissues with a favorable safety profile in mice was observed. Conclusion: UTAA06 exhibited extraordinary anticancer properties both in vitro and in vivo against solid tumors. Additionally, the outstanding manufacturing process of UTAA06 will facilitate the clinical application of this product. Citation Format: Licui Jiang, Changsong Qi, Fengtao You, Shufen Xiang, Ping Zhang, Min Wang, Hai Wu, Kunmin Yan, Jiequn Huang, Huimin Meng, Nan Yang, Shirley Zhang, Lin Shen, Lin Yang. A B7-H3-CAR-modified Vδ1 T cells showed potentanti-solid tumor potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5255.

Классические и активирующие химерные антигенные рецепторы PD-1 как элемент мультитаргетного подхода в лечении гематологических и солидных новообразований

Aim. To generate anti-PD-L1 CAR-T effectors carrying extracellular domain PD-1 as antigen-recognizing site and to study their cytolytic activity as well as to functionally assess the anti-PD-L1 CAR-T effectors in vitro with a view to apply them in multi-targeted tumor therapy.
 Materials & Methods. Chimeric antigen receptor PD-1 was constructed using molecular cloning of PD-1 antigen-recognizing region (12–170 amino acids) into mammalian expression plasmid vector adding activation and co-stimulatory domains. Primary Т-lymphocytes of healthy donor peripheral blood mononuclear fraction were derived by expanding monoclonal antibody combination on surface markers CD3/CD28. Anti-PD-L1 CAR-T effectors were obtained by lentiviral transduction of primary T-lymphocyte genome of a healthy donor. Chimeric antigen receptor PD-1 expression and transduction efficiency were assessed by flow cytofluorometry. Specific cytotoxicity of the anti-PD-L1 CAR-T effectors was analyzed in vitro by means of real-time cytotoxicity assay (RTCA) with HeLa_PD-L1 target cell line co-cultivation. The level of cytokines IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A was assessed by flow cytofluorometry using Human Th1/Th2/Th17 CBA Kit (BD, USA).
 Results. The efficiency of lentiviral transduction and the proportion of the anti-PD-L1 CAR-T effectors were 42 %. The specificity of cytotoxic response of the anti-PD-L1 CAR-T effectors with a low effector/tumor ratio (1:20) was verified during HeLa_PD-L1 co-cultivation by a 1.5-fold decrease in the cell index (CI = 0.738) versus control (CI = 1.0645). The increase in synthesis of cytokines IL-2 (1000 pg/mL), IL-6 (438.5 pg/mL), TNF-α (44 pg/mL), and IFN-γ (1034 pg/mL) during HeLa_PD-L1 target cell line co-cultivation confirms the functionality of the analyzed effector cells.
 Conclusion. Anti-PD-L1 chimeric antigen receptor was constructed and tested in vitro. Anti-PD-L1 CAR-T lymphocytes specifically recognize and promote the cytolysis of tumor target cells by increased secretion of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6, and IL-2. Chimeric antigen receptor PD-1 can be modified into chimeric switch receptor (CSR) by deleting CD3ζ-domain and can be used together with other CARs without predicted non-specific toxicity.

Optimisation of a primary human CAR-NK cell manufacturing pipeline.

Autologous chimeric antigen receptor (CAR) T-cell therapy of B-cell malignancies achieves long-term disease remission in a high fraction of patients and has triggered intense research into translating this successful approach into additional cancer types. However, the complex logistics involved in autologous CAR-T manufacturing, the compromised fitness of patient-derived Tcells, the high rates of serious toxicities and the overall cost involved with product manufacturing and hospitalisation have driven innovation to overcome such hurdles. One alternative approach is the use of allogeneic natural killer (NK) cells as a source for CAR-NK cell therapy. However, this source has traditionally faced numerous manufacturing challenges. To address this, we have developed an optimised expansion and transduction protocol for primary human NK cells primed for manufacturing scaling and clinical evaluation. We have performed an in-depth comparison of primary human NK cell sources as a starting material by characterising their phenotype, functionality, expansion potential and transduction efficiency at crucial timepoints of our CAR-NK manufacturing pipeline. We identified adult peripheral blood-derived NK cells to be the superior source for generating a CAR-NK cell product because of a higher maximum yield of CAR-expressing NK cells combined with potent natural, as well as CAR-mediated anti-tumor effector functions. Our optimised manufacturing pipeline dramatically improves lentiviral transduction efficiency of primary human NK cells. We conclude that the exponential expansion pre- and post-transduction and high on-target cytotoxicity make peripheral blood-derived NK cells a feasible and attractive CAR-NK cell product for clinical utility.

Optimal, Off-the-Shelf, CAR-iNKT Cell Platform-Based Immunotherapy for Multiple Myeloma

Autologous CAR-T cell immunotherapy targeting BCMA confers significantly improved progression free survival as compared to standard-of-care in relapsed/refractory multiple myeloma. However, despite >50% complete remission rates, patients continue to relapse. Of note, in current licenced BCMA CAR-T products, 4-1BB comprises the co-stimulatory domain of a 2 nd generation CAR. Expression of CD1d by myeloma plasma cells (PC) lends itself to immunotherapy with the CD1d-restricted, glycolipid-reactive iNKT cells which can be deployed ‘off-the-shelf’ without causing aGVHD. We hypothesised that ‘off-the-shelf’ anti-BCMA CAR-iNKT would be an alternative platform for the treatment of MM. We sought to identify the CAR design that would function optimally in conjunction with iNKT cells. To this end, we designed several different second (28z, 4-1BBz, OX40z) and third (28z-4-1BBz, 28z-OX40z) generation BCMA CARs and tested their anti-myeloma activity. Lentiviral transduction efficiency of iNKT cells was comparable for all five CARs and all five CAR-iNKT were able to kill two BCMA-expressing but not BCMA knock out myeloma cell lines in short- and long-term cytotoxicity assays. The level of cytotoxicity varied with donor, timing of the assay and cell lines, with the only consistent pattern seen with the 3 rdgeneration CD28-4-1BB CAR. This showed the least reactivity at low E:T ratios in both 4 and 16hrs assays for both donors tested. Testing cytotoxicity against primary myeloma PC (n=3) showed that the 3 rd gen CARs were less active than the three 2 nd gen counterparts. Short term proliferation assays using Incucyte Zoom demonstrated a higher proliferative potential of CD28z CAR-iNKT cells with CD28z-4-1BBz CAR showing the least proliferation. In a longer term 35-day assay, following stimulation with the iNKT cell ligand aGalCer, we found that CD28z CAR-iNKT cells showed the highest expansion. To further probe functional differences between the five CARs, we tested the corresponding CAR-iNKT cells in acoustic force avidity assays. These highlighted CD28z CAR-iNKT cells as having the highest avidity amongst CAR-iNKT cells with untransduced iNKT cells the lowest (Fig. 1A ). Using confocal microscopy, we explored how these differences in avidity impacted the ability of CAR-iNKT cells to shape the immune synapse (IS) and engage the cytolytic apparatus. We found that while conjugates of untransduced iNKT cells with myeloma cells all fell in the distal centrosome group, of the five CARs, CD28z and 4-1BBz CAR-iNKT conjugates recorded the highest and the lowest respectively proximal and docked conjugates, i.e., those signifying imminent or active discharge of the cytolytic granules. To delve further into the mechanism(s) of higher avidity and ability to established IS by CD28z CAR-iNKT, we compared the transcriptomes of CD28z vs 4-1BBz CAR-iNKT from three donors. We found 64 genes differentially expressed (14 up- and 50 down-regulated in CD28z CAR-iNKT, padj<0.05). Amongst the upregulated genes we identified, currently investigating genes that encode proteins that were previously implicated in the strength and duration of IS. To investigate the in vivo correlates of these functional assays, we deployed a xenograft myeloma model using MM1.S luciferase-expressing cells (Fig. 1B ). All five CAR iNKT cells were i.v transferred to myeloma-bearing mice at the limiting dose of 10 6 CAR+ cells/mouse and disease burden was monitored by bioluminescence. In line with the functional assays, we found that mice receiving CD28z CAR-iNKT cells demonstrated the lowest disease burden and survived the longest (p<0.01). While disease burden and survival of animals receiving CD28z-4-1BBz CAR-iNKT was the same as controls, 4-1BBz CAR-iNKT-treated mice showed significantly higher disease burden and shortened survival as compared to CD28z CAR-iNKT. Further disease burden assessment by flow-cytometry at sacrifice showed that CD28z CAR-iNKT-treated animals had the lowest (p<0.01) frequency of myeloma cells in their peripheral blood, bone marrow and spleen. Finally, in ongoing xenograft assays, we find that compared to 10 6 28z BCMA CAR-iNKT and 5x10 6 untransduced iNKT, 5x10 6 28z BCMA CAR-iNKT cells maintain better disease control (p=0.008, day7). We conclude that CD28z by enhancing effector-target avidity and strength of IS constitutes the optimal CAR design for clinical development of CAR-iNKT therapy of multiple myeloma.

B Cell Therapeutics

Plasma cells are specialized immune cells that can survive in the bone marrow for decades and continually secrete large amounts of antibodies, thus providing long-term, often lifelong protection from repeated exposures to a pathogen. The long-term survival and the capability to secrete high amounts of protein make plasma cells a potential candidate for stable delivery of therapeutic proteins and antibodies. However, engineering plasma cells or B cells has been challenging due to low lentiviral transduction efficiency, incompatibility with plasmid transfection and loss of episomal DNA expression over time. We have previously shown that human plasma cells can be engineered to secrete antibodies and proteins in in vitro culture systems via gene editing. In the current study, we have generated engineered murine plasma cells that can secrete the clotting factor IX (FIX). We hypothesize that the FIX-secreting plasma cells will engraft long-term in FIX-deficient immunocompetent mice and secrete therapeutic levels of FIX leading to protection from Hemophilia B. The standard of care for Hemophilia B is repeated intravenous infusion of recombinant FIX or AAV-based gene therapy. However, 30-60% humans have pre-existing immunity to AAV-based gene therapy and gene therapy can induce an immune response in people lacking pre-existing antibodies. Moreover, gene therapy is unsuitable for pediatric patients due to their rapid growth and proportional loss of AAV. Plasma cell-based therapy is lowly immunogenic and can be used for redosing in pediatric patients that require more product with age. Here, we will describe proof-of-concept studies showing that engineered murine plasma cells can engraft into immunocompetent mice without the need for conditioning and produce stable levels of protein for >10 weeks. We developed a three-step in vitro co-culture system that can be used to engineer and differentiate naïve B cells into plasma cells. These in vitro generated murine plasma cells had a comparable gene expression profile and phenotype to natural plasma cells and can engraft stably, in the absence of conditioning, into immunocompetent mice whereupon they secreted antibodies (Figure 1). Moreover, we show that plasma cells can be re-dosed to increase antibody levels without triggering a secondary immune response (Figure 1). For delivery of therapeutic proteins, we have developed adeno-associated virus-based homology directed repair reagents to edit murine B cells at several loci, including beta-2 microglobulin, Rosa-26, and the antibody heavy chain. By co-delivery of cis-linked fluorescent markers, we were able to sort and expand only edited cells, enabling enrichment of cells expressing a therapeutic protein. In proof-of-concept studies, we have used this approach to generate a human FIX-secreting plasma cell product with close to 90% edited cells at the end of the B cell culture. The enriched plasma cells generated 0.28 pg FIX/cell/day compared to 0.19 pg FIX/cell/day generated by the bulk edited cells (figure 2). We estimate that engraftment of 1 million edited plasma cells is sufficient to generate therapeutic levels of FIX upon (figure 2). We have developed reagents for a novel cell-based therapeutic tool with protein secretion capabilities and far-reaching applications. Out studies will provide insights into the feasibility and efficacy of a B cell based therapeutic product.

The RLR intrinsic antiviral system is expressed in neural retina and restricts lentiviral transduction of human Mueller cells

The retinoic acid-inducible gene I (RIG)-I-like receptor (RLR) family of RNA sensor proteins plays a key role in the innate immune response to viral nucleic acids, including viral gene delivery vectors, but little is known about the expression of RLR proteins in the retina. The purpose of this study was to characterize cell-specific expression patterns of RLR proteins in non-human primate (NHP) neural retina tissue and to examine if RLR pathway signaling restricts viral gene delivery transduction. Since RLR protein signaling converges at the mitochondrial antiviral signaling protein (MAVS), experiments were performed to determine if knockdown of MAVS affected FIVGFP transduction efficiency in the human Mueller cell line MIO-M1. Immunoblotting confirmed expression of RIG-I, melanoma differentiation-associated protein 5 (MDA5), laboratory of genetics and physiology 2 (LGP2), and MAVS proteins in MIO-M1 cells and NHP retina tissue. Double label immunofluorescence (IF) studies revealed RIG-I, LGP2, and MAVS were expressed in Mueller microglial cells in the NHP retina. In addition, LGP2 and MDA5 proteins were detected in cone and retinal ganglion cells (RGC). MDA5 was also present in a subset of calretinin positive amacrine cells, and in nuclei within the inner nuclear layer (INL). Knockdown of MAVS significantly increased the transduction efficiency of the lentiviral vector FIVGFP in MIO-M1 cells, compared to control cells. FIVGFP or AAVGFP challenge did not alter expression of the LGP2, MAVS, MDA5 or RIG-I genes in MIO-M1 cells or NHP retina tissue compared to media treated controls. Our data demonstrate that innate immune response proteins involved in viral RNA sensing, including MDA5, RIG-I, LGP2, and MAVS, are expressed in several cell types within the NHP neural retina. In addition, the MAVS protein restricts non-human lentiviral transduction efficiency in MIO-M1 cells.

Improving Lentiviral Transduction of Human Adipose-Derived Mesenchymal Stem Cells.

Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications. Polybrene is the most commonly used reagent to improve viral gene transfer efficiency in laboratory research; however, it is not approved for human use and has also been shown to impair MSC proliferation and differentiation. Therefore, there is a need for optimized transduction protocols that can also be adapted to clinical settings. LentiBOOST (LB) and protamine sulfate are alternative transduction enhancers (TEs) that can be manufactured to current Good Manufacturing Practice standards, are easily applied to existing protocols, and have been previously studied for the transduction of human CD34+ hematopoietic stem cells. In this study, we investigated these reagents for the enhancement of lentiviral transduction of adipose-derived MSCs. We found that the combination of LB and protamine sulfate could yield comparable or even superior transduction efficiency to polybrene, with no dose-dependent adverse effects on cell viability or stem cell characteristics. This combination of TEs represents a valuable clinically compatible alternative to polybrene with the potential to significantly improve the efficiency of lentiviral transduction of MSCs for gene therapy applications.

P423: ADAPTING CRISPR CAS9 DROPOUT SCREENS TO IN VIVO PDX MODELS OF ACUTE LEUKEMIAS

Background: Acute leukemias require better treatment and targeted therapies represent interesting future therapeutic options. Such therapies precisely inhibit molecules with essential function, also called vulnerabilities or dependencies, so that leukemia cells die once the target is inhibited. Aims: Here, we aimed at identifying therapeutic targets on a patient individual level and in the surrounding of a living organism. Towards this aim, we established CRISPR Cas9 dropout screens in PDX models of acute leukemias in vivo. Methods: Primary patient acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) samples were transplanted into immunocompromised NSG mice and re-passaged to develop serially transplantable PDX models. To overcome limited transduction efficiency inherent to PDX leukemia cells, lentiviruses were used. Results: We first elaborated on the maximum library size to be used in PDX models in vivo. As only a limited number of PDX cells home into mice, library size is limited to ensure the coverage of the library. We used, screening sgRNAs as genetic barcodes in Cas9 negative samples to determine the maximum library size upon next generation sequencing (NGS); a regular distribution of all sgRNAs recovered from mice at the end of the experiments indicated a suitable library size. We found that PDX ALL models tolerated a larger library compared to PDX AML models that might reflect lower intra-sample heterogeneity and higher leukemia stem cell frequency in ALL compared to AML. To perform knockout screens, a split-construct for Cas9 was used to reduce plasmid size and optimize lentiviral transduction efficiency into PDX cells; concomitantly split-GFP was used to enrich cells expressing both Cas9 split plasmids by flow cytometry. A customized library consisting of 146 target genes combined with positive and negative control genes was designed at 5 sgRNAs per gene using the CLUE platform (www.crispr-clue.de) (Becker et al., Nucleic Acids Res. 2020) and cloned into a lentiviral vector backbone. The sgRNA library vectors include either a puromycin-resistance cassette or an H-2Kk surface marker, each combined with a BFP fluorescent marker. This allows to determine transduction efficiency by FACS-based detection of BFP and to enrich the transduced PDX cells either by puromycin selection or by H-2Kk-MACS selection. Input samples were collected after library transduction and enrichment to >90% CRISPR/Cas9 sgRNA library positive cells, while remaining cells were injected into immunocompromised NSG mice, grown in vivo and re-isolated at advanced disease stage. Analysis of input and endpoint samples using NGS enabled to determine the frequency of sgRNAs and bioinformatically compare the abundance of different sgRNAs between the control and the samples of interest using MAGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout). A significant sgRNA dropout was characterized by a p-value below 0.05 and a FDR below 0.1. The screening experiments led to the top 10 depleted dropout genes for ALL and AML PDX samples. Besides individual dropouts of the different samples, also shared dropouts were detected. Especially sample overlapping dropouts might represent interesting starting points for new therapeutic options. Summary/Conclusion: In summary, we have established a CRISPR Cas9 screening pipeline, which allows investigating therapeutic targets on a patient-individual level in ALL PDX models and, for the first time, in AML PDX models in vivo.

Abstract 4202: Dual chlorotoxin and methylguanine-DNA methyltransferase (MGMT) γδ-T cells for drug resistant immunotherapy

Abstract High-grade gliomas are the most aggressive and refractory of brain tumors. Historical approaches to treating glioblastoma (GBM) using chimeric antigen receptor modified T cells (CAR-T) have been challenging, without a lasting benefit to patients, often due to tumor heterogeneity and a lack of tumor specific antigens. We report preliminary in vitro design and optimization of a γδ T cell-based CAR-T using Chlorotoxin (CLTX-CAR), a peptide isolated from the venom of the Israeli Deathstalker Scorpion that efficiently binds to GBM and other tumor cells with limited cross reactivity to normal tissues. This CLTX-CAR (CTLX-CAR-noZ) omits a CD3z signaling domain to maintain the natural innate targeting and tumor recognition properties of the γδ T cell through its endogenous T cell and NKG2D receptors, and CD16-based antibody-dependent cellular cytotoxicity (ADCC). This construct also includes our Drug Resistant Immunotherapy (DRI) platform that incorporates an additional p140K-MGMT encoding gene into the construct. This confers resistance to alkylating agents such as temozolomide (TMZ) that allows our DRI γδ T cells to remain viable and functional when combined with chemotherapy to force high-expression of natural killer group D ligands (NKG2DL) on the surface of tumor cells. As expected, the non-signaling, CLTX-CAR-noZ modified Jurkat cells did not result in T cell activation, as compared to the highly activated CD69 corresponding to signaling CLTX-CAR-CD3z transduced Jurkat cells. Conversely, CTLX-CAR-noZ transduced γδ T cells demonstrated higher cytotoxicity against U87MG and U251MG GBM cell lines over unmodified control γδ T cells. Dual-CLTX-CAR constructs lacking CD3z and using 2x CLTX peptides as the targeting domain (dCLTX-CAR-noZ) exhibited up to 90% lentiviral transduction efficiency into γδ T cells. When co-cultured in suspension with GBM cells in vitro, dCLTX-CAR-noZ γδ T cells demonstrated an average cytotoxicity of 62.1% against U87MG-GFP cells at an E:T ratio of 4:1 in comparison to 35.4% with non-transduced γδ T control cells. Time-lapsed microscopic recording over a period of 15hrs also verified killing of U251MG-GFP cells by dCLTX-CAR-noZ γδ T cells when cocultured with GBM cells at an E:T ratio of 2:1. Serial-killing of tumor cells by γδ T cells was documented. We are advancing this approach to xenograft animal models and as a clinical development candidate (INB-300) for both GBM and other extracranial tumors. Citation Format: Lei Ding, Yanjie Li, Evan Klumpp, Lawrence Lamb. Dual chlorotoxin and methylguanine-DNA methyltransferase (MGMT) γδ-T cells for drug resistant immunotherapy [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 4202.

CD9 and folate receptor overexpression are not sufficient for VSV-G-independent lentiviral transduction.

Extracellular vesicles have become a research focus for their potential as therapeutic vehicles that carry cargo substances. Extracellular vesicles may origin from the endosomal compartment and share several characteristics with the envelope of lentiviruses. A previous study reported that constitutive expression of the tetraspanin CD9, an extracellular vesicle marker, not only increases vesicle secretion from cells, but has also a positive effect on lentiviral transduction efficiency. Moreover, it was shown that expression of CD9 on the viral envelope in absence of viral glycoproteins was sufficient for the transduction of mammalian cells. In this study, we investigate the effect of CD9 and folate receptor alpha, a GPI-anchored protein, on biosynthesis and transduction efficiency of vesicles carrying lentiviral vectors. We demonstrate that neither CD9 nor FRα nor the combination of both were able to mediate a significant transduction of therapeutic vesicles carrying lentiviral RNA. Further studies are required to identify endogenous mammalian proteins that can be used for pseudotyping of viral envelopes to improve viral targeting without inducing immune responses.

Minimal impact of ZAP on lentiviral vector production and transduction efficiency

The antiviral protein ZAP binds CpG dinucleotides in viral RNA to inhibit replication. This has likely led to the CpG suppression observed in many RNA viruses, including retroviruses. Sequences added to retroviral vector genomes, such as internal promoters, transgenes, or regulatory elements, substantially increase CpG abundance. Because these CpGs could allow retroviral vector RNA to be targeted by ZAP, we analyzed whether it restricts vector production, transduction efficiency, and transgene expression. Surprisingly, even though CpG-high HIV-1 was efficiently inhibited by ZAP in HEK293T cells, depleting ZAP did not substantially increase lentiviral vector titer using several packaging and genome plasmids. ZAP overexpression also did not inhibit lentiviral vector titer. In addition, decreasing CpG abundance in a lentiviral vector genome did not increase its titer, and a gammaretroviral vector derived from murine leukemia virus was not substantially restricted by ZAP. Overall, we show that the increased CpG abundance in retroviral vectors relative to the wild-type retroviruses they are derived from does not intrinsically sensitize them to ZAP. Further understanding of how ZAP specifically targets transcripts to inhibit their expression may allow the development of CpG sequence contexts that efficiently recruit or evade this antiviral system.

A systems pharmacology model for gene therapy in sickle cell disease.

We developed a mathematical model for autologous stem cell therapy to cure sickle cell disease (SCD). Experimental therapies using this approach seek to engraft stem cells containing a curative gene. These stem cells are expected to produce a lifelong supply of red blood cells (RBCs) containing an anti‐sickling hemoglobin. This complex, multistep treatment is expensive, and there is limited patient data available from early clinical trials. Our objective was to quantify the impact of treatment parameters, such as initial stem cell dose, efficiency of lentiviral transduction, and degree of bone marrow preconditioning on engraftment efficiency, peripheral RBC numbers, and anti‐sickling hemoglobin levels over time. We used ordinary differential equations to model RBC production from progenitor cells in the bone marrow, and hemoglobin assembly from its constituent globin monomers. The model recapitulates observed RBC and hemoglobin levels in healthy and SCD phenotypes. Treatment simulations predict dynamics of stem cell engraftment and RBC containing the therapeutic gene product. Post‐treatment dynamics show an early phase of reconstitution due to short lived stem cells, followed by a sustained RBC production from stable engraftment of long‐term stem cells. This biphasic behavior was previously reported in the literature. Sensitivity analysis of the model quantified relationships between treatment parameters and efficacy. The initial dose of transduced stem cells, and the intensity of myeloablative bone marrow preconditioning are predicted to most positively impact long‐term outcomes. The quantitative systems pharmacology approach used here demonstrates the value of model‐assisted therapeutic design for gene therapies in SCD.

The Engineered MARCH8-Resistant Vesicular Stomatitis Virus Glycoprotein Enhances Lentiviral Vector Transduction.

Lentiviral vectors are one of the most commonly used viral delivery systems for gene therapy. Vesicular stomatitis virus-G envelope glycoprotein (VSV G)-pseudotyped lentiviral vectors have been widely used in clinical studies for treatment of virus infections and genetic deficient diseases. However, the efficiency of lentiviral vector transduction has been long recognized as a limiting factor in clinical gene therapy application, especially in transducing hematopoietic stem cells. MARCH8 (membrane-associated RING-CH 8), an E3 ubiquitin ligase, has been reported to target and downregulate VSV G. Results in this study show that MARCH8 induces ubiquitination and lysosome degradation of VSV G, and knockout of MARCH8 in virus-producing cells increases lentiviral vector transduction by elevating the level of VSV G protein. We then engineered VSV G mutant that has the lysine residues in the cytoplasmic domain substituted for arginine, and showed that this G mutant resists degradation by MARCH8, and allows the enhancement of transduction efficiency of lentiviral vector particles than the parental VSV G protein. This engineered VSV G mutant thus further advances the lentiviral vector system as a powerful tool in gene therapy.

Efficient lentiviral transduction method to gene modify cord blood CD8+ T cells for cancer therapy applications

Adoptive T cell therapy utilizing tumor-specific autologous T cells has shown promising results for cancer treatment. However, the limited numbers of autologous tumor-associated antigen (TAA)-specific T cells and the functional aberrancies, due to disease progression or treatment, remain factors that may significantly limit the success of the therapy. The use of allogeneic T cells, such as umbilical cord blood (CB) derived, overcomes these issues but requires gene modification to induce a robust and specific anti-tumor effect. CB T cells are readily available in CB banks and show low toxicity, high proliferation rates, and increased anti-leukemic effect upon transfer. However, the combination of anti-tumor gene modification and preservation of advantageous immunological traits of CB T cells represent major challenges for the harmonized production of T cell therapy products. In this manuscript, we optimized a protocol for expansion and lentiviral vector (LV) transduction of CB CD8+ T cells, achieving a transduction efficiency up to 83%. Timing of LV treatment, selection of culture media, and the use of different promoters were optimized in the transduction protocol. LentiBOOST was confirmed as a non-toxic transduction enhancer of CB CD8+ T cells, with minor effects on the proliferation capacity and cell viability of the T cells. Positively, the use of LentiBOOST does not affect the functionality of the cells, in the context of tumor cell recognition. Finally, CB CD8+ T cells were more amenable to LV transduction than peripheral blood (PB) CD8+ T cells and maintained a more naive phenotype. In conclusion, we show an efficient method to genetically modify CB CD8+ T cells using LV, which is especially useful for off-the-shelf adoptive cell therapy products for cancer treatment.

Establishing functional lentiviral vector production in a stirred bioreactor for CAR-T cell therapy

ABSTRACT As gene delivery tools, lentiviral vectors (LV) have broad applications in chimeric antigen receptor therapy (CAR-T). Large-scale production of functional LV is limited by the adherent, serum-dependent nature of HEK293T cells used in the manufacturing. HEK293T adherent cells were adapted to suspension cells in a serum-free medium to establish large-scale processes for functional LV production in a stirred bioreactor without micro-carriers. The results showed that 293 T suspension was successfully cultivated in F media (293 CD05 medium and SMM293-TII with 1:1 volume ratio), and the cells retained the capacity for LV production. After cultivation in a 5.5 L bioreactor for 4 days, the cells produced 1.5 ± 0.3 × 107 TU/mL raw LV, and the lentiviral transduction efficiency was 48.6 ± 2.8% in T Cells. The yield of LV equaled to the previous shake flask. The critical process steps were completed to enable a large-scale LV production process. Besides, a cryopreservation solution was developed to reduce protein involvement, avoid cell grafting and reduce process cost. The process is cost-effective and easy to scale up production, which is expected to be highly competitive.

Lentiviral delivery of combinatorial CAR/CRISPRi circuit into human primary T cells is enhanced by TBK1/IKK\u025b complex inhibitor BX795

BackgroundAdoptive transfer of engineered immune cells is a promising strategy for cancer treatment. However, low transduction efficiency particularly when large payload lentiviral vectors are used on primary T cells is a limitation for the development of cell therapy platforms that include multiple constructs bearing long DNA sequences. RB-340-1 is a new CAR T cell that combines two strategies in one product through a CRISPR interference (CRISPRi) circuit. Because multiple regulatory components are included in the circuit, RB-340-1 production needs delivery of two lentiviral vectors into human primary T cells, both containing long DNA sequences. To improve lentiviral transduction efficiency, we looked for inhibitors of receptors involved in antiviral response. BX795 is a pharmacological inhibitor of the TBK1/IKKɛ complex, which has been reported to augment lentiviral transduction of human NK cells and some cell lines, but it has not been tested with human primary T cells. The purpose of this study was to test if BX795 treatment promotes large payload RB-340-1 lentiviral transduction of human primary T cells.MethodsTo make the detection of gene delivery more convenient, we constructed another set of RB-340-1 constructs containing fluorescent labels named RB-340-1F. We incorporated BX795 treatment into the human primary T cell transduction procedure that was optimized for RB-340-1F. We tested BX795 with T cells collected from multiple donors, and detected the effect of BX795 on T cell transduction, phenotype, cell growth and cell function.ResultsWe found that BX795 promotes RB-340-1F lentiviral transduction of human primary T cells, without dramatic change in cell growth and T cell functions. Meanwhile, BX795 treatment increased CD8+ T cell ratios in transduced T cells.ConclusionsThese results indicate that BX795 treatment is effective, and might be a safe approach to promote RB-340-1F lentiviral transduction of human primary T cells. This approach might also be helpful for other T cell therapy products that need delivery of complicated platform via large payload lentiviral vectors.

Construction of a novel lentiviral vector knocking down PD-1 via microRNA and its application in CAR-T cells

By inserting microRNAs into the intron of EF1α promoter, we constructed a novel lentiviral vector knocking down PD-1 gene via microRNA and applied it to CAR-T cells. Lentiviral transduction efficiency and PD-1-silencing efficiency were detected by flow cytometry. PD-1 expression was detected by Western blotting. Relative expression of microRNA was measured by Q-PCR. Cytotoxicity of CAR-T cells based on this vector was tested by luciferase bioluminescence and flow cytometry. Compared with lentiviral vector with microRNA transcribed by U6 promotor, the transduction efficiency of lentiviral vector with microRNA which was inserted into the intron of EF1α promoter was more significant, and the knockdown rate of PD-1 was more than 90%, which was validated by flow cytometry and Western blotting. And the relative expression level of microRNA in Jurkat cells transduced with this novel lentiviral vector was shown by Q-PCR. Compared with normal CAR-T cells, CAR-T cells based on this vector showed stronger cytotoxicity against PD-L1 positive Raji cells. We successfully constructed a novel lentiviral vector that knocked down PD-1 via microRNA and verified the superiority of its transduction efficiency and knockdown efficiency of PD-1. CAR-T cells based on this vector can exert a more powerful cytotoxicity, thus providing theoretical support for the subsequent treatment of PD-L1 positive tumors.

Purification of Human CD34+CD90+ HSCs Reduces Target Cell Population and Improves Lentiviral Transduction for Gene Therapy

Hematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant, and infectious diseases. We have shown in a nonhuman primate gene therapy and transplantation model that the CD34+CD90+ cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. In this study, we show the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy. Alternative HSC enrichment strategies include the purification of CD133+ cells or CD38low/– subsets of CD34+ cells from human blood products. We directly compared these strategies to the isolation of CD90+ cells using a good manufacturing practice (GMP) grade flow-sorting protocol with clinical applicability. We show that CD90+ cell selection results in about 30-fold fewer target cells in comparison to CD133+ or CD38low/– CD34+ hematopoietic stem and progenitor cell (HSPC) subsets without compromising the engraftment potential in vivo. Single-cell RNA sequencing confirmed nearly complete depletion of lineage-committed progenitor cells in CD90+ fractions compared to alternative selections. Importantly, lentiviral transduction efficiency in purified CD90+ cells resulted in up to 3-fold higher levels of engrafted gene-modified blood cells. These studies should have important implications for the manufacturing of patient-specific HSC gene therapy and gene-engineered cell products.

An improved strategy for CRISPR/Cas9 gene knockout and subsequent wildtype and mutant gene rescue.

A fluorescence marker mOrange was inserted to the popular pLentiCrispr-V2 to create pLentiCrispr-V2-mOrange (V2mO) that contained both a puromycin selection and a fluorescent marker, making viral production and target transduction visible. Lentiviruses packaged with this plasmid and appropriate guide RNAs (gRNAs) successfully knocked out the genes RhoA, Gli1, and Gal3 in human gastric cancer cell lines. Cas9-gRNA editing efficiency could be estimated directly from Sanger electropherograms of short polymerase chain reaction products around the gRNA regions in Cas9-gRNA transduced cells. Single cloning of transduced target cell pools must be performed to establish stable knockout clones. Rescue of wildtype (RhoA and Gal3) and mutant (RhoA.Y42C) genes into knockout cells was successful only when cDNAs, where gRNAs bind, were modified by three nucleotides while the amino acid sequences remained unchanged. Stringent on-target CRISPR/Cas9 editing was observed in Gal3 gene, but not in RhoA gene since RhoA.Y42C already presented a nucleotide change in gRNA5 binding site. In summary, our improved strategy added these advantages: adding visual marker to the popular lentiviral system, monitoring lentiviral production and transduction efficiencies, cell-sorting Cas9+ cells in target cells by fluorescence-activated cell sorting, direct estimation of gene editing efficiency of target cell pools by short PCR electropherograms around gRNA binding sites, and successful rescue of wildtype and mutant genes in knockout cells, overcoming Cas9 editing by modifying cDNAs.

Process for an efficient lentiviral cell transduction.

The combination of lentiviruses with techniques such as CRISPR-Cas9 has resulted in efficient and precise processes for targeted genome modification. An often-limiting aspect, however, is the efficiency of cell transduction. Low efficiencies with particular cell types and/or the high complexity of lentiviral libraries can cause insufficient representation. Here, we present a protocol that yielded substantial increases in transduction efficiency in various cell lines in comparison to several other procedures.