Self-inactivating Lentiviral Vector Research Articles

Optimized human CYP4B1 in combination with the alkylator prodrug 4-ipomeanol serves as a novel suicide gene system for adoptive T-cell therapies.

Engineering autologous or allogeneic T cells to express a suicide gene can control potential toxicity in adoptive T-cell therapies. We recently reported the development of a novel human suicide gene system that is based on an orphan human cytochrome P450 enzyme, CYP4B1, and the naturally occurring alkylator prodrug 4-ipomeanol. The goal of this study was to systematically develop a clinically applicable self-inactivating lentiviral vector for efficient co-expression of CYP4B1 as an ER-located protein with two distinct types of cell surface proteins, either MACS selection genes for donor lymphocyte infusions after allogeneic stem cell transplantation or chimeric antigen receptors for retargeting primary T cells. The U3 region of the myeloproliferative sarcoma virus in combination with the T2A site was found to drive high-level expression of our CYP4B1 mutant with truncated CD34 or CD271 as MACS suitable selection markers. This lentiviral vector backbone was also well suited for co-expression of CYP4B1 with a codon-optimized CD19 chimeric antigen receptor (CAR) construct. Finally, 4-ipomeanol efficiently induced apoptosis in primary T cells that co-express mutant CYP4B1 and the divergently located MACS selection and CAR genes. In conclusion, we here developed a clinically suited lentiviral vector that supports high-level co-expression of cell surface proteins with a potent novel human suicide gene.

458. Development of a Stable Producer Cell Line for Scalable Lentiviral Vector Production for Gene Therapy of Hemoglobinopathie

Current manufacturing of clinical grade lentiviral vectors (LVVs) for gene therapy applications commonly relies on transient transfection of adherent 293T cells. Improvements in production efficiency and scalability would provide value in meeting the needs for increased amounts of vector required for clinical development. An inducible producer cell line grown in suspension culture represents a potentially more scalable manufacturing process for LVV production which eliminates the need for costly plasmid and transfection reagents. We have engineered a packaging cell line by introducing doxycycline-inducible Gag-Pol, Rev, and VSVG envelope genes into a suspension cell line. Packaging cell clones were isolated by single cell sorting and screened by qPCR for the presence of the delivered genes. Virus production was assessed by transient transfection of a lentiviral vector and doxycycline treatment. A titer of up to 5.0E6 transduction units per milliliter (TU/mL) was observed with packaging cell line stability demonstrated after greater than four months of continuous passage. Next, a self-inactivating lentiviral vector was excised from its plasmid backbone and ligated in vitro to a linear neomycin resistance cassette and used to transfect the lentiviral packaging cell line to generate a panel of producers. Following two weeks of G418 drug selection, adherent colonies were plucked and screened for viral production followed by single cell sorting to isolate individual producer clones. From five different plucked colonies with titers greater than 3.0E6 TU/mL, more than 100 clones were screened and 7 were identified that produced a harvest titer greater than 1.0E7 TU/mL. Four of these clones were successfully re-adapted to suspension and scaled up to 3L culture. LVV generated from individual producer clones was compared for the ability to transduce adult mobilized CD34+ hematopoietic stem cells (HSCs). Interestingly, the vector copy number (VCN) in CD34+ HSCs for the LVV derived from the producer cells varied between clones. Clones that produced the highest VCN have been chosen for further characterization in order to better understand the differences in HSC transduction. These lentiviral producer cell lines represent an important first step toward the creation of a manufacturing process that can better support clinical and commercial development of HSC lentiviral gene therapy.

530. Lentiviral Vector Particles Pseudotyped with Wild-Type Baboon Endogenous Retrovirus (BAEV) Glycoprotein Outperform VSV-G Particles in Transducing Human CD34+ Cells Isolated from Cytokine-Mobilized Peripheral Blood or Bone Marrow

Lentiviral vector gene therapy for hematopoietic disorders caused by single gene mutation or deficiency has demonstrated success in clinical trials. However, efficient gene delivery is still challenging, requiring high multiplicity of infection (MOI) to achieve average vector copy numbers of at least 1 per cell. Conditions utilizing high MOI result in improved transduction rates and adequate copy number but these benefits can coincide with potential insertional mutagenesis. Attempting to achieve optimal gene transfer at reduced MOI, we directly compared self-inactivating (SIN) lentiviral vector particles encoding eGFP under control of a murine stem cell virus (MSCV) promoter/enhancer pseudotyped with envelope glycoproteins for BaEV or vesicular stomatitis virus (VSV). BaEV proteins were either full-length (BaWT) or lacked the terminal R-peptide (BaR-less), a version previously reported to improve infectious titer (TU, transducing units per mL) using transient production procedures. Using a standardized four-plasmid transient production protocol, cells transfected with R-less expression plasmids produced syncitia; an issue that resolved using BaWT. Titers were significantly higher for virus packaged with BaWT compared to R-less (BaR-less: 3.6 × 105 ± 6.6 × 104 N=12; BaWT 2.4 × 106 ± 4.5 × 105; n=10; p<0.001) determined by flow cytometry analysis of GFP expression in transduced HEK-293T cells. However, these values were 10-to 20-fold less than those observed for VSV-G (3.5 × 107 ± 4.5 × 106). Human CD34+ hematopoietic stem/progenitor cells isolated from bone marrow (BM) or cytokine-mobilized peripheral blood (mPB) of healthy donors were prestimulated and transduced overnight on retronectin-coated plates with BaWT particles at an MOI of 0.5, 1, 2, or 4. The following day, cells were either plated in methylcellulose to assess colony-formation (CFU), maintained in liquid culture (Bulk), or transplanted into NOD-scid IL2Rgnull (NSG) mice pre-conditioned with busulfan (n=10 mice/1×106 cells each). Gene transfer efficiency was gauged by expression of GFP following flow cytometry analysis of cells maintained in culture or visual inspection of colonies growing in methylcellulose using an inverted fluorescence microscope after 6 or 12 days, respectively. Transduction efficiency increased with viral MOI reaching peak levels using an MOI of 2 (Bulk, 93% GFP+; CFU, 98% GFP+). Six NSG mice survived to 14 weeks post-transplant and demonstrated engraftment of CD45+ cells ranging from 19 to 54% (39 ± 13%; mean ± SD) and GFP marking ranging from 32 to 59% (41 ± 10%) determined by flow cytometry. Finally, we compared GFP-encoding particles pseudotyped with VSV-G or BaWT normalizing for levels of p24 quantified by ELISA (BaWT, 1155.0 pg/ml, 3.3 × 106 TU; VSV-G, 1164.9 pg/ml, 5.1 × 107 TU) to account for differences in calculated titer. Prestimulated mPB CD34+ cells were transduced overnight and placed into methylcellulose or transplanted into 15 NSG mice each. Methylcellulose colonies were analyzed for GFP after 12 days of growth (BaWT, 95.6%; VSV-G, 76.6%). Transplanted mice will be analyzed at 16-18 weeks post-transplant to measure engraftment and GFP expression. We believe the stability of producer cells expressing BaWT, the high efficiency of transduction at low MOI, and the ability to transduce adequate numbers of cells with unconcentrated virus supernatant warrant continued optimization of transient production methods and development of a stable producer cell line utilizing BaWT envelope as an alternative to VSV-g.

2. Identification and Ranking of Different Chromatin Insulators to Block Vector-Driven Enhancer-Mediated Insertional Mutagenesis In Vivo

Aimed at refining the safety profile of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications we investigated the impact of chromatin insulators (CI) on vector-mediated genotoxicity. Specifically, we studied four recently identified CI whose function is mediated by CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates, and cloned these CI in the LTRs of a SIN. LV with a strong enhancer/promoter in internal position (CI. SIN. LV).We took advantage of two sensitive in vivo genotoxicity assays based on the systemic injection of LVs in newborn tumor-prone Cdkn2a−/− and Cdkn2a+/− mice, that allow to measure vector-induced genotoxicity as accelerated tumor onset proportional to the genotoxic potential of the tested LV.CI. SIN. LVs displayed slightly not statistically significant improvement in the median survival time vs. the uninsulated SIN. LV counterpart (ranging from 193.5 to 214 days vs. 186 days, respectively) in Cdkn2a−/− mice.In Cdkn2a+/− mice, two insulated vectors significantly improved the median survival time, which resulted non-statistically different from Mock mice (450 and 511 vs. 505.5 days respectively), while the other two CI. SIN. LVs studied resulted to be still slightly genotoxic (median survival time: 412 and 429.5 days). To gain more insights on the safety profile of these LVs we retrieved and analyzed the vector integration sites (IS) (n>14000 IS) and identified common integration sites (CIS) in the murine tumors generated in our experimental framework and in both murine models.In Cdkn2a−/− mice, uninsulated SIN. LV-induced tumors harbored activating integrations targeting Map3k8 oncogene, while tumors obtained in mice treated with two out of four different insulated LVs significantly reduced the frequency of tumors with Map3k8-activating insertions. The reduced targeting frequency of Map3k8 was accompanied by a skewing of integrations inactivating Pten, Rasa1 or other tumor-suppressors, an escape genotoxicity mechanism on which insulators cannot act.In Cdkn2a−/+ mice we identified different predominant CIS genes targeted by the different insulated vectors. These data show that heterozygous Cdkn2a−/+ mice allow discriminating between more subtle shades of genotoxicity of the different vector designs and are therefore instrumental to understand the different molecular mechanisms of insertional mutagenesis and ways to avoid them. Interestingly by comparing the results from both in vivo assays we observed that one CI displayed superior safety profile in terms of significant improvement in the median survival time and/or in terms of reduced oncogenic CIS identified.In summary we validated new human-origin insulator elements able to block SIN. LV genotoxicity in vivo. Overall, these data highlight the importance of stringent in vivo genotoxicity testing of improved vector versions and support the use of CI for future gene therapy applications.

455. Automated Lentiviral Transduction of T Cells with CARS Using the CliniMACS Prodigy

T lymphocytes are exhibiting enormous potential in early phase clinical trials in patients with haematological malignancies. However, the complex procedures involved in the ex-vivo modification of T cells is labour intensive and currently limited to a small number of centres with the required infrastructure and expertise. To simplify procedures and widen applicability for clinical therapies, we have adopted the CliniMACS Prodigy platform to automate these multifaceted cell manufacturing processes. We found efficient lentiviral transduction of human T cells in a GMP compliant manner and demonstrate the feasibility of implementing this device in the manufacture of chimeric antigen receptor (CAR) based T cell immunotherapies. Eight automated T cell Transduction (TCT) processes have been performed using a self-inactivating third generation lentiviral vector encoding a CD19 specific CAR (CAR19), three of which using a clinical grade vector for final stage validation studies. Either fresh or cryopreserved peripheral blood mononuclear cells from non-mobilised leukapheresis from healthy donors were loaded onto the CliniMACS Prodigy using single use closed tubing sets. All cells were cultured in TexMACS media and activated with TransAct™. Transduction occurred 24-28hours post activation and cells were expanded for up to 8 days in the CentriCult-Unit enabling stable cell culture conditions and automated cell feeding. Finally, cells were harvested and cryopreserved to assess the functional capabilities of CAR19 T cells. Small scale comparison transductions were run in parallel to assess the efficiency of the automated T-cell modification process. The mean T cell expansion during automated cell cultivation was 16.2x (range 5.4-28.4x) with an average yield over 8 days of 14.5×108 total lymphocytes from a starting lymphocyte count of 1×108. This was comparable to cell expansion achieved in manual small scale experiments under the same activation conditions, 18.1x (range 11.5-27.5x). Successful transduction was also observed in the automated system with a mean transduction efficiency of 49.1% (range 23.9-64.9% CAR19+ T cells) which was again similar to transduction efficiencies achieved in manual small scale controls (mean of 51.8%). Flow cytometry analysis of the final product showed a high purity of CD45+CD3+ T cells (mean = 94%) with a relatively high frequency of CD8+ T cells (mean 48.9%). Further immunophenotyping revealed the bulk of the T cell product to be a mix of stem cell memory and central memory based on CD45RA, CD62L and CD95 expression with minimal expression of the T cell exhaustion marker PD-1. Additionally, CAR19 T cells generated using the automated procedure, were functional in cytotoxic activity both in vitro and in an in vivo mouse model. Importantly, these data are comparable to data generated from previous GMP manufacture of CAR19 T cells using the WAVE bioreactor with X-Vivo15 media and magnetic beads conjugated with anti-CD3/CD28 antibodies with the added advantage of increased simplicity in manufacture. In summary, we have demonstrated the feasibility of the CliniMACS Prodigy platform for the generation of CAR+ T cells for adoptive immunotherapy. Automated activation, transduction and expansion resulted in clinically relevant doses of CAR19 T cells with greatly reduced ‘hands-on’ operator time. Given the closed-system nature of the device, and automated features, the CliniMACS Prodigy should widen applicability of T-cell engineering beyond centres with highly specialised infrastructures.

529. Lentiviral Vectors with a Reduced Splicing Interference Potential Have a Significantly Improved Safety Profile In Vivo

Genotoxicity assays based on systemic vector injection into newborn tumor-prone Cdkn2a−/− and Cdkn2a+/− mice has shown that self-inactivating (SIN) lentiviral vector (LV) harboring strong or moderate enhancer/promoters in internal position caused acceleration in hematopoietic tumor onset compared to control mice. Integration site (IS) analysis in vector-induced tumors showed that oncogene activation or tumor suppressor inactivation occurs by mechanisms of aberrant splicing and/or enhancer-mediated overexpression of cellular genes. Although oncogene activation may be reduced by the use of SIN design, moderate cellular promoters and insulator sequences, how to reduce genotoxic splicing-capture events and aberrant transcript formation triggered by vector integration is still unclear. Here, we specifically designed SINLVs harboring sequences complementary to microRNAs (mirT sequence) which are active in hematopoietic cells (mir223 and mir142-3p) within the SIN LTR (mirsT-LTR.LV) or in the vector backbone and outside the gene expression cassette (mirT-LV). In our rationale, the mirT sequences when incorporated in an aberrantly generated mRNA would be selectively degraded through the miRNA pathway. Thus, by taking advantage of our in vivo models, we assessed the genotoxicity of these LVs with mirT sequences. Systemic injection of mirsT-LTR.LV (N=34) and mirT-LV (N=39) in Cdkn2a−/− mice did not cause any significant acceleration in hematopoietic tumor onset compared to un-injected mice (N=37) or mice injected with a SINLV that does not harbor mirT sequences (N=24). Similar results have been obtained after injection of the same vectors in Cdkn2a+/− mice (N=29 mirsT-LTR.LV, N=25 mirT-LV, N=40 un-injected and N=15 injected control mice). To gain additional information on the safety profile of these vectors, we performed IS analysis (N>10,000) in tumor-derived DNA. By this analysis, we previously found that Map3k8 activation by LV insertions was the major mechanism of genotoxicity when prototypical SINLVs were injected into Cdkn2a−/− mice. Now, we found that mice treated with mirsT-LTR.LV and mirT-LV did not show any Map3k8 activating insertions, suggesting that the new vectors are efficient in preventing its activation and confirming their superior safety profile. Furthermore, as expected, Pten was the most frequently targeted gene in tumors derived from Cdkn2a−/− mice injected with the LVs harboring mirT sequences. Pten insertions mainly targeted exons, suggesting the potential inactivation of its transcription unit. Finally, we found that Sfi1 was the major Common Insertion Site (CIS) in Cdkn2a+/− mice injected with LVs harboring mirT sequences. This CIS gene however appears to be the product of an intrinsic bias of LV integration, rather than the result of a selection process. Overall, our studies showed that these new advanced design LVs have a significantly improved safety profile and could represent the vector design of choice in future gene therapy applications.

210. High Expression of Second Generation CD19 CAR with a 4-1BB Costimulatory Domain from a Retroviral Vector Impairs CAR T Cell Expansion by Enhancing Fas-Mediated Apoptosis

Recent clinical trials in patients with B cell malignancies demonstrated that CD19 CAR T cells could expand in vivo and eliminate target cells. Two main variants of the CD19 CAR have been successful - a retroviral construct with CD28 and CD3 zeta signaling domains (28. zeta) and a lentivirus with 4-1BB. zeta. While infused CD28. zeta CAR T cells rapidly expanded and produced immediate anti-tumor activity, 4-1BB. zeta CAR T cells persisted longer and had sustained anti-tumor activity. Although these differences may represent differences in the costimulation provided by each CAR, the choice of viral vector may also contribute to differences in performance. We compared the in vitro expansion and anti-tumor activity of T cells transduced with either 28. zeta or 4-1BB. zeta CD19 CAR using a standard retroviral vector SFG. We demonstrate that retroviral transduction of T cells with a 4-1BB. zeta CD19 CAR reduced T cell expansion 18 fold after 2 weeks of culture and impaired anti-tumor activity compared to cells transduced with a similar vector encoding CD28. zeta CD19 CAR. High expression of the 4-1BB. zeta CD19 CAR led to upregulation of Fas and relocation of intracellular FasL to the cell surface, resulting in co-localization of both proteins on the plasma membrane. Consistent with this finding, we observed a 5-fold increase in apoptosis of 4-1BB. zeta CAR T cells which was completely reversed by chemical blockade of caspase-8, a key initiator caspase activated by Fas signaling. We also observed a gradual downregulation of 4-1BB. zeta CD19 CAR levels on surviving T cells suggesting that high CAR expression is toxic for T cells. In fact, reducing the level of 4-1BB. zeta CD19 CAR expression by inserting an IRES sequence upstream of the CAR normalized Fas levels on T cells and restored T cell expansion and anti-tumor activity. Similarly, transducing T cells with a self-inactivating lentiviral vector resulted in reduced levels of 4-1BB. zeta CD19 CAR expression allowed excellent expansion, similar to that of T cells with 28. zeta CD19 CAR. We also observed improved cytotoxicity of T cells with lower expression of 4-1BB. zeta CAR in co-culture assays with CD19+ cell lines Daudi (5-fold reduction in remaining tumor cells with lenti-CAR and 22-fold for IRES-CAR) and Raji (18-fold reduction for both lenti- and IRES-CAR constructs) and sustained performance in sequential killing assays.These studies reveal a mechanism that limits expansion and function of T cells expressing high levels of 4-1BB. zeta CD19 CAR and provide a basis for rational choice of expression systems and design of chimeric molecules that incorporate the 4-1BB signaling domain.

402. Bioengineering of Peripheral Blood Derived Gamma Delta T Cells in a Serum-Free Expansion Medium

To date the main source of effector cells in preclinical and clinical immunotherapy trials has been αβ T cells. Although cellular products utilizing these cells have shown some promise, there is a need for better control of antitumor immunogenicity, especially in the context of chimeric antigen receptor technologies. Therefore, as an alternative, we are investigating the use of a T cell subset, γΔ T cells. These are a potentially attractive cellular therapeutic as they can be expanded to clinically useful levels and have potent MHC independent intrinsic anti-tumorigenicity driven by their recognition of stress antigens on tumor cells. Our efforts have focused on development of a clinically reliable expansion and manufacturing process of genetically engineered γΔ T cells. In the current study we describe the optimization of culture conditions in serum free media (SFM) that can support robust expansion followed by genetic modification of the expanded cells. Peripheral blood mononuclear cells (PBMC) isolated from healthy donors were cultured in several SFMs including: OpTmizer, X-Vivo, SCGM, and AIM V as well as commonly used media containing serum, RPMI1640 supplemented with either 10% FBS or human serum. Complete growth media also included 2 mM L-glutamine, zoledronic acid (5µM added only at the start of culture), and IL-2 (either 100 or 1000 lU/ml added twice weekly). Of the SFM cultures, robust γΔ T cell expansion was only observed in OpTmizer supplemented with high-dose IL-2, which resulted in expansion and γΔ T cell percentages to levels sufficient for clinical use. Specifically, in OpTmizer, the percentage of γΔ T cells increased from a mean of 2.4% (0.8%-3.6%) in the starting product to 66.4% (35%-94.5%, 6 of 9 cultures were >70% γΔ T cell), resulting in 80-fold expansion at 14 days. The SFM-expanded γΔ T cells were evaluated for their anti-tumor cytotoxicity and shown to be equally effective at killing both hematologic and solid cancer cell lines as γΔ T cells expanded in serum containing media. Transduction efficiency was tested using GFP encoding self-inactivating lentiviral vectors (LV). These studies demonstrated that SIV and HIV transduce expanded γΔ T cells equally, and that compared to EF1α, cassettes employing an MSCV promoter provide greater transgene expression. The timing of LV addition was optimized with respect to i) γΔ T cell expansion, ii) LDL Receptor (LDL-R) expression, and iii) LV dose (3-4 consecutive transductions over 48 hours). The greatest transduction efficiencies were achieved in cultures with the greatest expansion. As expected a range of transduction efficiencies was observed for various donors with a mean of approximately 20%, which is similar to LV transduction levels observed for αβ T cell transductions with similar multiplicity of infection and rates of expansion. Therefore, an optimized strategy to expand and transduce γΔ T cells with demonstrated anti-cancer properties was developed. The utilization of SFM to expand these cells to clinically useful doses and redirection toward tumor cells via bioengineering can result in a clinically safe, widely applicable, and potentially more efficacious cellular immunotherapy.

759. Reduction of HLH-like Manifestations in Murine Model of Munc13-4 Deficiency Following Lentiviral Gene Transfer into Hematopoietic Stem and Progenitor Cells

Patients with mutations in UNC13D gene, coding for Munc13-4 protein, suffer from type 3 Familial hemophagocytic lymphohistiocytosis (FHL3), a life-threatening disorder of the immune system which represents 25% of all FHLs. Munc13-4 controls docking of lytic granules before they fused with the plasma membrane in cytotoxic T and NK lymphocytes and it's defect results in defective cytotoxic function of these cells. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, is partially successful even when a compatible donor is available because of the important inflammatory background of patients. In this context gene therapy could be a promising therapeutic option especially for those patients without any compatible donor. In this study, we took advantages from a murine model of FHL3, the Jinx mice, to investigate the feasibility of HSPC gene therapy for this pathology. Jinx mice do not spontaneously develop clinical features of hemophagocytic lymphohistiocytosis (HLH), but do so when infected with lymphocytic choriomeningitis virus (LCMV). We generated and used a self-inactivated lentiviral vector to complement HSC from Unc13d -/- (Jinx) mice and transplanted them back into the irradiated Jinx recipients. This transplantation led to the complete reconstitution of the immune system at levels comparable to that of control mice. The recipients were then challenged with LCMV. While Jinx mice reconstituted with GFP expressing HSPC developed leukopenia, anemia and body weight loss, characteristic of HLH in this murine model, gene corrected Jinx recipients developed only mild or no HLH manifestations. This reduction in HLH manifestation correlated with a significant reduction of virus titer in the liver and serum level of IFN-g and inflammatory cytokines. All these ameliorations might be explained by the restoration of cytotoxic function of CTLs as demonstrated in an in-vitro degranulation assay. Overall, this study provides data supporting the potential of HSC gene therapy in a FHL immune dysregulation such as UNC13D deficiency.

Gene Therapy for X-Linked Severe Combined Immunodeficiency: Where Do We Stand?

More than 20 years ago, X-linked severe combined immunodeficiency (SCID-X1) appeared to be the best condition to test the feasibility of hematopoietic stem cell gene therapy. The seminal SCID-X1 clinical studies, based on first-generation gammaretroviral vectors, demonstrated good long-term immune reconstitution in most treated patients despite the occurrence of vector-related leukemia in a few of them. This gene therapy has successfully enabled correction of the T cell defect. Natural killer and B cell defects were only partially restored, most likely due to the absence of a conditioning regimen. The success of these pioneering trials paved the way for the extension of gene-based treatment to many other diseases of the hematopoietic system, but the unfortunate serious adverse events led to extensive investigations to define the retrovirus integration profiles. This review puts into perspective the clinical experience of gene therapy for SCID-X1, with the development and implementation of new generations of safer vectors such as self-inactivating gammaretroviral or lentiviral vectors as well as major advances in integrome knowledge.

Production of Retrovirus-Based Vectors in Mildly Acidic pH Conditions.

Gene transfer vectors based on retroviridae are increasingly becoming a tool of choice for biomedical research and for the development of biotherapies in rare diseases or cancers. To meet the challenges of preclinical and clinical production, different steps of the production process of self-inactivating γ-retroviral (RVs) and lentiviral vectors (LVs) have been improved (e.g., transfection, media optimization, cell culture conditions). However, the increasing need for mass production of such vectors is still a challenge and could hamper their availability for therapeutic use. Recently, we observed that the use of a neutral pH during vector production is not optimal. The use of mildly acidic pH conditions (pH 6) can increase by two- to threefold the production of RVs and LVs pseudotyped with the vesicular stomatitis virus G (VSV-G) or gibbon ape leukemia virus (GALV) glycoproteins. Here, we describe the production protocol in mildly acidic pH conditions of GALVTR- and VSV-G-pseudotyped LVs using the transient transfection of HEK293T cells and the production protocol of GALV-pseudotyped RVs produced from a murine producer cell line. These protocols should help to achieve higher titers of vectors, thereby facilitating experimental research and therapeutic applications.

Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach.

Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4+ T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.

RD-MolPack technology for the constitutive production of self-inactivating lentiviral vectors pseudotyped with the nontoxic RD114-TR envelope

To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications.

Gene Therapy Using a Self-Inactivating Lentiviral Vector Improves Clinical and Laboratory Manifestations of Wiskott-Aldrich Syndrome

The X-linked disorder Wiskott-Aldrich Syndrome (WAS), characterized by thrombocytopenia, eczema, recurrent infections, autoimmunity and cancer, is typically treated with allogeneic transplantation. Somatic gene therapy (GT) using autologous CD34+ cells is a promising treatment alternative for high-risk patients lacking matched allogeneic donors, as it avoids graft versus host disease. GT using a γ-retroviral vector with intact viral enhancers was efficacious but had a high rate of leukemia due to insertional oncogenesis. We report here preliminary results of 4 WAS patients who underwent GT using a self-inactivating lentiviral (SIN-LV) vector, in which viral enhancers have been deleted and the human WAS cDNA is controlled by a 1.6kB fragment of the human WAS promoter (w1.6_hWASP_WPRE SIN-LV). WASP expression per cell induced by this vector was lower than in normal cells when examined in murine models and human trials. We hypothesized that the SIN-LV would readily correct immune abnormalities due to selective advantage for WAS protein (WASP) expressing T cells whereas correction of myeloid and platelet abnormalities would require high vector copy number (VCN) in the infused cells.Patients with severe WAS (clinical scores 3-5) were enrolled at a median age of 32 months (17 months-8 years). Patients 1 and 3 had detectable but low WASP expression. Patients 2 and 4 carried mutations that abrogated WASP expression but had evidence of somatic reversion in T and/or NK cells. CliniMACS purified CD34+ mobilized peripheral blood or bone marrow cells were transduced with the vector and infused after busulfan (12-15mg/kg) and fludarabine (120mg/m2) conditioning. CD34+ cell doses ranged from 6.3-24.91 x 106 cells/kg. VCN of the infused cells was variable (3.37, 1.34, 0.54, 1.01 copies/cell). Busulfan exposure was myeloablative or near-myeloablative in patients 1, 3, and 4 (81.2, 77.2, 84.5 mg*h/L) and submyeloablative in patient 2 (48.8 mg*h/L).All patients are alive with median follow-up of 13.5 months (9-24 months). All patients had improvement in eczema, remain platelet transfusion independent and have had no severe bleeding events. WASP expression in T cells was increased post-GT over baseline. Selective advantage for WASP expressing T cells was apparent in patients 1, 2 and 4, who had higher VCN in T cells at 6 months post-GT (0.93-2.21) than in B (0.48-1.7) or myeloid (0.13-0.89) cells. The presence of revertants in patients 2 and 4 did not appear to interfere with T cell reconstitution. In contrast patient 3 who had the highest WASP expression at baseline and the lowest VCN of infused cells (0.5 copies/cell), had the lowest VCN in T cells at 8 months (0.1 copies/cell). Defective T cell proliferation in response to anti-CD3 stimulation, characteristic of WAS, was improved post-GT. Next generation sequencing of T cell receptors in sorted naïve, memory and regulatory T cells revealed profound abnormalities of diversity, decreased entropy and marked clonal expansions pre-GT; most of these improved at 6 months post-GT. Cytoskeletal function in myeloid cells was highly abnormal pre-GT, as shown by absence of podosome formation in monocyte-derived dendritic cells (0-1% vs. 61% in controls). Podosome formation at 6 months post GT was improved but subnormal (4-40%). Only patient 1, who received the highest cell dose, the highest VCN, and myeloablative busulfan exposure had robust platelet reconstitution (pre-GT 24 versus 110 x 109/L 6 months post-GT) and high level gene marking in myeloid cells 0.89 copies/cell. At the same timepoint, patients 2, 3 and 4 had platelet counts of 20-30 x 109/L and correspondingly lower VCN in myeloid cells (0.13-0.27 copies/cell).No severe adverse events related to GT have occurred to date, with relatively short follow-up. Integration site analysis of sorted cells showed highly polyclonal reconstitution, with distributions of integration acceptor sites as expected for the lentiviral vector backbone. In summary, GT using a SIN-LV that induces expression of WASP at levels below wild type improved the clinical and laboratory manifestations of WAS, with better reconstitution in patients receiving cells with high VCN. DisclosuresOff Label Use: Off-label use of CliniMACS purified CD34+ cells.

Update of Results from the Northstar Study (HGB-204): A Phase 1/2 Study of Gene Therapy for Beta-Thalassemia Major Via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex-Vivo with a Lentiviral Beta AT87Q-Globin Vector (LentiGlobin BB305 Drug Product)

Background: Hematopoietic stem cell (HSC) gene transfer has the potential to induce globin production and mitigate or eliminate blood transfusions in patients with β-thalassemia major. Previously reported early results in subjects with β-thalassemia major participating in the ongoing HGB-205 and HGB-204 (Northstar) studies suggest that transplantation with autologous CD34+ cells transduced with a replication-defective, self-inactivating LentiGlobin BB305 lentiviral vector containing an engineered βA-T87Q-globin gene (LentiGlobin BB305 Drug Product) has a positive safety profile and leads to βA-T87Q globin production and can lead to transfusion independence. Here we provide an update on subjects treated with the LentiGlobin BB305 Drug Product in the Northstar study.Subjects and Methods: HSCs from transfusion-dependent β-thalassemia major subjects are mobilized by a combination of G-CSF and plerixafor, collected via apheresis, and CD34+ cells are selected and transduced with LentiGlobin BB305 lentiviral vector to produce drug product. Subjects undergo myeloablation with busulfan before LentiGlobin BB305 Drug Product infusion. Subjects are monitored for hematologic recovery, vector copy number, βA-T87Q-globinexpression, adverse events, and transfusion requirements after drug product infusion. Integration site analysis (ISA) and replication-competent lentivirus (RCL) assays are performed as part of the safety monitoring.Results: As of 31 July 2015, 10 subjects with transfusion-dependent β-thalassemia (β0/β0 [n=5], β0/βE [n=3], β0/β+ [n=1], and 1 heterozygous β0 genotype) have been infused with drug product. Before enrollment, subjects had received a median of 170 ml/kg/year (range: 137 to 233 ml/kg/year) of red blood cell (RBC) transfusions. The median age of the subjects was 26 years (range: 18 to 35 years), with 8 females and 2 males, and subjects received a median of 7.9 x 106 CD34+ cells/kg (range: 5.3 to 15.0 x106/kg) with median vector copy number of 0.8 (range: 0.3 to 1.5 copies/diploid genome).All subjects engrafted after drug product infusion; median time to engraftment was Day +17 (range +13 to +29) for neutrophils and Day +30 (range: +17 to +35) for platelets. The toxicity profile observed was consistent with autologous transplantation. To date, no ≥ Grade 3 drug-product-related adverse events have been observed, and there is no evidence of clonal dominance or replication competent lentivirus after a median follow-up of 198 days (range: 65 to 492 days) post-infusion. All subjects have detectable vector-derived HbAT87Q with a median peak level of 5.4 g/dL (range: 2.4 to 8.9 g/dL) ≥ 3 months post-infusion. The 7 subjects (3 β0/β0, 2 β0/βE, 1 β0/β+ and 1 heterozygous β0 genotype) monitored for at least 6 months post-infusion are making a median of 5.2 g/dL (range: 1.9 to 8.2 g/dL) of HbAT87Q with total Hb ranging from 8.5 to 11.1 g/dL at their last visit. Of these 7 subjects, 2 β0/β0 subjects have received a single RBC transfusion post-discharge, 1 β0/β0 subject remains transfusion dependent, and all 4 non-β0/β0 subjects have been RBC transfusion-free for ≥ 90 days (median 287 days of transfusion independence, range 171 to 396 days).Conclusion: Ten subjects with β-thalassemia major in the Northstar study have been infused with LentiGlobin BB305 Drug Product without ≥ Grade 3 drug product-related adverse events or evidence of clonal dominance. To date, LentiGlobin derived HbAT87Q is detectable in all infused subjects leading to transfusion independence or reduction in transfusion needs in almost all subjects. Gene therapy with the LentiGlobin BB305 is a promising modality for the treatment of patients with β-thalassemia major. DisclosuresWalters:ViaCord and AllCells, Inc: Other: Medical director. Kwiatkowski:ISIS: Membership on an entity's Board of Directors or advisory committees; Shire Pharmaceuticals and Sideris Pharmaceuticals: Consultancy; Sideris Pharmaceuticals: Consultancy; Novartis: Research Funding. Schiller:Sunesis: Honoraria, Research Funding. von Kalle:bluebird bio, Inc.: Consultancy. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Petrusich:bluebird bio, Inc.: Employment, Equity Ownership. Soni:bluebird bio, Inc.: Employment, Equity Ownership. Thompson:bluebird bio, Inc.: Consultancy, Research Funding.

Initial Results from Study Hgb-206: A Phase 1 Study Evaluating Gene Therapy By Transplantation of Autologous CD34+ Stem Cells Transduced Ex Vivo with the Lentiglobin BB305 Lentiviral Vector in Subjects with Severe Sickle Cell Disease

Background: Hematopoietic stem cell (HSC) gene transfer has the potential to reduce or eliminate the symptoms of severe sickle cell disease (SCD). Previously reported early results in a single subject with severe SCD from the ongoing study HGB-205 suggested that transplantation with autologous CD34+ cells transduced with a replication-defective, self-inactivating LentiGlobin BB305 lentiviral vector containing an engineered βA-T87Q -globin gene (LentiGlobin BB305 Drug Product) is well-tolerated and yields robust production of anti-sickling HbAT87Q at 6 months post-transplant. Here we provide initial data on subjects with severe SCD enrolled in the multi-center HGB-206 Study.Subjects and Methods: Subjects with severe SCD undergo HSC collection via bone marrow harvest. CD34+ cells are selected and transduced with LentiGlobin BB305 lentiviral vector. Subjects undergo myeloablation with intravenous busulfan, followed by infusion of LentiGlobin BB305 transduced cells. Subjects are monitored for hematologic engraftment, vector copy number (VCN), βA-T87Q -globinexpression and adverse events. Integration site analysis (ISA) and replication-competent lentivirus (RCL) assays are also performed. Prophylactic pRBC transfusions are continued in subjects with SCD who are on chronic transfusion pre-transplant to maintain HbS <30%, followed by gradual taper over time.Results: As of 31 July 2015, LentiGlobin BB305 drug product has been manufactured for 2 subjects with severe SCD, and 1 subject has been infused. Additional subjects are undergoing screening. Patient data are presented in Table 1. To date, the safety profile is consistent with autologous transplantation, without ≥ Grade 3 LentiGlobin BB305 Drug Product related adverse events.Follow-up of the infused subject and available data on additional subjects who may have undergone drug product infusion in the coming months will be presented.Conclusions: LentiGlobin BB305 transduced cells have been manufactured for 2 subjects with severe SCD, and 1 subject has been infused with the drug product. LentiGlobin BB305 gene therapy is a promising approach to decrease the HbS levels in patients with severe sickle cell disease. Table 1Preliminary dosing parameters and transplantation outcomesSubjectAge (years)/ Sex (M/F)GenotypeVCN in Drug ProductaCD34+ cell dose (x106/kg)Day of Neutrophil EngraftmentDrug Product- related Adverse EventsLast Study Visit (Months Post-infusion)206-113-130125/FβS/βS0.5/0.62.7NANAPending infusion206-114-130342/MβS/βS1.32.9Day +16None1MAs of 31 July 2015.F=female; M= Male for gender, and months post-infusion for visit; NA, not applicable; VCN, vector copy number;aIf more than one drug product was manufactured, the VCN of each drug product lot is presented. DisclosuresWalters:ViaCord and AllCells, Inc: Other: Medical director. Thompson:bluebird bio, Inc.: Consultancy, Research Funding. Kwiatkowski:ISIS: Membership on an entity's Board of Directors or advisory committees; Shire Pharmaceuticals and Sideris Pharmaceuticals: Consultancy; Sideris Pharmaceuticals: Consultancy; Novartis: Research Funding. von Kalle:bluebird bio, Inc.: Consultancy. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Sandler:bluebird bio, Inc.: Employment, Equity Ownership. Soni:bluebird bio, Inc.: Employment, Equity Ownership.

Preclinical Development and Validation of a Hematopoietic Stem Cell-Based Lentivector Treatment for Hemophilia a

Hemophilia A is a monogenic bleeding disorder currently treated by lifelong infusions of factor VIII (fVIII). The development of gene-based treatments for hemophilia A has been hampered by several biological hurdles including the low level expression of fVIII from genetically engineered target cells, the large transgene size, and transgene product immunogenicity. We now show that these major hurdles are overcome by i) using a bioengineered FVIII transgene (ET3) that expresses at 10 - 100-fold higher levels compared to other human FVIII sequences, ii) targeting hematopoietic stem cells (HSC) ex vivo using a self-inactivating (SIN) lentiviral vector (LV) encoding ET3, and iii) using a non-myeloablative conditioning regimen prior to HSC transplantation that results in transgene product associated immune tolerance. The clinical trial design and preclinical data were favorably reviewed by the RAC, Institutional Biosafety Committee, and FDA. However upon full-scale clinical production of the SIN-ET3-LV, an additional hurdle was discovered that resulted in low LV manufacturing titer, which we showed is due to promoter competition between the CMV/HIV-LTR and the internal promoter driving ET3 production (eg. EF1a). We show that high-titer recombinant LV encoding ET-3 can be achieved by replacing the EF1a internal promoter with one that has low activity in the 293T producer cells. CD68 is a monocyte-restricted promoter that is not active in 293T producer cell lines and thus minimizes promoter interference. Using a SIN-CD68-ET3-LV expression vector, high titer virus was generated using a laboratory scale production strategy as well as a 6 L scalable clinical LV production platform. Our scalable manufacturing process generated titers >2 x 109/ml, which is sufficient to support an ex vivo HSC transduction/transplantation pilot/phase I clinical trial. Late stage preclinical testing using clinically processed vector showed that the high titer virus efficiently transduces hematopoietic cell lines, as determined by quantitative PCR, and Southern analysis, which confirmed integrated transgene stability over 8 weeks. Efficient and stable gene transfer into murine Sca-1+ cells harvested from hemophilia A mice was also achieved. A dose response of LV addition to Sca-1 cells and FVIII expression was observed in transplanted mice. Expression of >40% normal FVIII levels was achieved at multiplicity of infections (MOIs) as low 20, and long term FVIII expression was observed post HSCT. No difference in the engraftment of genetically modified cells as compared to mock-modified cells was observed. In addition, efficient, stable and dose responsive gene marking (up to 0.6 VCN/cell) of human mobilized peripheral blood(mPB)-derived CD34+ cells was reproducibly achieved. Transduction of mPB-CD34+ cells at MOIs ranging from 1 to 125 had no effect on methylcellulose colony forming unit growth when comparing SIN-CD68-ET3-LV to non-modified cells. In addition, there was no effect on mPB-CD34+ cell engraftment of NSG mice when comparing modified and mock modified cells. Furthermore, next generation sequencing-based insertion site analysis showed integration of SIN-CD68-ET3-LV to be similar, with respect to integration events near oncogenes, to that of other vectors in current clinical trials. Collectively, this comprehensive, preclinical data set provides support for regulatory submissions to conduct a pilot/phase I clinical trial of a SIN-CD68-ET3-LV transduced-HSC transplantation product for the treatment of hemophilia A. DisclosuresDoering:Bayer Healthcare: Consultancy, Honoraria, Research Funding; Expression Therapeutics: Equity Ownership. Denning:Expression Therapeutics: Employment. Spencer:Expression Therapeutics: Equity Ownership.

Automated Lentiviral Transduction of T Cells with Cars Using the Clinimacs Prodigy

BACKGROUNDGenetically modified T cells have enormous potential for the treatment of relapsed and refractory haematopoietic malignancies. CD19-positive B-cell malignancies including acute lymphoblastic leukaemia (ALL), chronic lymphocytic leukaemia (CLL) or B cell non-Hodgkin lymphomas (NHL) have been shown to be an excellent target for adoptive immunotherapy with T cells expressing CD19-specific chimeric antigen receptors (CARs). The increasing need for genetically modified T cells is hampered by the limited number of centres with the required infrastructure and expertise to produce this complex therapeutic product. Ex vivo modification of T cells requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, Miltenyi Biotec has developed the CliniMACS Prodigy platform and is automating complex cell manufacturing processes. These have now been adapted for lentiviral transduction of T cells and we show the feasibility and effectiveness of the device for adoptive immunotherapy using chimeric antigen receptors.METHODSA self-inactivating third generation lentiviral vector encoding a CAR specific for CD19 (CAR19) was used for automated T-cell transductions (TCT). Using closed single-use tubing sets (TS520), fresh or cryopreserved peripheral blood mononuclear cells from non-mobilised leukapheresis collected from healthy donors were loaded onto the CliniMACS Prodigy, washed and activated in TexMACS media with TransAct, a polymeric nanomatrix activation reagent agonist for CD3 and CD28. Cells were transduced 24-48h after activation and expanded in the CentriCult-Unit of the tubing set, allowing for stable culture conditions as well as automated feeding and media exchange. Small and large scale comparison transductions were run in parallel to assess the efficiency of the automated T-cell modification. Finally, cells were harvested and cryopreserved to assess the functional capabilities of CAR19 T cells.RESULTSThree automated TCT runs were performed and continuously monitored to assess cell expansion, transduction efficiency and the phenotype of the final cell product. On average, expansion during automated cultivation was 11.7x (range: 5.4 - 22.8x) which was comparable to the expansion achieved in small scale controls (12.3x ± 1.2x). The average yield from the automated process was 11.8x108 total lymphocytes/run (ranging between 4 - 23.2x108 lymphocytes/run). Notably, this was comparable to existing CAR19 T cell manufacturing processes using a WAVE-Bioreactor. In all three runs in the Prodigy, successful transduction was observed with an average transduction efficiency of 32% CAR19-positive cells (range: 22- 45%). Again, this was similar to transduction efficiencies (32% CAR19-positive; range: 27-40%) in previous WAVE-production campaigns using X-Vivo15 media and magnetic beads conjugated with anti-CD3/CD28 antibodies for T-cell activation (Dynabeads). Flow cytometry analysis of the final cell product showed a high purity of CD45+/CD3+ cells (90%) as well as a relatively high frequency of CD8-positive cytotoxic T cells (56%). Immunophenotyping revealed high expression of CD45RA, CD62L, CD27 and CD95 with moderate expression of CCR7. Importantly, no significant difference in PD-1 expression was observed between automatically and manually processed cells. Finally, functional analysis showed cytotoxic activity as well as IFN-γ/TNF-α production upon co-cultivation with CD19-expressing target cells.CONCLUSIONIn summary, we have demonstrated the feasibility of the CliniMACS Prodigy for the generation of CAR+ T cells for adoptive immunotherapy. Automated activation, transduction and expansion resulted in clinically relevant doses of CAR19 T cells with very little 'hands-on' operator time. Given the closed-system nature of the device, and automated features, the CliniMACS Prodigy should widen applicability of T-cell engineering beyond centres with highly specialised infrastructures. DisclosuresMock*:Miltenyi Biotec GmbH: Research Funding. Nickolay*:Miltenyi Biotec GmbH: Research Funding. Peggs:Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership. Johnston:Miltenyi Biotec GmbH: Employment. Kaiser:Miltenyi Biotec GmbH: Employment. Pule:CELLECTIS: Research Funding; AUTOLUS: Employment, Equity Ownership, Research Funding; AMGEN: Honoraria; UCLB: Patents & Royalties. Thrasher:Miltenyi Biotec GmbH: Research Funding; Autolus Ltd: Consultancy, Equity Ownership, Research Funding. Qasim:Cellectis: Research Funding; Miltenyi Biotec GmbH: Research Funding; Autolus Ltd: Consultancy, Equity Ownership, Research Funding; Cell Medica: Research Funding.

Expansion and Bioengineering of Peripheral Blood-Derived Anti-Cancer Gd T Cells in Serum Free Media

Conventional immunotherapy relies on expansion and, in some settings, transduction of αβ T cells. Success is dependent on several highly variable patient-specific parameters including i) cell number obtained from harvest and ex vivo expansion, ii) targeting capacity of cells to tumors, iii) ability of infused T cells to survive and function in a immune evading tumor microenvironment (e.g., down-regulation of major histocompatibility complex (MHC) on the tumor cell surface), and iv) on-target and off-target adverse events such as graft vs host disease (GvHD). Following the the early success of a few anti-leukemia products, some progress has been made on expanding the applicability to a wider variety of hematologic and solid neoplasms but rapid progress has been hindered due to lack of efficacy and significant adverse event profiles. Thus, alternative cellular immunotherapy approaches such as gd T cells are being pursued. Although gd T cells represent a small fraction (2-5%) of circulating peripheral blood mononuclear cells (PBMC), they are ideal candidates for immunotherapy applications because they i) possess intrinsic anti-tumorigenicity, ii) require no priming to mediate tumor cell lysis in an MHC-independent manner, iii) direct tumor killing via recognition of stress-responsive ligands frequently up-regulated by tumor cells, and, as we show, iv) can be expanded to clinical cell doses in cGMP serum free media (SFM). Although tremendous effort has been devoted to αβ T cell processing, little focus has been directed toward the clinical development of genetically engineered gd T cells. Herein, we describe the optimization of drug and cytokine dosing and, culture in SFM, robust expansion and genetic modification necessary for clinical applications. Peripheral blood mononuclear cells (PBMC) isolated from healthy donors were cultured in several SFMs, including: OpTmizer, X-Vivo, SCGM, and AIM V as well as commonly used media containing serum, such as RPMI1640 supplemented with either 10% FBS or human serum. Complete growth media also included 2mM L-glutamine (if not present in the commercial product), Zoledronic Acid (5µM added only at the start of culture), and IL-2 (either 100 or 1000 IU/ml added twice weekly). Of the SFM cultures, PBMC isolated from 7 of 8 donors had robust expansion only in OpTmizer (supplemented with high-dose IL-2) with a concomitant rise in gd T cell percentage to levels sufficient for clinical use. Specifically, the percentage of gd T cells increased from a mean of 3% (2.4-3.6%) to 61% (21-84%), resulting in an 88 fold expansion at 14 days. While gd T cell numbers were lower in SFM compared to serum-containing RPMI, the final product starting with PBMC harvested during a single apheresis procedure when cultured in OpTmizer can yield therapeutic doses. The expanded gd T cells were evaluated for their transduction efficiency using a 3rd generation self-inactivating lentiviral vector (LV) to introduce a reporter gene encoding EGFP. Two different LVs were tested: HIV and SIV harboring the transgene under the control of EF1α and MSCV promoters, respectively. The timing of LV addition was optimized with respect i) gd T cell expansion, ii) LDL Receptor (LDLR) expression (a known receptor utilized by VSV-G pseudotyped LV) and iii) LV dose (3-4 consecutive transductions over 48 hours). Transductions remained consistent using approximately 30% LV concentrated stock by volume. As expected, greater transduction was achieved in cultures with the greatest expansion. Interestingly, higher transduction efficiency was observed in gd T cell populations with the highest LDLR levels. In gd T cells, an MSCV promoter provided greater GFP expression compared to an EF1α promoter. As expected a range of transduction efficiencies was observed for various donors spanning from 13-33%, which is similar to LV transduction levels observed for αβ T cell transductions with similar multiplicity of infection and expansion levels. Therefore, an optimized method of gd T cell expansion and transduction was developed that can be tested in early phase clinical trials. The absence of MHC-restriction affords the opportunity, with appropriate elimination of the αβ T cell component, for use in the allogeneic setting with limited risk of GvHD, and the use of SFM provides clinically safer, widely applicable, and potentially more efficacious cellular immunotherapy. DisclosuresDoering:Expression Therapeutics: Equity Ownership; Bayer Healthcare: Consultancy, Honoraria, Research Funding. Spencer:Expression Therapeutics: Equity Ownership.

Chemoprotection of murine hematopoietic cells by combined gene transfer of cytidine deaminase (CDD) and multidrug resistance 1 gene (MDR1).

BackgroundHematologic toxicity represents a major side effect of cytotoxic chemotherapy frequently preventing adequately dosed chemotherapy application and impeding therapeutic success. Transgenic (over)expression of chemotherapy resistance (CTX-R) genes in hematopoietic stem- and progenitor cells represents a potential strategy to overcome this problem. To apply this concept in the context of acute myeloid leukemia and myelodysplasia, we have investigated the overexpression of the multidrug resistance 1 (MDR1) and the cytidine deaminase (CDD) gene conferring resistance to anthracyclines and cytarabine (Ara-C), the two most important drugs in the treatment of these diseases.MethodsState-of-the-art, third generation, self-inactivating (SIN) lentiviral vectors were utilized to overexpress a human CDD-cDNA and a codon-optimized human MDR1-cDNA corrected for cryptic splice sites from a spleen focus forming virus derived internal promoter. Studies were performed in myeloid 32D cells as well as primary lineage marker negative (lin−) murine bone marrow cells and flow cytometric analysis of suspension cultures and clonogenic analysis of vector transduced cells following cytotoxic drug challenge were utilized as read outs.ResultsEfficient chemoprotection of CDD and MDR1 transduced hematopoietic 32D as well as primary lin− cells was proven in the context of Ara-C and anthracycline application. Both, CTX-R transduced 32D as well as primary hematopoietic cells displayed marked resistance at concentrations 5–20 times the LD50 of non-transduced control cells. Moreover, simultaneous CDD/MDR1 gene transfer resulted in similar protection levels even when combined Ara-C anthracycline treatment was applied. Furthermore, significant enrichment of transduced cells was observed upon cytotoxic drug administration.ConclusionsOur data demonstrate efficient chemoprotection as well as enrichment of transduced cells in hematopoietic cell lines as well as primary murine hematopoietic progenitor cells following Ara-C and/or anthracycline application, arguing for the efficacy as well as feasibility of our approach and warranting further evaluation of this concept.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0260-4) contains supplementary material, which is available to authorized users.