Clinical-grade Lentiviral Vectors Research Articles

Establishment and Characterization of a Stable Producer Cell Line Generation Platform for the Manufacturing of Clinical-Grade Lentiviral Vectors.

Background/Objectives: To date, nearly 300 lentiviral-based gene therapy clinical trials have been conducted, with eight therapies receiving regulatory approval for commercialization. These advances, along with the increased number of advanced-phase clinical trials, have prompted contract development and manufacturing organizations (CDMOs) to develop innovative strategies to address the growing demand for large-scale batches of lentiviral vectors (LVVs). Consequently, manufacturers have focused on optimizing processes under good manufacturing practices (GMPs) to improve cost-efficiency, increase process robustness, and ensure regulatory compliance. Nowadays, the LVV production process mainly relies on the transient transfection of four plasmids encoding for the lentiviral helper genes and the transgene. While this method is efficient at small scales and has also proven to be scalable, the industry is exploring alternative processes due to the high cost of GMP reagents, and the batch-to-batch variability predominantly attributed to the transfection step. Methods: Here, we report the development and implementation of a reliable and clinical-grade envisioned platform based on the generation of stable producer cell lines (SCLs) from an initial well-characterized lentiviral packaging cell line (PCL). Results: This platform enables the production of VSV-G-pseudotyped LVVs through a fully transfection-free manufacturing process. Our data demonstrate that the developed platform will facilitate successful technological transfer to large-scale LVV production for clinical application. Conclusions: With this simple and robust stable cell line generation strategy, we address key concerns associated with the costs and reproducibility of current manufacturing processes.

T Cell Activators Exhibit Distinct Downstream Effects on Chimeric Antigen Receptor T Cell Phenotype and Function.

T cell activation is an essential step in chimeric Ag receptor (CAR) T (CAR T) cell manufacturing and is accomplished by the addition of activator reagents that trigger the TCR and provide costimulation. We explore several T cell activation reagents and examine their effects on key attributes of CAR T cell cultures, such as activation/exhaustion markers, cell expansion, gene expression, and transduction efficiency. Four distinct activators were examined, all using anti-CD3 and anti-CD28, but incorporating different mechanisms of delivery: Dynabeads (magnetic microspheres), TransAct (polymeric nanomatrix), Cloudz (alginate hydrogel), and Microbubbles (lipid membrane containing perfluorocarbon gas). Clinical-grade lentiviral vector was used to transduce cells with a bivalent CD19/CD22 CAR, and cell counts and flow cytometry were used to monitor the cells throughout the culture. We observed differences in CD4/CD8 ratio when stimulating with the Cloudz activator, where there was a significant skewing toward CD8 T cells. The naive T cell subset expressing CD62L+CCR7+CD45RA+ was the highest in all donors when stimulating with Dynabeads, whereas effector/effector memory cells were highest when using the Cloudz. Functional assays demonstrated differences in killing of target cells and proinflammatory cytokine secretion, with the highest killing from the Cloudz-stimulated cells among all donors. This study demonstrates that the means by which these stimulatory Abs are presented to T cells contribute to the activation, resulting in differing effects on CAR T cell function. These studies highlight important differences in the final product that should be considered when manufacturing CAR T cells for patients in the clinic.

Long-term stability of clinical-grade lentiviral vectors for cell therapy

The use of lentiviral vectors in cell and gene therapy is steadily increasing, both in commercial and investigational therapies. Although existing data increasingly support the usefulness and safety of clinical-grade lentiviral vectors used in cell manufacturing, comprehensive studies specifically addressing their long-term stability are currently lacking. This is significant considering the high cost of producing and testing GMP-grade vectors, the limited number of production facilities, and lengthy queue for production slots. Therefore, an extended shelf life is a critical attribute to justify the investment in large vector lots for investigational cell therapies. This study offers a thorough examination of essential stability attributes, including vector titer, transduction efficiency, and potency for a series of clinical-grade vector lots, each assessed at a minimum of 36months following their date of manufacture. The 13 vector lots included in this study were used for cell product manufacturing in 16 different clinical trials, and at the time of the analysis had a maximum storage time at -80°C of up to 8years. The results emphasize the long-term durability and efficacy of GMP-grade lentiviral vectors for use in exvivo cell therapy manufacturing.

Assessment of Transduction of CD34+ Human Hematopoietic Stem Cells from Patients with Severe Hemophilia-Α with Lentiviral Vector Carrying a High Expression FVIII Transgene (CD68-ET3-LV)

Background: Alternative strategies are needed for the large number of people with hemophilia who are ineligible for AAV based gene therapy due to age or high levels of anti-AAV neutralizing antibodies. High inter-individual variability of expression and ill sustained factor levels are also challenges with AAV based gene therapy for hemophilia A, in particular. We have developed a third-generation lentiviral vector mediated hematopoietic stem cell-based gene therapy for hemophilia A. (Doering et al Human Gene Therapy 2018; 29: 1183-1201) This vector (CD68-ET3-LV) has a high expression FVIII transgene with a CD68 promoter targeting expression in monocytic cells predominantly. We report here successful transduction of severe hemophilia A patient derived human hematopoietic stem cells with this vector. Methods: Mobilized peripheral blood stem cells were collected by apheresis from three patients with severe hemophilia A without inhibitors who also received prophylactic clotting factor replacement therapy during this period. CD34+ hematopoietic stem cells (HSCs) were enriched on the CliniMACS Plus® system (Miltenyi Biotec, Bergish Gladbach, Germany) using the CliniMACS CD34® reagent system. Purified HSC were then transduced with this CD68-ET3-LV as follows - HSCs cultured on retronectin coated surfaces in xenofree media with cytokines were exposed to clinical grade CD68-ET3-LV vector in two ways - a double transduction protocol without any enhancer and a single transduction with an enhancer. After completion of this step, the product was assessed for viability and vector copy number (VCN) by trypan blue labelling and Q-PCR performed on genomic DNA from CFU cells, respectively. Transduced HSCs were also assessed for their engraftment potential. CD68-ET3-LV vector transduced HSC (1x10 6) were transplanted into NBSGW mice via tail vein injection. Engraftment was assessed at 16 weeks after transplantation. This protocol was approved by the Institutional Review Board of the Christian Medical College, Vellore, India. Results: G-CSF (10 ug/kg/day) based peripheral blood stem cell mobilization was well tolerated by all participants. The apheresis procedures which were done with plasma FVIII levels in the normal range after prophylactic replacement therapy were unremarkable. The total collection of mobilized CD34+ cells from the three donors was 268±80.5x10 6 CD34+ cells. An aliquot of 2x10 6 CD34+ cells/ml was used in the transduction experiments. The data on viability and vector copy number post transduction shown in the tables 1 and 2 confirm that viability was not affected by the manipulation of these HSCs in both double and single transduction methods. The vector copy number (VCN) in the genomic DNA obtained from CFU cells collected from CD34+ HSCs cultured in MethoCult H4434™ (Stem Cell Technologies™, Vancouver, Canada) varied from 0.62 to 1.21 in double transduction (n=3) and nearly doubled in the single transduction method with an enhancer to VCNs of 1.5 and 2.4 in two samples tested. Transplantation studies in NBSGW mice showed successful engraftment of human CD34+ HSCs with a mean engraftment of 80.99 ± 4.2% of CD45+ multilineage hematopoietic cells in the bone marrow 16 weeks after transplantation. Conclusion: To the best of our knowledge, this is the first report of transduction of mobilized peripheral blood CD34+ HSCs from patients with severe hemophilia A using a clinical grade lentiviral vector with a high expression FVIII transgene. These data establish feasibility and safety of the procedure. The transduction protocol showed good efficiency with very high viability, significant VCN and good engraftment of these gene modified human HSCs in a mouse model. These protocols are currently being evaluated in a first in human phase 1 clinical trial of gene therapy for severe hemophilia A without inhibitors.

Development of a perfusion process for continuous lentivirus production using stable suspension producer cell lines.

The large-scale production of clinical-grade lentiviral vectors (LVs) for gene therapy applications is a remaining challenge. The use of adherent cell lines and methods like transient transfection are cost-intensive and hamper process scalability as well as reproducibility. This study describes the use of two suspension-adapted stable packaging cell lines, called GPRGs and GPRTGs, for the development of a scalable and serum-free LV production process. Both stable packaging cell lines are based on an inducible Tet-off system, thus requiring doxycycline removal for initiation of the virus production. Therefore, we compared different methods for doxycycline removal and inoculated three independent 5 L bioreactors using a scalable induction method by dilution, an acoustic cell washer and manual centrifugation. The bioreactors were inoculated with a stable producer cell line encoding for a LV carrying a clinically relevant gene. LV production was performed in perfusion mode using a cell retention device based on acoustic wave separation. Comparable cell-specific productivities were obtained with all three methods and cumulative functional yields up to 6.36 × 1011 transducing units per bioreactor were generated in a 234-h long process, demonstrating the usability of stable Tet-off cell lines for an easily scalable suspension process. Remarkably, cell viabilities >90% were maintained at high cell densities without compromising productivity throughout the whole process, allowing to further extend the process time. Given its low effects of toxicity during virus production, the presented cell lines are excellent candidates to develop a fully continuous LV production process to overcome the existing bottlenecks in LV manufacturing.

Clinical grade lentiviral vector purification and quality control requirements.

Lentiviral vectors have been proven to be a powerful tool in gene therapies that includes the ability to perform long-term gene editing in both dividing and non-dividing cells. In order to meet the rising demand for clinical-grade lentiviral vectors for future clinical trials and requirements by regulatory agencies, new methods and technologies were developed, including the rapid optimization of production and purification processes. However, gaps still exist in achieving ideal yields and recovery rates in large-scale manufacturing process steps. The downstream purification process is a critical step required to obtain a sufficient quantity and high-quality lentiviral vectors products, which is challenged by the low stability of the lentiviral vector particles and large production volumes associated with the manufacturing process. This review summarizes the most recent and promising technologies and enhancements used in the large-scale purification process step of lentiviral vector manufacturing and aims to provide a significant contribution towards the achievement of providing sufficient quantity and quality of lentiviral vectors in scalable processes.

Development of a cGMP-compliant process to manufacture donor-derived, CD45RA-depleted memory CD19-CAR T cells.

Autologous chimeric antigen receptor (CAR) T cells targeting the CD19 antigen have demonstrated a high complete response rate in relapsed/refractory B-cell malignancies. However, autologous CAR T cell therapy is not an option for all patients. Here we optimized conditions for clinical-grade manufacturing of allogeneic CD19-CAR T cells using CD45RA-depleted donor memory T cells (Tm) for a planned clinical trial. Tm were activated using the MACS GMP T Cell TransAct reagent and transduced in the presence of LentiBOOST with a clinical-grade lentiviral vector that encodes a 2nd generation CD19-CAR with a 41BB.zeta endodomain. Transduced T cells were transferred to a G-Rex cell culture device for expansion and harvested on day 7 or 8 for cryopreservation. The resulting CD19-CAR(Mem) T cells expanded on average 34.2-fold, and mean CAR expression was 45.5%. The majority of T cells were CD4+ and had a central memory or effector memory phenotype, and retained viral specificity. CD19-CAR(Mem) T cells recognized and killed CD19-positive target cells in vitro and had potent antitumor activity in an ALL xenograft model. Thus we have successfully developed a current good manufacturing practice-compliant process to manufacture donor-derived CD19-CAR(Mem) T cells. Our manufacturing process could be readily adapted for CAR(Mem) T cells targeting other antigens.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Production of Lentiviral Vectors Using Suspension Cells Grown in Serum-free Media

Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option.

Improvement ofDe NovoCholesterol Biosynthesis Efficiently Promotes the Production of Human Immunodeficiency Virus Type 1-Derived Lentiviral Vectors

The use of lentiviral vectors (LVs) for gene transfer in research, technological, or clinical applications requires the production of large amounts of vector. Mass production of clinical-grade LVs remains a challenge and limits certain perspectives for therapeutic use. Some improvements in LV production protocols have been possible by acting on multiple steps of the production process. The addition of animal-derived cholesterol to the culture medium of producer cells is known to increase the infectivity of LVs. To avoid the use of this animal-derived product in clinical settings, an alternative approach is to increase de novo the production of cholesterol by overexpressing a crucial cholesterogenic enzyme, namely, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). This project evaluates the impact of such an approach on the production, infectivity, and stability of LVs. We demonstrated that the overexpression of human HMGCR isoform 1 (hHMGCR1) in LV producer cells efficiently increased de novo cholesterol biosynthesis and enhanced by 2- to 3-fold the physical and infectious titers of LVs. We also observed that LVs produced in hHMGCR1-overexpressing cells were comparable in stability to LVs produced under classical conditions and were capable of transducing human CD34+ hematopoietic stem/progenitor cells efficiently. Interestingly, we also showed that LV production in the absence of fetal calf serum (FCS) but under hHMGCR1-overexpressing conditions allowed a viral production yield comparable to that achieved under classical conditions in high FCS content, leading the way to the establishment of new LV production protocols on adherent cells without serum.

Preclinical Testing of the Safety and Tolerability of Lentiviral Vector-Mediated Above-Normal Alpha-L-Iduronidase Expression in Murine and Human Hematopoietic Cells Using Toxicology and Biodistribution Good Laboratory Practice Studies.

In order to support the clinical application of hematopoietic stem cell (HSC) gene therapy for mucopolysaccharidosis I (MPS I), biosafety studies were conducted to assess the toxicity and tumorigenic potential, as well as the biodistribution of HSCs and progenitor cells (HSPCs) transduced with lentiviral vectors (LV) encoding the cDNA of the alpha-iduronidase (IDUA) gene, which is mutated in MPS I patients. To this goal, toxicology and biodistribution studies were conducted, employing Good Laboratory Practice principles. Vector integration site (IS) studies were applied in order to predict adverse consequences of vector gene transfer and to obtain HSC-related information. Overall, the results obtained in these studies provided robust evidence to support the safety and tolerability of high-efficiency LV-mediated gene transfer and above-normal IDUA enzyme expression in both murine and human HSPCs and their in vivo progeny. Taken together, these investigations provide essential safety data to support clinical testing of HSC gene therapy in MPS I patients. These studies also underline criticisms associated with the use of currently available models, and highlight the value of surrogate markers of tumorigenicity that may be further explored in the future. Notably, biological evidence supporting the efficacy of gene therapy on MPS I disease and its feasibility on patients' HSCs were also generated, employing clinical-grade LVs. Finally, the clonal contribution of LV-transduced HSPCs to hematopoiesis along serial transplantation was quantified in a minimum of 200-300 clones, with the different level of repopulating cells in primary recipients being reflected in the secondary.

288. Development of Cocal Glycoprotein Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T Cells

Lentiviral vectors (LVs) are routinely used for stable gene transfer and have demonstrated great promise in hematopoietic stem cell gene therapy and also immunotherapy using genetically modified T cells. LVs are commonly pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G), which confers broad tropism to the vector and allows for vector concentration by centrifugation. However, the use of VSV-G has several limitations, such as susceptibility to inactivation by human serum complement making it unsuitable for in vivo delivery. Furthermore, VSV-G is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. In this study, we first validate the use of cocal vesiculovirus envelope to pseudotype LVs by demonstrating that cocal LVs transduce hematopoietic stem cells and CD4+ T cells more efficiently than VSV-G LVs. We also provide evidence that cocal and VSV-G envelopes use the same receptor for cell entry. We then describe the development of two high-titer, cocal-pseudotyped, LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T cell receptor (TCR). The different 3rd generation lentiviral helper genes were sequentially introduced in HEK293T cells by co-transfection with plasmids encoding antibiotic resistance genes followed by selection to allow for stable protein expression. Cells expressing the cocal envelope produced over 10-times more infectious LV particles as compared to VSV-G expressing cells. High-titer cocal producer cells were isolated by screening for best single clones, which were capable of generating concentrated titers above 108 infectious units per mL. We found that these producer cells were stable after serial passages for over 3 months, with no drop in titer detected over time. The resulting GFP and WT1-TCR vectors performed at least as well as identical vectors made with our standard transient transfection protocol for the transduction of CD34+ and CD4+ T cells, respectively. Cocal LV producer cells were also adapted for growth in suspension, serum-free culture, which will facilitate efforts for the scaling up of vector production. In summary, we have successfully developed two independent LV producer cells lines with clinically usable titers. The broad applicability of our cocal packaging cell line offers a promising tool toward the generation of large-scale, clinical grade LV.

235. Improved Ex Vivo Gene Therapy Using Highly Purified Hematopoietic Stem and Progenitor Cells

Ex vivo gene addition into CD34+ hematopoietic stem and progenitor cells (HSPC) followed by autologous transplantation has proven a safe and efficacious therapy for immunodeficiencies, storage diseases and hemoglobinopathies. According to xenograft models and population size estimates of vector-marked cells in gene therapy-treated patients, less than 0.01% of infused CD34+ cells drive long-term (LT) repopulation. Advances in clinical-grade cell sorting technology may make HSC-enriched CD34+ subpopulations accessible for gene therapy, with advantages in terms of lentiviral vector (LV) cost, safety (lowering of integration load) and, potentially, efficacy. By differentially marking mobilized peripheral blood (mPB) CD34 subpopulations distinguished by increasing levels of CD38 expression in order to quantitatively assess their hematopoietic output in an NSG xenograft model over 6 months, we previously mapped most (>90%) LT repopulating capacity to CD34+CD38- cells (lowest 10% CD38 staining), while CD34+CD38int cells drove short-term (ST) reconstitution during the first 2 months after transplantation. We now characterize these subpopulations in terms of CD90 expression, a marker, which has been used in conjunction with CD34 for HSC purification in past clinical trials. CD34+ mPB cells were sorted into CD38-90+ (5% of CD34+), CD38-90- (5%), CD38+90+ (30%) and CD38+90- (60%) fractions and exhaustively transduced with GFP-, OFP-, BFP- and mCherry-expressing LVs, respectively. Differentially marked subfractions were pooled maintaining their original proportions and transplanted into NSG mice. ST engraftment mainly came from CD38+ cells, with equal contribution from the CD90+ and CD90- compartment. LT engraftment was almost exclusively derived from CD34+CD38- cells, of which 70% came from CD90+ and 30% from CD90- cells. Hence, CD34+CD38- is a more sensitive and specific marker combination than CD34+CD90+ to purify LT-HSC. CD34+CD38- cells can be purified by a sequential bead-based selection (CD34 selection of CD38-depleted cells) potentially applicable to clinical practice. We show that CD34+CD38- cells can be efficiently transduced with clinical grade LVs using shortened ex vivo manipulation protocols, reaching similar gene marking levels as with the standard protocol currently used in clinical trials that comprises a double dose of LV. Transduction was stable for at least 5 months when serially measured in xenotransplanted mice, and mice showed multi-lineage hematopoiesis indistinguishable from CD34+ grafts. Based on these results, we are aiming towards clinical development of a new gene therapy protocol based on CD34+CD38-HSPC efficiently transduced with minimum ex vivo culture time (<36h). Our platform will substantially improve the efficacy, safety and feasibility of future ex vivo gene therapy studies.

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.

286. Genome Editing of Inducible Cell Lines for Scalable Production of Improved Lentiviral Vectors for Human Gene Therapy

Lentiviral vectors (LVs) represent efficient and versatile vehicles for gene therapy. Current manufacturing of clinical-grade LVs mostly relies on transient transfection of plasmids expressing the multiple vector components. This method is labor and cost intensive and becomes challenging when facing the need of scale-up and standardization. The development of stable LV producer cell lines will greatly facilitate overcoming these hurdles. We have generated an inducible LV packaging cell line, carrying the genes encoding for third-generation vector components stably integrated in the genome under the control of tetracycline-regulated promoters. These LV packaging cells are stable in culture even after single-cell cloning and can be scaled up to large volumes. In order to minimize the immunogenicity of LVs for in vivo administration, we set out to remove the highly polymorphic class-I major histocompatibility complexes (MHC-I) expressed on LV packaging cells and incorporated in the LV envelope. We performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of all MHC-I to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. The resulting B2M-negative cells were devoid of surface-exposed MHC-I and produced MHC-free LVs. These LVs retain their infectivity on all tested cells in vitro and efficiently transduced the mouse liver upon intravenous administration. Strikingly, the MHC-free LVs showed significantly reduced immunogenicity in a T-cell activation assay performed on human primary T cells co-cultured with syngeneic monocytes exposed to LV, from several (n=7) healthy donors. To reproducibly generate LV-producer cell lines from these cells, we insert the LV genome of interest in the AAVS1 locus, chosen for robust expression, exploiting engineered nucleases and homology-directed repair. By this strategy, we have obtained several independent producer cell lines for LVs that express marker or therapeutic genes and are devoid of plasmid DNA contamination. LVs produced by these cells reproducibly show titer and infectivity within the lower bound range of standard optimized transient transfection, and effectively transduce relevant target cells, such as hematopoietic stem/progenitor cells and T cells ex vivo and the mouse liver in vivo. Overall, we provide evidence that rationally designed targeted genome engineering can be used to improve the yield, quality, safety and sustainability of LV production for clinical use.

464. RCL-Pooling Assay: A Simplified Method for the Detection of Replication Competent Lentiviruses in Vector Batches Using Sequential Pooling

Non-replicative recombinant HIV-1 derived lentiviral vectors (LV) are increasingly used in gene therapy clinical trials for various genetic diseases, infectious diseases or cancer. Before they are used in man, preparations of LV must undergo extensive biochemical and biological quality control testing. In particular, the legislation stipulates that the absence of replication-competent lentiviruses (RCL) must be demonstrated with suitable methods, on representative fractions of batches. Current standard and widely used methods based on cell culture are challenging because high titers of vector batches achieved translate into high volumes of cell culture that have to be tested. Since vector batch titers and sizes are continuously-increasing due to the improvement of production and purification methods, it is necessary to modify the current cell culture method. Here, we propose a practical optimization of the p24-decrease-based culture assay developed by Escarpe et al. (2003) using a pairwise pooling strategy enabling the test of higher vector inoculum volumes. These modifications significantly decrease material handling, operator time, leading to a cost effective method, while maintaining optimal sensibility of the RCL testing. This optimized RCL-pooling assay ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.

"RCL-Pooling Assay": A Simplified Method for the Detection of Replication-Competent Lentiviruses in Vector Batches Using Sequential Pooling.

Nonreplicative recombinant HIV-1-derived lentiviral vectors (LV) are increasingly used in gene therapy of various genetic diseases, infectious diseases, and cancer. Before they are used in humans, preparations of LV must undergo extensive quality control testing. In particular, testing of LV must demonstrate the absence of replication-competent lentiviruses (RCL) with suitable methods, on representative fractions of vector batches. Current methods based on cell culture are challenging because high titers of vector batches translate into high volumes of cell culture to be tested in RCL assays. As vector batch size and titers are continuously increasing because of the improvement of production and purification methods, it became necessary for us to modify the current RCL assay based on the detection of p24 in cultures of indicator cells. Here, we propose a practical optimization of this method using a pairwise pooling strategy enabling easier testing of higher vector inoculum volumes. These modifications significantly decrease material handling and operator time, leading to a cost-effective method, while maintaining optimal sensibility of the RCL testing. This optimized "RCL-pooling assay" ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.

466. Automated Generation of Genetically Modified Human CD34+ Cells in a Functionally Closed System

Hereditary genetic disorders have been shown to be effectively treated by re-introduction of the wild-type gene product via viral modification of autologous CD34+ progenitor cells. However, current manufacturing processes are performed manually or at best by using semi-automated procedures. A standardized production of cellular therapeutic agents and their genetic modification is a major requirement to move cellular and gene therapies from their current translational setting into routine clinical use. A closed and highly automated manufacturing procedure would lead to large improvements in product performance, product safety and operator safety. We have recently developed a functionally closed and fully automated cell processing device, the CliniMACS Prodigy®, which enables complex cellular products to be manufactured.In this functionally closed system, the following steps have been automated: concentration and washing of blood products, magnetic labeling and enrichment of CD34+ cells, cultivation and pre-activation of the enriched CD34+ cells, transduction with lentiviral vectors and resuspension of the final cell product in buffer for infusion.Automated cell preparation, cell labeling and magnetic CD34+ cell separation was achieved from both mobilized and non-mobilized apheresis samples as well as bone marrow aspirates. CD34+ cells were enriched to purities exceeding 90%. Small scale experiments (10e5-10e7 purified CD34+ cells) performed with a newly developed automated transduction process, incorporating a single transduction cycle, resulted in high transduction efficiencies (up to 90%). A scale-up of the procedure was performed using larger numbers of cells (>10e7) enriched from mobilized apheresis material. CD34+ cells were transduced with a clinical-grade lentiviral vector using a two hit protocol already described in a clinical setting and used as standard for a control experiment run in parallel (Scaramuzza et al. 2013, Mol. Ther). Equivalent gene marking efficiencies were obtained. Moreover, gene modified cells maintained full engraftment potential as demonstrated by biodistribution studies in NSG mice.These preliminary data demonstrate that automated cell processing and genetic manipulation of haematopoetic progenitor cells can be efficiently performed in a closed system. Work in progress aims to further optimize this procedure and improve the efficiency of CD34+ cell transduction to levels superior to those currently achieved by manual protocols.

81. Development of a Second Generation Stable Lentiviral Packaging Cell Line in Support of Clinical Gene Transfer Protocols

Production of lentiviral vectors for clinical gene transfer trials is challenging due to the cost of acquiring GMP-grade plasmids and the labor intensive nature of making vectors via transient transfection. To facilitate clinical grade lentiviral vector production, we developed a packaging cell line, GPRG, which was used to make a fully stable producer, GPRG-EF1α-hgcOPT. This stable producer is presently supporting two clinical trials for the treatment of X-SCID. While initial results in one of these trials have been positive, the output and potency of this line mandates repeated production runs to supply enough vector for larger subjects, producing only enough for 2-3 cases per 6 day run. A second generation packaging cell line, GPRTG, has been developed and tested as a future platform for lentiviral gene transfer trials. This new cell line expresses codon-optimized HIV-1 gagpol, rev and tat. Expression of Rev, Tat and Vesicular Stomatitis Virus G proteins (VSV-G) are regulated by a Tet-Off/On system. We have improved the performance of the GPRTG cell line relative to GPRG by including the expression of HIV-1 Tat while decreasing the expression of Rev and by the independent re-insertion of the Rev, Tat and VSV-G packaging components. We generated a second fully stable producer, GPRTG-EF1α-hgcOPT, expressing the identical insulated SIN vector as in the first generation line. Both cell lines produce high titer vector preparations (>107 tu/ml) and are stable for at least 3 months of continuous passage. Although the vector titers are similar, GPRTG-EF1α-hgcOPT generated vector shows increased transduction efficiency of hCD34+ cells. PCR analysis of bulk CFU-GMs indicates a 2-fold increase in vector positive colonies and 3-5 fold increase in vector copy number with vector from GPRTG-EF1α-hgcOPT as compared to vector generated from GPRG-EF1α-hgcOPT when used at similar MOIs. We hypothesize that the basis for enhanced transduction efficiency of GPRTG derived vector is due to the expression of Tat and reduced expression of Rev each of which may affect vector transduction potency. In support of this hypothesis, our data show that transiently prepared vectors made with Rev only or in the presence of highly abundant Rev transduce hCD34+ cells less efficiently. We are currently employing the second generation cell line, GPRTG, to support future and on-going clinical protocols to treat other rare hematologic disorders including Wiskott-Aldrich Syndrome and Chronic Granulomatous Disease.

6. Targeted Genome Editing of Cell Lines for Improved and Scalable Production of Lentiviral Vectors for Human Gene Therapy

Lentiviral vectors (LVs) represent efficient and versatile vehicles for gene therapy. The manufacturing of clinical-grade LVs relies on transient transfection of vector components. This method is labor and cost intensive and becomes challenging when facing the need of scale-up and standardization. The development of stable LV producer cell lines will greatly facilitate overcoming these hurdles. We have generated an inducible LV packaging cell line, carrying the genes encoding for third-generation vector components stably integrated in the genome under the control of tetracycline-regulated promoters. In order to minimize the immunogenicity of LVs for in vivo administration, we set out to remove the highly polymorphic and antigenic class-I major histocompatibility complex (MHC-I) expressed on LV packaging cells and subsequently incorporated on the LV envelope. We performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of the MHC-I to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. We generated B2M-negative cells devoid of surface-exposed MHC-I, which retain the ability to produce LVs. In order to insert the LV genome of interest in the packaging cell line, we performed site-specific integration in predetermined loci of the genome of these cells, chosen for robust expression, exploiting artificial nucleases and homology-directed repair. In several independent iterations of this process, we generated producer cell lines both for LV expressing marker genes and a therapeutic gene, i.e. coagulation factor IX (FIX), the gene mutated in hemophilia B. We show that these LV producer cells are stable in culture and can produce several liters of LV-containing conditioned medium. These LVs have comparable or only slightly lower infectious titer and specific infectivity than LVs produced by state-of-the-art transient transfection process and can transduce therapeutically relevant target cells, such as hematopoietic stem/progenitor cells and T lymphocytes to high efficiency. Moreover, we intravenously administered FIX-expressing LVs produced by the cell line to hemophilia B mice and established therapeutic levels of circulating FIX. These data indicate that site-specific integration is an efficient, rapid and reproducible method to generate LV producer cells, starting from a universal stable inducible LV packaging cell line. Overall, we provide evidence that rationally designed targeted genome engineering can be used to improve the quality, safety and sustainability of LV production for clinical use.