Production Of Lentiviral Vectors Research Articles

Effects of an indole derivative on cell proliferation, transfection, and alternative splicing in production of lentiviral vectors by transient co-transfection.

Lentiviral vectors derived from human immunodeficiency virus type I are widely used to deliver functional gene copies to mammalian cells for research and gene therapies. Post-transcriptional splicing of lentiviral vector transgene in transduced host and transfected producer cells presents barriers to widespread application of lentiviral vector-based therapies. The present study examined effects of indole derivative compound IDC16 on splicing of lentiviral vector transcripts in producer cells and corresponding yield of infectious lentiviral vectors. Indole IDC16 was shown previously to modify alternative splicing in human immunodeficiency virus type I. Human embryonic kidney 293T cells were transiently transfected by 3rd generation backbone and packaging plasmids using polyethyleneimine. Reverse transcription-quantitative polymerase chain reaction of the fraction of unspliced genomes in human embryonic kidney 293T cells increased up to 31% upon the indole's treatment at 2.5 uM. Corresponding yield of infectious lentiviral vectors decreased up to 4.5-fold in a cell transduction assay. Adjusting timing and duration of IDC16 treatment indicated that the indole's disruption of early stages of transfection and cell cycle had a greater effect on exponential time course of lentiviral vector production than its reduction of post-transcriptional splicing. Decrease in transfected human embryonic kidney 293T proliferation by IDC16 became significant at 10 uM. These findings indicated contributions by early-stage transfection, cell proliferation, and post-transcriptional splicing in transient transfection of human embryonic kidney 293T cells for lentiviral vector production.

Genetic alteration of SJ293TS cells and modification of serum-free media enhances lentiviral vector production

Successful cell and gene therapy clinical trials have resulted in the United States Food and Drug Administration and European Medicines Agency approving their use for treatment of patients with certain types of cancers and monogenetic diseases. These novel therapies, which rely heavily on lentiviral vectors to deliver therapeutic transgenes to patient cells, have driven additional investigations, increasing demand for both pre-clinical and current Good Manufacturing Practices-grade viral vectors. To better support novel studies by improving current production methods, we report the development of a genetically modified HEK293T-based cell line that is null for expression of both Protein Kinase R and Beta-2 microglobulin and grows in suspension using serum-free media, SJ293TS-DPB. Absence of Protein Kinase R increased anti-sense lentiviral vector titers by more than 7-fold, while absence of Beta-2 microglobulin, a key component of Major Histocompatibility Complex class I molecules, has been reported to reduce the immunogenicity of lentiviral particles. Furthermore, we describe an improved methodology for culturing SJ293TS-DPB which facilitates expansion, reduces handling, and increases titers by 2-fold compared to previous methods. SJ293TS-DPB stably produced lentiviral vectors for over four months and generated lentiviral vectors that efficiently transduce healthy human donor T cells and CD34+ hematopoietic stem cells.

Quasi-perfusion studies for intensified lentiviral vector production using a continuous stable producer cell line

Quasi-perfusion culture was employed to intensify lentiviral vector (LV) manufacturing using a continuous stable producer cell line in an 8-day process. Initial studies aimed to identify a scalable seeding density, with 3, 4, and 5 x 104 cells cm-2 providing similar specific productivities of infectious LV. Seeding at 3 x 104 cells cm-2 was selected, and the quasi-perfusion was modulated to minimise inhibitory metabolite accumulation and vector exposure at 37 °C. Similar specific productivities of infectious and physical LV were achieved at 1, 2, and 3 vessel volumes per day (VVD), with 1 VVD selected to minimise downstream processing volumes. The optimised process was scaled 50-fold to 1,264 cm2 flasks, achieving similar LV titres. However, scaling up beyond this to a 6,320 cm2 multilayer flask reduced titres, possibly from suboptimal gas exchange. Across three independent processes in 25 cm2 to 6,320 cm2 flasks, reproducibility was high with a coefficient of variation of (7.7 ± 2.9)% and (11.9 ± 3.0)% for infectious and physical LV titres, respectively. The optimised flask process was successfully transferred to the iCELLis™ Nano (Cytiva) fixed-bed bioreactor, with quasi-perfusion at 1 VVD yielding 1.62 x 108 TU.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to4.64×107TU mL-1in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38×107TU mL-1in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of>1×107TUmL-1day-1for up to 29 consecutive days in a non-optimized 5L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Challenges in the analysis of pharmaceutical lentiviral vector products by orthogonal and complementary physical (nano)particle characterization techniques

Lentiviral vectors (LVVs) are used as a starting material to generate chimeric antigen receptor (CAR) T cells. Therefore, LVVs need to be carefully analyzed to ensure safety, quality, and potency of the final product. We evaluated orthogonal and complementary analytical techniques for their suitability to characterize particulate matter (impurities and LVVs) in pharmaceutical LVV materials at development stage derived from suspension and adherent manufacturing processes. Microfluidic resistive pulse sensing (MRPS) with additional manual data fitting enabled the assessment of mode diameters for particles in the expected LVV size range in material from adherent production. LVV material from a suspension process, however, contained substantial amounts of particulate impurities which blocked MRPS cartridges. Sedimentation-velocity analytical ultracentrifugation (SV-AUC) resolved the LVV peak in material from adherent production well, whereas in more polydisperse samples from suspension production, presence of particulate impurities masked a potential signal assignable to LVVs. In interferometric light microscopy (ILM) and nanoparticle tracking analysis (NTA), lower size detection limits close to ∼ 70 nm resulted in an apparent peak in particle size distributions at the expected size for LVVs emphasizing the need to interpret these data with care. Interpretation of data from dynamic light scattering (DLS) was limited by insufficient size resolution and sample polydispersity. In conclusion, the analysis of LVV products manufactured at pharmaceutical scale with current state-of-the-art physical (nano)particle characterization techniques was challenging due to the presence of particulate impurities of heterogeneous size. Among the evaluated techniques, MRPS and SV-AUC were most promising yielding acceptable results at least for material from adherent production.

High yield sterile filtration process for highly concentrated lentiviral vectors.

The development and manufacture of biopharmaceuticals are subject to strict regulations that specify the required minimum quality of the products. A key measure to meet these quality requirements is the integration of a sterile filtration step into the commercial manufacturing process. Whereas common procedures for most biologics exist, this is challenging for lentiviral vector (LVV) production for ex vivo gene therapy. LVVs nominal size is more than half the pore size (0.2µm) of filters used for sterile filtration. Hence, highly concentrated virus solutions are prone to filter clogging if aggregation of viruses occurs or impurities attach to the viruses. Several filters were screened aiming to identify those which allow filtering highly concentrated stocks of LVVs of up to 1E+9 transducing unitsmL-1 , which corresponds to 4.5E+12 particlesmL-1 . In addition, the effect of endonuclease treatment upstream of the purification process on filter performance was studied. In summary, three suitable filters were identified in a small-scale study (<15mL) with virus yields >80% and the process was successfully scaled-up to a final scale of 100mL LVV stock solution.

Lentiviral Vectors in CAR-T Cell Manufacturing: Biological Principles, Manufacturing Process, and Frontier Development

Chimeric antigen receptor T cell (CAR-T) therapy, as the preeminent strategy in immunotherapy, has conferred benefits upon numerous cancer patients. Currently, there are six CAR-T therapies approved by the Food and Drug Administration (FDA), while the China National Medical Products Administration (NMPA) has approved three. The manufacturing process for CAR-T cells (CAR-Ts) entails using lentiviral vectors (LVVs), which are efficacious in transducing functional genes to both dividing and nondividing cells. Also, LVVs exert their capacity to integrate relatively large segments of DNA. Despite this fact, LVV-based CAR-Ts production can prove prohibitively expensive, and the yield of LVV can be further enhanced. In recent years, in-vivo generation of CAR-Ts has attracted significant attention since it stands to eliminate the ex-vivo isolation and activation process for T cells, thereby decreasing the cost and time involved in production. In view of this, this review summarizes the biological principles of LVVs and their manufacturing process, which encompasses lentiviral vector production, T cell isolation and activation, and CAR-Ts production. Additionally, the cutting-edge developments associated with LVVs are outlined herein, offering direction for the production of the next generation of CAR-Ts.

Exploring nutrient supplementation and bioprocess optimization to improve the production of lentiviral vectors in serum-free medium suspension cultures.

The use of lentiviral vectors (LV) in gene therapy has been growing in recent years. To meet the increasing clinical demand, LV production platforms will benefit from improved productivity and scalability to enable cost-effective manufacture of LV-based therapies. Here we report the adaptation of 293T cells to serum-free suspension cultures and the improvement of LV yields through transfection parameters optimization, process intensification and medium supplementation with nutrient boosters. Cells were sequentially adapted to different serum-free culture media, transfection parameters were optimized and the two best-performing conditions were selected to explore process intensification by increasing cell density at the time of transfection. LV production at higher cell densities increased volumetric titers up to 12-fold and lipid supplementation was the most efficient metabolic optimization strategy further enhancing LV productivity by 3-fold. Furthermore, cell concentration was identified and validated as an important source of transfection variability impairing cellular uptake of DNA polyplexes, impacting transfection efficiency and reducing LV titers down to 6-fold. This work contributes to improving LV-based gene therapy by establishing new scalable manufacturing platforms and providing key metabolic insights, unveiling important bioreaction parameters to improve vector yields.

Slow and steady wins the race: Optimizing lentiviral vector production for efficient clinical-scale manufacturing

Slow and steady wins the race: Optimizing lentiviral vector production for efficient clinical-scale manufacturing

Identification of a psychiatric risk gene NISCH at 3p21.1 GWAS locus mediating dendritic spine morphogenesis and cognitive function

BackgroundSchizophrenia and bipolar disorder (BD) are believed to share clinical symptoms, genetic risk, etiological factors, and pathogenic mechanisms. We previously reported that single nucleotide polymorphisms spanning chromosome 3p21.1 showed significant associations with both schizophrenia and BD, and a risk SNP rs2251219 was in linkage disequilibrium with a human specific Alu polymorphism rs71052682, which showed enhancer effects on transcriptional activities using luciferase reporter assays in U251 and U87MG cells.MethodsCRISPR/Cas9-directed genome editing, real-time quantitative PCR, and public Hi-C data were utilized to investigate the correlation between the Alu polymorphism rs71052682 and NISCH. Primary neuronal culture, immunofluorescence staining, co-immunoprecipitation, lentiviral vector production, intracranial stereotaxic injection, behavioral assessment, and drug treatment were used to examine the physiological impacts of Nischarin (encoded by NISCH).ResultsDeleting the Alu sequence in U251 and U87MG cells reduced mRNA expression of NISCH, the gene locates 180 kb from rs71052682, and Hi-C data in brain tissues confirmed the extensive chromatin contacts. These data suggested that the genetic risk of schizophrenia and BD predicted elevated NISCH expression, which was also consistent with the observed higher NISCH mRNA levels in the brain tissues from psychiatric patients compared with controls. We then found that overexpression of NISCH resulted in a significantly decreased density of mushroom dendritic spines with a simultaneously increased density of thin dendritic spines in primary cultured neurons. Intriguingly, elevated expression of this gene in mice also led to impaired spatial working memory in the Y-maze. Given that Nischarin is the target of anti-hypertensive agents clonidine and tizanidine, which have shown therapeutic effects in patients with schizophrenia and patients with BD in preliminary clinical trials, we demonstrated that treatment with those antihypertensive drugs could reduce NISCH mRNA expression and rescue the impaired working memory in mice.ConclusionsWe identify a psychiatric risk gene NISCH at 3p21.1 GWAS locus influencing dendritic spine morphogenesis and cognitive function, and Nischarin may have potentials for future therapeutic development.

Optimizing and developing a scalable, chemically defined, animal component-free lentiviral vector production process in a fixed-bed bioreactor

Lentiviral vectors (LVVs) play a critical role in gene delivery for exvivo gene-modified cell therapies. However, the lack of scalable LVV production methods and the high cost associated with them may limit their use. In this work, we demonstrate the optimization and development of a scalable, chemically defined, animal component-free LVV production process using adherent human embryonic kidney 293T cells in a fixed-bed bioreactor. The initial studies focused on the optimization of the culture process in 2D static cultures. Process changes such as decreasing cell seeding density on day 0 from 2.5×104 to 5×103 cells/cm2, delaying the transient transfection from 24 to 120h post-seeding, reducing plasmid DNA to 167ng/cm2, and adding 5mM sodium butyrate 6h post-transfection improved functional LVV titers by 26.9-fold. The optimized animal component-free production process was then transferred to the iCELLis Nano bioreactor, a fixed-bed bioreactor, where titers of 1.2×106 TU/cm2 were achieved when it was operated in perfusion. In this work, comparable functional LVV titers were obtained with FreeStyle 293 Expression medium and the conventional Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum both at small and large scale.

Serum-free lentiviral vector production is compatible with medium-resident nuclease activity arising from adherent HEK293T host cells engineered with a nuclease-encoding transgene

At present lentiviral vector production for cell and gene therapy commonly involves transient plasmid transfection of mammalian cells cultivated in serum-containing media and addition of exogenous nuclease to reduce host cell and plasmid DNA impurities. Switching from serum-containing media to chemically-defined, serum free media, and minimising the number of process additions, are both increasingly regarded as necessary steps for simplifying and potentially automating lentiviral vector bioprocessing in future. Here we adapted human embryonic kidney 293T (HEK293T) cells to grow in serum-free media and also modified these cells with transgenes designed to encode a secreted nuclease activity. Stable transfection of HEK293T cells with transgenes encoding the Staphylococcus aureus nuclease B (NucB) open reading frame with either its native secretion signal peptide, the murine Igκ chain leader sequence or a novel viral transport fusion protein, all resulted in qualitatively detectable nuclease activity in serum-free media. Serum-free transient transfection of human embryonic kidney HEK293T cells stably harbouring the transgene for NucB with its native secretion signal produced active lentivirus in the presence of medium-resident nuclease activity. This lentivirus material was able to transduce the AGF-T immortal T cell line with a green fluorescent protein reporter payload at a level of 2.05 × 105 TU/mL (±3.34 × 104 TU/mL). Sufficient nuclease activity was present in 10 μL of this unconcentrated lentivirus material to degrade 1.5 μg DNA within 2 h at 37 °C, without agitation - conditions compatible with lentivirus production. These observations demonstrate that lentiviral vector production, by transient transfection, is compatible with host cells harbouring a nuclease transgene and evidencing nuclease activity in their surrounding growth media. This work provides a solid basis for future investigations, beyond the scope of this present study, in which commercial and academic groups can apply this approach to therapeutic payloads and potentially omit exogenous nuclease bioprocess additions.

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.

Process intensification for lentiviral vector manufacturing using tangential flow depth filtration

For gene therapies to become more accessible and affordable treatment options, process intensification is one possible strategy to increase the number of doses generated per batch of viral vector. Process intensification for lentiviral vector manufacturing can be achieved by enabling perfusion in the production bioreactor when applied in tandem with a stable producer cell line, allowing for significant expansion of cells and production of lentiviral vectors without the need for transfer plasmids. Tangential flow depth filtration was used to achieve an intensified lentiviral vector production by enabling perfusion to expand cell density and allow for continuous separation of lentiviral vectors from producer cells. Hollow-fiber depth filters made of polypropylene with 2- to 4-μm channels demonstrated high filter capacity, extended functional life, and efficient separation of lentiviral vectors from producer cells and debris when used for this intensified process. We anticipate that process intensification with tangential flow depth filtration at 200-L scale from a suspension culture can produce on the order of magnitude of 10,000 doses per batch of lentiviral vectors required for CAR T or TCR cell and gene therapy that would require approximately 2×109 transducing units per dose.

Machine learning and metabolic modelling assisted implementation of a novel process analytical technology in cell and gene therapy manufacturing

Process analytical technology (PAT) has demonstrated huge potential to enable the development of improved biopharmaceutical manufacturing processes by ensuring the reliable provision of quality products. However, the complexities associated with the manufacture of advanced therapy medicinal products have resulted in a slow adoption of PAT tools into industrial bioprocessing operations, particularly in the manufacture of cell and gene therapy products. Here we describe the applicability of a novel refractometry-based PAT system (Ranger system), which was used to monitor the metabolic activity of HEK293T cell cultures during lentiviral vector (LVV) production processes in real time. The PAT system was able to rapidly identify a relationship between bioreactor pH and culture metabolic activity and this was used to devise a pH operating strategy that resulted in a 1.8-fold increase in metabolic activity compared to an unoptimised bioprocess in a minimal number of bioreactor experiments; this was achieved using both pre-programmed and autonomous pH control strategies. The increased metabolic activity of the cultures, achieved via the implementation of the PAT technology, was not associated with increased LVV production. We employed a metabolic modelling strategy to elucidate the relationship between these bioprocess level events and HEK293T cell metabolism. The modelling showed that culturing of HEK293T cells in a low pH (pH 6.40) environment directly impacted the intracellular maintenance of pH and the intracellular availability of oxygen. We provide evidence that the elevated metabolic activity was a response to cope with the stress associated with low pH to maintain the favourable intracellular conditions, rather than being indicative of a superior active state of the HEK293T cell culture resulting in enhanced LVV production. Forecasting strategies were used to construct data models which identified that the novel PAT system not only had a direct relationship with process pH but also with oxygen availability; the interaction and interdependencies between these two parameters had a direct effect on the responses observed at the bioprocess level. We present data which indicate that process control and intervention using this novel refractometry-based PAT system has the potential to facilitate the fine tuning and rapid optimisation of the production environment and enable adaptive process control for enhanced process performance and robustness.

Lentiviral Transduction of Nonhuman Primate Hematopoietic Stem and Progenitor Cells.

The nonhuman primate (NHP) animal model is an important predictive preclinical model for developing gene and cell therapies. It is also an experimental animal model used to study hematopoietic stem and progenitor cell (HSPC) biology, with the capability of serving as a step for the translation of the basic research concepts from small animals to humans. Lentiviral vectors are currently the standard gene delivery vehicles for transduction of HSPCs in the clinical setting. They have proven to be less genotoxic and more efficient than the previously used murine γ-retroviruses. Transplantation of lentiviral vector-transduced HSPCs into autologous macaques has been well developed over the past two decades. In this chapter, we provide detailed methodologies for lentiviral vector transduction of rhesus macaque HSPCs, including production and titration of lentiviral vector, purification of CD34+ HSPCs, and lentiviral vector transduction and assessment.

Genetic Modification of Primary Human Myeloid Cells to Study Cell Migration, Activation, and Organelle Dynamics.

Myeloid dendritic cells (DCs) and macrophages are mononuclear phagocytes with key roles in the immune system. As antigen-presenting cells, they link innate detection of microbes with programming adaptive immune responses. Myeloid DCs and macrophages also play critical roles in development, promote tissue homeostasis, and direct repair in response to injury and inflammation. As cellular migration and organelle dynamics are intimately connected with these processes, it is necessary to develop tools to track myeloid cell behavior and function. Here, we build on previously established protocols to isolate primary human myeloid cells from peripheral blood and report an optimized method for their genetic modification with lentiviral vectors to study processes related to cell migration, activation, and organelle dynamics. Specifically, we provide a protocol for delivering genetically encoded fluorescent markers into primary monocyte-derived DCs (MDDCs) and monocyte-derived macrophages (MDMs) to label mitochondria, peroxisomes, and whole cells. We describe the isolation of primary CD14+ monocytes from peripheral blood using positive selection with magnetic beads and, alternatively, isolation based on plastic adherence. Isolated CD14+ cells can be transduced with lentiviral vectors and subsequently cultured in the presence of cytokines to derive MDDCs or MDMs. This protocol is highly adaptable for cotransduction with vectors to knock down or overexpress genes of interest. These tools enable mechanistic studies of genetically modified myeloid cells through flow cytometry, fluorescence microscopy, and other downstream assays. © 2022 Wiley Periodicals LLC. Basic Protocol: Transduction of MDDCs and MDMs with lentiviral vectors encoding fluorescent markers Alternate Protocol 1: Isolation of monocytes by plastic adhesion Alternate Protocol 2: Transduction of MDDCs and MDMs with lentiviral vectors to knock down or overexpress genes of interest Support Protocol 1: Production and purification of lentiviral vectors for transduction into primary human myeloid cells Support Protocol 2: Flow cytometry of MDDCs and MDMs Support Protocol 3: Fixed and live-cell imaging of fluorescent markers in MDMs and MDDCs.

Production of Lentiviral Vectors Using a HEK-293 Producer Cell Line and Advanced Perfusion Processing.

The field of lentiviral vector (LV) production continues to face challenges in large-scale manufacturing, specifically regarding producing enough vectors to meet the demand for treating patients as well as producing high and consistent quality of vectors for efficient dosing. Two areas of interest are the use of stable producer cell lines, which facilitates the scalability of LV production processes as well as making the process more reproducible and robust for clinical applications, and the search of a cell retention device scalable to industrial-size bioreactors. This manuscript investigates a stable producer cell line for producing LVs with GFP as the transgene at shake flask scale and demonstrates LV production at 3L bioreactor scale using the Tangential Flow Depth Filtration (TFDF) as a cell retention device in perfusion mode. Cumulative functional yields of 3.3 x 1011 and 3.9 x 1011 transducing units were achieved; the former over 6 days of LV production with 16.3 L of perfused media and the latter over 4 days with 16 L. In comparing to a previously published value that was achieved using the same stable producer cell line and the acoustic filter as the perfusion device at the same bioreactor scale, the TFDF perfusion run produced 1.5-fold higher cumulative functional yield. Given its scale-up potential, the TFDF is an excellent candidate to be further evaluated to determine optimized conditions that can ultimately support continuous manufacturing of LVs at large scale.

Transition from serum-supplemented monolayer to serum-free suspension lentiviral vector production for generation of chimeric antigen receptor T cells

Background aimsLentiviral vectors (LVs) have been used extensively in gene therapy protocols because of their high biosafety profile and capacity to stably express a gene of interest. Production of these vectors for the generation of chimeric antigen receptor (CAR) T cells in academic and research centers is achieved using serum-supplemented static monolayer cultures. Although efficient for pre-clinical studies, this method has a number of limitations. The main hurdles are related to its incompatibility with robust and controlled large-scale production. For this reason, cell suspension culture in bioreactors is desirable. Here the authors report the transition of LV particle production from serum-supplemented monolayer to serum-free suspension culture with the objective of generating CAR T cells. MethodsA self-inactivating LV anti-CD19 CAR was produced by transient transfection using polyethylenimine (PEI) in human embryonic kidney 293 T cells previously adapted to serum-free suspension culture. ResultsLV production of 8 × 106 transducing units (TUs)/mL was obtained in serum-supplemented monolayer culture. LV production in the serum-free suspension conditions was significantly decreased compared with monolayer production. Therefore, optimization of the transfection protocol was performed using design of experiments. The results indicated that the best condition involved the use of 1 μg of DNA/106 cells, 1 × 106 cells/mL and PEI:DNA ratio of 2.5:1. This condition used less DNA and PEI compared with the standard, thereby reducing production costs. This protocol was further improved with the addition of 5 mM of sodium butyrate and resulted in an increase in production, with an average of 1.5 × 105 TUs/mL. LV particle functionality was also assessed, and the results indicated that in both conditions the LV was capable of inducing CAR expression and anti-tumor response in T cells, which in turn were able to identify and kill CD19+ cells in vitro. ConclusionsThis study demonstrates that the transition of LV production from small-scale monolayer culture to scalable and controllable bioreactors can be quite challenging and requires extensive work to obtain satisfactory production.

Thinking big: the need for innovation in the production of lentivirus-based cell and gene therapies

<b>Elisa Manzotti</b>, Founder, BioInsights, speaks to <b>Hanna Leinonen, John Moscariello, Lee DaviesScott Cross </b>and <b>Will Junker</b> <div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/H Leinonen copy.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Hanna Leinonen</h3><h5 class="author-title">Senior Scientist, Kuopio Center for Gene and Cell Therapy</h5> Hanna M. Leinonen holds a PhD in Molecular Medicine from the University of Eastern Finland (2014). She has 15 years’ experience on lentiviral vectors and 6 years’ experience on bioprocessing. She is currently working as a Senior Scientist in Kuopio Center for Gene and Cell Therapy (Finland) focusing on development of upstream processing for viral vectors (lentivirus, adenovirus, AAV). </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/26118d9d52f34a12859025a3d5f4aabc.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">John Moscariello</h3><h5 class="author-title">Executive Director, Viral Vector and Gene Editing Process Development, Bristol Myers Squibb</h5> John Moscariello currently serves as Executive Director of Viral Vector and Gene Editing Process Development at Bristol-Myers Squib (BMS). At BMS, John’s group develops viral vector processes that enable rapid timelines to generate clinical viral vectors as well as the development and characterization of commercial viral vector processes. Prior to his work at BMS, John was the Vice President of Process Development at AGC Biologics where his team was responsible cell line development, upstream and downstream process development, analytical and formulation development, and technical support for AGC Biologic’s commercial manufacturing facility and supported development activities from generating processes for Toxicology/Phase 1 supply up to and including commercialization and post-approval process support. John is very active in the biotechnology community. He is on the Scientific Advisory Board for various conferences, including the BioProcess International conference series, and the CBI conference series on achieving efficient facilities and the next frontier of single-use technologies. John obtained his Ph.D. in Chemical and Biological Engineering from the University of Wisconsin-Madison, and his B.Eng. in Chemical Engineering from the University of Delaware. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Lee Davies 1.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Lee Davies</h3><h5 class="author-title">Director, Process Research &amp; Development, Oxford Biomedica</h5><p class="oneComWebmail-MsoNormal"><i>As Director of Process Research and Development, Lee helps drive development, innovation and improvements in&nbsp;</i><i>world-class lentiviral vector manufacturing processes,&nbsp;</i><i>applying scientific and engineering principles to all aspects of upstream and downstream process development, including process optimisation and scale-up, technology transfer to GMP manufacturing and process characterisation to support chemistry, manufacturing and controls (CMC) regulatory submissions.&nbsp;</i><i>As part of&nbsp;the</i><i>&nbsp;Process Research and Development team, Lee works</i><i>&nbsp;at the&nbsp;cutting edge of gene therapy, ensuring its applicability to the needs of the wider gene and cell therapy community. The team’s mission covers the&nbsp;</i><i>life cycle of gene therapies, taking</i><i>&nbsp;processes from the early phase development work through to&nbsp;fully optimised large scale, GMP-ready processes, manufacturing and commercialisation.&nbsp;</i><i>Prior to joining Oxford Biomedica 6 years ago, Lee worked at the University of Oxford as a postdoctoral researcher and obtained a D.Phil in Gene Therapy for Cystic Fibrosis.</i> </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Scott_122321_WebReady copy.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Scott Cross</h3><h5 class="author-title">Senior Principal, Dark Horse Consulting</h5> Scott has over 20 years experience working with viral vectors, vaccines, and biologics in GMP environments. He has been responsible for cleanroom design, build out and commissioning of vector facilities, as well as the oversight of viral vector, cell therapy, fill finish and manufacturing support departments. Scott started his career at Merck and Co. where he worked on the development of an Adenovirus-based HIV vaccine and later, the development, optimization, and validation of release assays for live virus vaccines. He then moved to Indiana University (IU) where he managed the IU Vector Production Facility (IUVPF), overseeing GMP production and testing of Lentiviral and Retroviral vectors for the IUVPF and the National Gene Vector Laboratories (NGVL). While at the IUVPF he also managed the design, build and commissioning of a new viral vector GMP production and testing facility. After leaving the IUVPF, Scott joined Cincinnati Children’s Hospital Medical Center (CCHMC) managing the GMP Vector Production Facility, Viral Vector Core, and the Aseptic Processing Labs. Scott then moved to the University of Florida where he was the Director of Cell Therapy, Fill Finish and Manufacturing Support Operations for Florida Biologix and Brammer Bio. He was also responsible for the design of two new fill finish suites. Most recently, Scott served as Vice President of Vector Operations at Orchard Therapeutics where he was one of the original ten members and responsible for viral vector development, GMP vector production, plasmid production, oversight of 10 CDMOs, due diligence, and facility design. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Will Junker headshot copy.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Will Junker</h3><h5 class="author-title">Head of Vector Manufacturing Quality, Kite Pharma</h5> Will Junker is Head of Vector Quality and oversees the operation of QC, QA and QE to bring Kite’s vector manufacturing facility on line through licensure. Before this role, Will built Kite’s Quality Engineering function globally. His 35 year career in BioPharma includes 13 years at AstraZeneca and 15 years at Aventis/Sanofi as well as a few years at Baxter BioScience in technical/quality roles of increasing responsibility. </div></div></div>