Progress and Perspectives in the Development of Lentiviral Vector Producer Cells

After two decades of clinical trials, gene therapy demonstrated effectiveness in the treatment of a series of diseases. Currently, several gene therapy products are approved and used in the clinic. Lentiviral vectors (LVs) are one of the most used transfer vehicles to deliver genetic material and the vector of choice to modify hematopoietic cells to correct primary immunodeficiencies, hemoglobinopathies, and leukodystrophies. LVs are also widely used to modify T cells to treat cancers in immunotherapies (e.g., chimeric antigen receptors T cell therapies, CAR-T). In genome editing, LVs are used to deliver sequence-specific designer nucleases and DNA templates. The approval LV gene therapy products (e.g., Kymriah, for B-cell Acute lymphoblastic leukemia treatment; LentiGlobin, for β-thalassemia treatment) reinforced the need to improve their bioprocess manufacturing. The production has been mostly dependent on transient transfection. Production from stable cell lines facilitate GMP compliant processes, providing an easier scale-up, reproducibility and cost-effectiveness. The establishment of stable LV producer cell lines presents, however, several difficulties, with the cytotoxicity of some of the vector proteins being a major challenge. Genome editing technologies pose additional challenges to LV producer cells. Herein the major bottlenecks, recent achievements, and perspectives in the development of LV stable cell lines are revised.     

A simple, versatile and efficient method to genetically modify human monocyte-derived dendritic cells with HIV-1-derived lentiviral vectors

Lentiviral vectors derived from the human immunodeficiency type 1 virus (HIV-1 LV) are among the finest tools available today for the genetic modification of human monocyte-derived dendritic cells (MDDCs). However, this process is largely inefficient because MDDCs show a strong resistance to HIV-1 transduction. Here we describe a step-by-step protocol from the production of LVs to cell transduction that allows the efficient genetic modification of MDDCs. This protocol can be completed in 23 d from the initial phase of LV production to the final analysis of the results of MDDC transduction. The method relies on the simultaneous addition of HIV-1 LVs along with noninfectious virion-like particles carrying Vpx, a nonstructural protein encoded by the simian immunodeficiency virus (Vpx-VLPs). When thus provided in target cells, Vpx exerts a strong positive effect on incoming LVs by counteracting the restriction present in MDDCs; accordingly, 100% of cells can be transduced with low viral inputs. Vpx-VLPs will improve the efficiency of LV-mediated transduction of MDDCs with vectors for both ectopic gene expression and depletion studies.     

Molecular Determinants of Vectofusin-1 and Its Derivatives for the Enhancement of Lentivirally Mediated Gene Transfer into Hematopoietic Stem/Progenitor Cells

Gene delivery into hCD34+ hematopoietic stem/progenitor cells (HSPCs) using human immunodeficiency virus, type 1-derived lentiviral vectors (LVs) has several promising therapeutic applications. Numerous clinical trials are currently underway. However, the efficiency, safety, and cost of LV gene therapy could be ameliorated by enhancing target cell transduction levels and reducing the amount of LV used on the cells. Several transduction enhancers already exist, such as fibronectin fragments or cationic compounds. Recently, we discovered Vectofusin-1, a new transduction enhancer, also called LAH4-A4, a short histidine-rich amphipathic peptide derived from the LAH4 family of DNA transfection agents. Vectofusin-1 enhances the infectivity of lentiviral and γ-retroviral vectors pseudotyped with various envelope glycoproteins. In this study, we compared a family of Vectofusin-1 isomers and showed that Vectofusin-1 remains the lead peptide for HSPC transduction enhancement with LVs pseudotyped with vesicular stomatitis virus glycoproteins and also with modified gibbon ape leukemia virus glycoproteins. By comparing the capacity of numerous Vectofusin-1 variants to promote the modified gibbon ape leukemia virus glycoprotein-pseudotyped lentiviral vector infectivity of HSPCs, the lysine residues on the N-terminal extremity of Vectofusin-1, a hydrophilic angle of 140° formed by the histidine residues in the Schiffer-Edmundson helical wheel representation, hydrophobic residues consisting of leucine were all found to be essential and helped to define a minimal active sequence. The data also show that the critical determinants necessary for lentiviral transduction enhancement are partially different from those necessary for efficient antibiotic or DNA transfection activity of LAH4 derivatives. In conclusion, these results help to decipher the action mechanism of Vectofusin-1 in the context of hCD34+ cell-based gene therapy.     

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.     

Plasmacytoid Dendritic Cells Contribute to Systemic but Not Local Antiviral Responses to HSV Infections

Plasmacytoid dendritic cells (pDC) produce type I interferons (IFN-I) and proinflammatory cytokines in response to viruses; however, their contribution to antiviral immunity in vivo is unclear. In this study, we investigated the impact of pDC depletion on local and systemic antiviral responses to herpes simplex virus (HSV) infections using CLEC4C-DTR transgenic mice. We found that pDC do not appear to influence viral burden or survival after vaginal HSV-2 infection, nor do they seem to contribute to virus-specific CD8 T cell responses following subcutaneous HSV-1 infection. In contrast, pDC were important for early IFN-I production, proinflammatory cytokine production, NK cell activation and CD8 T cell responses during systemic HSV-2 and HSV-1 infections. Our data also indicate that unlike pDC, TLR3-expressing cells are important for promoting antiviral responses to HSV-1 regardless of the route of virus administration.     

Induction of primary anti-HIV CD4 and CD8 T cell responses by dendritic cells transduced with self-inactivating lentiviral vectors

In this study, we demonstrate that a minimal self-inactivating (SIN) lentiviral vector (LV) that does not encode any human immunodeficiency virus (HIV) genes is able to induce HIV-specific CD4 and CD8 T cell responses after transduction of dendritic cells (DCs). The LV-DC-primed T cells displayed HIV-specific lytic degranulation, as illustrated by acquisition of CD107a/b expression on the cell surface and up-regulation of active caspase 3. HIV-specific cytotoxic T lymphocyte (CTL) response was consistently detected using different assays, and T cell receptors specific to three prominent HIV epitopes, SL9 (Gag peptide: SLYNTVATL), IV9 (Pol peptide: ILKEPVHGV), and MA10 (In peptide: MASDFNLPPV) were detected using HLA-A0201 peptide-tetramers. These results demonstrate that DCs transduced with the minimal SIN-LV can efficiently induce HIV-specific CD4 and CD8 T cell responses. Since LVs are popular gene transfer tools, our results have fundamental implications for future LV applications and DC vaccine development.     

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.     

Visualization of DC-SIGN-Mediated Entry Pathway of Engineered Lentiviral Vectors in Target Cells

Dendritic cells (DCs) are potent antigen-presenting cells and therefore have enormous potential as vaccine targets. We have previously developed an engineered lentiviral vector (LV) that is pseudotyped with a mutated Sindbis virus glycoprotein (SVGmu), which is capable of targeting DCs through Dendritic Cell-specific ICAM3-grabbing Nonintegrin (DC-SIGN), a receptor that is predominantly expressed by DCs. In this study, we aimed to elucidate the internalization and trafficking mechanisms of this viral vector system through direct visualization of GFP-Vpr-tagged viral particles in target DCs, which was further corroborated by drug inhibition and dominant-negative mutants of cellular proteins that regulate the endocytic traffic. We demonstrated that our engineered LVs enter the cell via receptor-mediated clathrin- and dynamin-dependent endocytosis. Microtubule networks were also involved in a productive infection. Viral vector fusion was low-pH-dependent and occurred in the early endosomal stage of the intracellular transport. Autophagy was also examined for its effect on transduction efficiency, and we observed that enhanced autophage activity reduced vector infectivity, while suppressed autophagy boosted transduction efficiency. This study shed some light on the internalization and trafficking mechanisms of DC-directed LVs and offers some strategies to further improve the efficiency of LV-mediated gene therapy.     

Regulation of Porcine Plasmacytoid Dendritic Cells by Cytokines

Plasmacytoid dendritic cells (pDC) are the most potent producers of type-I interferon (IFN) and represent the main interferon (IFN)-α source in response to many viruses. Considering the important roles played by type I IFN’s, not only as antiviral effectors but also as potent alarming cytokine of the immune system, we investigated how such responses are regulated by various cytokines. To this end, we stimulated enriched pDC in the presence or absence of particular cytokines with a strong activator, CpG DNA, or a weak activator of pDC, foot-and-mouth disease virus (FMDV). Alternatively, we pre-incubated pDC for 16 h before stimulation. The pro-inflammatory cytokines tested Interleukin (IL)-6, IL17A, tumour necrosis factor (TNF)-α did not influence IFN-α responses except TNF-α, which promoted responses induced by FMDV. The haematopoietic cytokines Fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) had enhancing effects on pDC activation at least in one of the protocols tested. IFN-β and IFN-γ were the most potent at enhancing FMDV-induced IFN-α, up to 10-fold. Interestingly, also the Th2 cytokine IL-4 was an efficient promoter of pDC activity, while IL-10 was the only negative regulator of IFN-α in pDC identified. The cytokines enhancing IFN-α responses also promoted pDC survival in cell culture with the exception of GM-CSF. Taken together this work illustrates how the cytokine network can influence pDC activation, a knowledge of relevance for improving vaccines and therapeutic interventions during virus infections, cancers and autoimmune diseases in which pDC play a role.     

Measles virus glycoprotein-pseudotyped lentiviral vectors are highly superior to vesicular stomatitis virus G pseudotypes for genetic modification of monocyte-derived dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells capable of promoting or regulating innate and adaptive immune responses against non-self antigens. To better understand the DC biology or to use them for immune intervention, a tremendous effort has been made to improve gene transfer in these cells. Lentiviral vectors (LVs) have conferred a huge advantage in that they can transduce nondividing cells such as human monocyte-derived DCs (MDDCs) but required high amounts of viral particles and/or accessory proteins such as Vpx or Vpr to achieve sufficient transduction rates. As a consequence, these LVs have been shown to cause dramatic functional modifications, such as the activation or maturation of transduced MDDCs. Taking advantage of new pseudotyped LVs, i.e., with envelope glycoproteins from the measles virus (MV), we demonstrate that MDDCs are transduced very efficiently with these new LVs compared to the classically used vesicular stomatitis virus G-pseudotyped LVs and thus allowed to achieve high transduction rates at relatively low multiplicities of infection. Moreover, in this experimental setting, no activation or maturation markers were upregulated, while MV-LV-transduced cells remained able to mature after an appropriate Toll-like receptor stimulation. We then demonstrate that our MV-pseudotyped LVs use DC-SIGN, CD46, and CD150/SLAM as receptors to transduce MDDCs. Altogether, our results show that MV-pseudotyped LVs provide the most accurate and simple viral method for efficiently transferring genes into MDDCs without affecting their activation and/or maturation status.     

Tailored HIV-1 Vectors for Genetic Modification of Primary Human Dendritic Cells and Monocytes

Monocyte-derived dendritic cells (MDDCs) play a key role in the regulation of the immune system and are the target of numerous gene therapy applications. The genetic modification of MDDCs is possible with human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LVs) but requires high viral doses to bypass their natural resistance to viral infection, and this in turn affects their physiological properties. To date, a single viral protein is able to counter this restrictive phenotype, Vpx, a protein derived from members of the HIV-2/simian immunodeficiency virus SM lineage that counters at least two restriction factors present in myeloid cells. By tagging Vpx with a short heterologous membrane-targeting domain, we have obtained HIV-1 LVs incorporating high levels of this protein (HIV-1-Src-Vpx). These vectors efficiently transduce differentiated MDDCs and monocytes either as previously purified populations or as populations within unsorted peripheral blood mononuclear cells (PBMCs). In addition, these vectors can be efficiently pseudotyped with receptor-specific envelopes, further restricting their cellular tropism almost uniquely to MDDCs. Compared to conventional HIV-1 LVs, these novel vectors allow for an efficient genetic modification of MDDCs and, more importantly, do not cause their maturation or affect their survival, which are unwanted side effects of the transduction process. This study describes HIV-1-Src-Vpx LVs as a novel potent tool for the genetic modification of differentiated MDDCs and of circulating monocyte precursors with strong potential for a wide range of gene therapy applications.     

Advances in Lentivirus Purification

Lentiviral vectors (LVs) have been increasingly used as a tool for gene and cell therapies since they can stably integrate the genome in dividing and nondividing cells. LV production and purification processes have evolved substantially over the last decades. However, the increasing demands for higher quantities with more restrictive purity requirements are stimulating the development of novel materials and strategies to supply the market with LV in a cost-effective manner. A detailed review of each downstream process unit operation is performed, limitations, strengths, and potential outcomes being covered. Currently, the majority of large-scale LV manufacturing processes are still based on adherent cell culture, although it is known that the industry is migrating fast to suspension cultures. Regarding the purification strategy, it consists of batch chromatography and membrane technology. Nevertheless, new solutions are being created to improve the current production schemes and expand its clinical use.     

Heterologous human immunodeficiency virus type 1 lentiviral vectors packaging a simian immunodeficiency virus-derived genome display a specific postentry transduction defect in dendritic cells

Heterologous lentiviral vectors (LVs) represent a way to address safety concerns in the field of gene therapy by decreasing the possibility of genetic recombination between vector and packaging constructs and the generation of replication-competent viruses. Using described LVs based on human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus MAC251 (SIV(MAC251)), we asked whether heterologous virion particles in which trans-acting factors belonged to HIV-1 and cis elements belonged to SIV(MAC251) (HIV-siv) would behave as parental homologous vectors in all cell types. To our surprise, we found that although the heterologous HIV-siv vector was as infectious as its homologous counterpart in most human cells, it was defective in the transduction of dendritic cells (DCs) and, to a lesser extent, macrophages. In DCs, the main postentry defect was observed in the formation of two-long-terminal-repeat circles, despite the fact that full-length proviral DNA was being synthesized and was associated with the nucleus. Taken together, our data suggest that heterologous HIV-siv vectors display a cell-dependent infectivity defect, most probably at a post-nuclear entry migration step. As homologous HIV and SIV vectors do transduce DCs, we believe that these results underscore the importance of a conserved interaction between cis elements and trans-acting viral factors that is lost or suboptimal in heterologous vectors and essential only in the transduction of certain cell types. For gene therapy purposes, these findings indicate that the cellular tropism of LVs can be modulated not only through the use of distinct envelope proteins or tissue-specific promoters but also through the specific combinatorial use of packaging and transfer vector constructs.     

Blocking TLR7- and TLR9-mediated IFN-α Production by Plasmacytoid Dendritic Cells Does Not Diminish Immune Activation in Early SIV Infection

Persistent production of type I interferon (IFN) by activated plasmacytoid dendritic cells (pDC) is a leading model to explain chronic immune activation in human immunodeficiency virus (HIV) infection but direct evidence for this is lacking. We used a dual antagonist of Toll-like receptor (TLR) 7 and TLR9 to selectively inhibit responses of pDC but not other mononuclear phagocytes to viral RNA prior to and for 8 weeks following pathogenic simian immunodeficiency virus (SIV) infection of rhesus macaques. We show that pDC are major but not exclusive producers of IFN-α that rapidly become unresponsive to virus stimulation following SIV infection, whereas myeloid DC gain the capacity to produce IFN-α, albeit at low levels. pDC mediate a marked but transient IFN-α response in lymph nodes during the acute phase that is blocked by administration of TLR7 and TLR9 antagonist without impacting pDC recruitment. TLR7 and TLR9 blockade did not impact virus load or the acute IFN-α response in plasma and had minimal effect on expression of IFN-stimulated genes in both blood and lymph node. TLR7 and TLR9 blockade did not prevent activation of memory CD4+ and CD8+ T cells in blood or lymph node but led to significant increases in proliferation of both subsets in blood following SIV infection. Our findings reveal that virus-mediated activation of pDC through TLR7 and TLR9 contributes to substantial but transient IFN-α production following pathogenic SIV infection. However, the data indicate that pDC activation and IFN-α production are unlikely to be major factors in driving immune activation in early infection. Based on these findings therapeutic strategies aimed at blocking pDC function and IFN-α production may not reduce HIV-associated immunopathology.     

Plasmacytoid dendritic cells promote host defense against acute pneumovirus infection via the TLR7-MyD88-dependent signaling pathway

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants. In human infants, plasmacytoid dendritic cells (pDC) are recruited to the nasal compartment during infection and initiate host defense through the secretion of type I IFN, IL-12, and IL-6. However, RSV-infected pDC are refractory to TLR7-mediated activation. In this study, we used the rodent-specific pathogen, pneumonia virus of mice (PVM), to determine the contribution of pDC and TLR7 signaling to the development of the innate inflammatory and early adaptive immune response. In wild-type, but not TLR7- or MyD88-deficient mice, PVM inoculation led to a marked infiltration of pDC and increased expression of type I, II, and III IFNs. The delayed induction of IFNs in the absence of TLR7 or MyD88 was associated with a diminished innate inflammatory response and augmented virus recovery from lung tissue. In the absence of TLR7, PVM-specific CD8(+) T cell cytokine production was abrogated. The adoptive transfer of TLR7-sufficient, but not TLR7-deficient pDC to TLR7 gene-deleted mice recapitulated the antiviral responses observed in wild-type mice and promoted virus clearance. In summary, TLR7-mediated signaling by pDC is required for appropriate innate responses to acute pneumovirus infection. It is conceivable that as-yet-unidentified defects in the TLR7 signaling pathway may be associated with elevated levels of RSV-associated morbidity and mortality among otherwise healthy human infants.