Use Of Lentiviral Vectors Research Articles

Emerging gene therapy approaches for the treatment of severe combined immunodeficiency (SCID): a comprehensive review

Background. Severe Combined Immunodeficiency (SCID) is a life-threatening condition resulting from various genetic mutations that impair immune cell development. Traditional treatment via hematopoietic stem cell transplantation (HSCT) has limitations, prompting exploration into gene therapy as a promising alternative. Objectives. This review aims to evaluate emerging gene therapy approaches for SCID, emphasizing the use of lentiviral vectors and CRISPR/Cas9 technology, and to assess their efficacy and safety in comparison to traditional HSCT. Materials and Methods. A comprehensive literature search was conducted using PubMed and Google Scholar to identify peer-reviewed articles, clinical trials, and observational studies on gene therapy for SCID published in the last 10 years. Studies were included based on their relevance to gene therapy interventions, and outcomes related to efficacy and safety were analyzed. Discussions. Gene therapy has shown significant promise in SCID treatment, particularly through the use of lentiviral vectors and CRISPR/Cas9 for precise genetic correction. Clinical trials demonstrate improved immunological reconstitution and patient outcomes, with reduced side effects compared to HSCT. However, challenges such as optimizing protocols, ensuring long-term safety, and equitable access to treatments remain. Ongoing research and trials continue to advance our understanding, offering hope for more effective, personalized SCID therapies in the future.

Therapeutic Gene Editing for Hemoglobinopathies.

In the last ten years, a consistent number of clinical studies have evaluated different gene approaches for the treatment of patients with sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT). Initial studies of gene therapy for hemoglobinopathies involved the use of lentiviral vectors to add functional copies of the gene encoding β-globin in defective CD34 cells; more recently, gene editing techniques have been used involving either CRISPR-Cas9, transcription activation-like effector protein nuclease, zinc finger nuclease, and base editing to either induce fetal hemoglobin production at therapeutic levels or to genetically repair the underlying molecular defect causing the disease. Here, we review recent gene editing studies that have started the development of a new era in the treatment of hemoglobinopathies and, in general, monoallelic hereditary diseases.

HIV-1 gp120 amplifies astrocyte elevated gene-1 activity to compromise the integrity of the outer blood-retinal barrier.

This study aims to investigate the functions and mechanistic pathways of Astrocyte Elevated Gene-1 (AEG-1) in the disruption of the blood-retinal barrier (BRB) caused by the HIV-1 envelope glycoprotein gp120. We utilized ARPE-19 cells challenged with gp120 as our model system. Several analytical techniques were employed to decipher the intricate interactions at play. These included PCR, Western blot, and immunofluorescence assays for the molecular characterization, and transendothelial electrical resistance (TEER) measurements to evaluate barrier integrity. We observed that AEG-1 expression was elevated, whereas the expression levels of tight junction proteins ZO-1, Occludin, and Claudin5 were downregulated in gp120-challenged cells. TEER measurements corroborated these findings, indicating barrier dysfunction. Additional mechanistic studies revealed that the activation of NFκB and MMP2/9 pathways mediated the AEG-1-induced barrier destabilization. Through the use of lentiviral vectors, we engineered cell lines with modulated AEG-1 expression levels. Silencing AEG-1 alleviated gp120-induced downregulation of tight junction proteins and barrier impairment while concurrently inhibiting the NFκB and MMP2/9 pathways. Conversely, overexpression of AEG-1 exacerbated these pathological changes, further compromising the integrity of the BRB. Gp120 upregulates the expression of AEG-1 and activates the NFκB and MMP2/9 pathways. This in turn leads to the downregulation of tight junction proteins, resulting in the disruption of barrier function.

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.

Preclinical proof of concept of a tetravalent lentiviral T-cell vaccine against dengue viruses.

Dengue virus (DENV) is responsible for approximately 100 million cases of dengue fever annually, including severe forms such as hemorrhagic dengue and dengue shock syndrome. Despite intensive vaccine research and development spanning several decades, a universally accepted and approved vaccine against dengue fever has not yet been developed. The major challenge associated with the development of such a vaccine is that it should induce simultaneous and equal protection against the four DENV serotypes, because past infection with one serotype may greatly increase the severity of secondary infection with a distinct serotype, a phenomenon known as antibody-dependent enhancement (ADE). Using a lentiviral vector platform that is particularly suitable for the induction of cellular immune responses, we designed a tetravalent T-cell vaccine candidate against DENV ("LV-DEN"). This vaccine candidate has a strong CD8+ T-cell immunogenicity against the targeted non-structural DENV proteins, without inducing antibody response against surface antigens. Evaluation of its protective potential in the preclinical flavivirus infection model, i.e., mice knockout for the receptor to the type I IFN, demonstrated its significant protective effect against four distinct DENV serotypes, based on reduced weight loss, viremia, and viral loads in peripheral organs of the challenged mice. These results provide proof of concept for the use of lentiviral vectors for the development of efficient polyvalent T-cell vaccine candidates against all DENV serotypes.

Time-dependent sorption behavior of lentiviral vectors during anion-exchange chromatography.

Use of lentiviral vectors (LVs) in clinical Cell and Gene Therapy applications is growing. However, functional product loss during capture chromatography, typically anion-exchange (AIEX), remains a significant unresolved challenge for the design of economic processes. Despite AIEX's extensive use, variable performance and generally low recovery is reported. This poor understanding of product loss mechanisms highlights a significant gap in our knowledge of LV adsorption and other types of vector delivery systems. This work demonstrates HIV-1-LV recovery over quaternary-amine membrane adsorbents is a function of time in the adsorbed state. Kinetic data for product loss in the column bound state was generated. Fitting a second order-like rate model, we observed a rapid drop in functional recovery due to increased irreversible binding for vectors encoding two separate transgenes ( = 12.7 and 18.7 min). Upon gradient elution, a two-peak elution profile implicating the presence of two distinct binding subpopulations is observed. Characterizing the loss kinetics of these two subpopulations showed a higher rate of vector loss in the weaker binding peak. This work highlights time spent in the adsorbed state as a critical factor impacting LV product loss and the need for consideration in LV AIEX process development workflows.

Combined therapy with methotrexate nanoconjugate and dendritic cells with downregulated IL-10R expression modulates the tumor microenvironment and enhances the systemic anti-tumor immune response in MC38 murine colon carcinoma.

Understanding the negative impact of the tumor microenvironment on the creation of an effective immune response has contributed to the development of new therapeutic anti-cancer strategies. One such solution is combined therapy consisting of chemotherapeutic administration followed by dendritic cell (DC)-based vaccines. The use of cytostatic leads to the elimination of cancer cells, but can also modulate the tumor milieu. Moreover, great efforts are being made to increase the therapeutic outcome of immunotherapy, e.g. by enhancing the ability of DCs to generate an efficient immune response, even in the presence of immunosuppressive cytokines such as IL-10. The study aimed to determine the effectiveness of combined therapy with chemotherapeutic with immunomodulatory potential - HES-MTX nanoconjugate (composed of methotrexate (MTX) and hydroxyethyl starch (HES)) and DCs with downregulated expression of IL-10 receptor stimulated with tumor antigens (DC/shIL-10R/TAg) applied in MC38 murine colon carcinoma model. With the use of lentiviral vectors the DCs with decreased expression of IL-10R were obtained and characterized. During in vivo studies MC38-tumor bearing mice received MTX or HES-MTX nanoconjugate as a sole treatment or combined with DC-based immunotherapy containing unmodified DCs or DCs transduced with shRNA against IL-10R (or control shRNA sequence). Tumor volume was monitored during the experiment. One week after the last injection of DC-based vaccines, tumor nodules and spleens were dissected for ex vivo analysis. The changes in the local and systemic anti-tumor immune response were estimated with the use of flow cytometry and ELISA methods. In vitro studies showed that the downregulation of IL-10R expression in DCs enhances their ability to activate the specific anti-tumor immune response. The use of HES-MTX nanoconjugate and DC/shIL-10R/TAg in the therapy of MC38-tumor bearing mice resulted in the greatest tumor growth inhibition. At the local anti-tumor immune response level a decrease in the infiltration of cells with suppressor activity and an increase in the influx of effector cells into MC38 tumor tissue was observed. These changes were crucial to enhance the effective specific immune response at the systemic level, which was revealed in the greatest cytotoxic activity of spleen cells against MC38 cells.

Adenine base editor–mediated correction of the common and severe IVS1-110 (G>A) β-thalassemia mutation

Abstractβ-Thalassemia (BT) is one of the most common genetic diseases worldwide and is caused by mutations affecting β-globin production. The only curative treatment is allogenic hematopoietic stem/progenitor cells (HSPCs) transplantation, an approach limited by compatible donor availability and immunological complications. Therefore, transplantation of autologous, genetically-modified HSPCs is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors are not equally effective in all patients and CRISPR/Cas9 nuclease-based strategies raise safety concerns. Thus, base editing strategies aiming to correct the genetic defect in patients’ HSPCs could provide safe and effective treatment. Here, we developed a strategy to correct one of the most prevalent BT mutations (IVS1-110 [G>A]) using the SpRY-ABE8e base editor. RNA delivery of the base editing system was safe and led to ∼80% of gene correction in the HSPCs of patients with BT without causing dangerous double-strand DNA breaks. In HSPC-derived erythroid populations, this strategy was able to restore β-globin production and correct inefficient erythropoiesis typically observed in BT both in vitro and in vivo. In conclusion, this proof-of-concept study paves the way for the development of a safe and effective autologous gene therapy approach for BT.

Targeted Base Editing Strategies for Beta-Hemoglobinopathies

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult hemoglobin β-chain. In β-thalassemia, the reduced (β+) or absent (β0) production of β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent RBC sickling, vaso-occlusive crises, organ damage and reduced life expectancy. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors or CRISPR/Cas9 nuclease are not equally effective in all the patients and/or raise safety concerns. Base editing (BE) allows the introduction of point mutations (C>T by cytidine base editors, CBEs; A>G by adenine base editors, ABEs) without generating dangerous double strand breaks. In this study, we have developed BE strategies aiming either to correct the genetic defect or to target disease modifiers in patient cells, and provide a safe and effective treatment for β-hemoglobinopathies. First, we exploited the capacity of ABEs in combination with single guide RNAs (gRNAs) to correct prevalent β-thalassemic mutations (the CD39 and IVS2-1 β0 mutations and the IVS1-110 β+ mutation). RBCs derived from edited HSPCs exhibited high β-globin levels. The delayed erythroid differentiation typically observed in β-thalassemic cell cultures was corrected by our treatment. Measurement of Annexin+ cells by flow cytometry showed a substantially reduced apoptotic rate in edited β-thalassemic samples compared to untreated controls. Finally, off-target activity was minimal and by-stander edits (additional base conversions occurring close to the target base) did not impact the β-globin expression. In parallel, we used BE to target disease modifiers such as the fetal γ-globin genes. In fact, the co-inheritance of genetic mutations causing a sustained fetal β-like γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin (HbF), HPFH) reduces the clinical severity of β-hemoglobinopathies. HPFH mutations in the promoter of the two γ-globin genes, HBG1 and HBG2 eitherdisrupt the binding sites (BS) for transcriptional repressors (e.g., LRF) or generate BS for transcriptional activators (e.g., KLF1). BE allowed the introduction of HPFH mutations in the HBG promoters, which disrupt the LRF BS (via CBE) or generate the KLF1 BS (via ABE). This led to therapeutically relevant HbF levels in RBCs differentiated from edited β-thalassemia or SCD HSPCs, and rescued the pathological phenotypes (correcting ineffective erythropoiesis in β-thalassemia and the sickling phenotype in SCD RBCs). Finally, serial xenotransplantation experiments showed BE in long-term HSCs; however, the efficiency was reduced compared to input HSPCs. RNA-seq analysis showed no alteration for ABE-treated HSPCs, but activation of the p53 pathway and dysregulation of genes involved in HSC biology in CBE-treated samples. To increase the fitness of edited HSCs and editing efficiency, we optimized the RNA delivery of the BE machinery and used histone deacetylase, RNase and apoptosis inhibitors. Moreover, we developed a strategy to enrich edited cells. By way of example, two rounds of mRNA electroporation led to increased BE efficiency with minimal toxicity. Furthermore, highly processive base editors outperformed the original enzymes even upon only one round of electroporation. In conclusion, we developed efficient BE approaches to either correct highly prevalent mutations or induce HbF expression. Overall, our treatments led to correction of the pathological phenotypes and optimal BE efficiencies to finally enable the clinical development of base-edited HSPCs for β-hemoglobinopathies.

Two Is Better Than One: Fetal Hemoglobin Reactivation and Alpha-Globin Downregulation to Correct the β-Hemoglobinopathy Phenotype through Base Editing

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult hemoglobin β-chain. In β-thalassemia, the reduced (β+) or absent (β0) production of β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent RBC sickling, vaso-occlusive crises, organ damage and reduced life expectancy. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors or CRISPR/Cas9 nuclease are not equally effective in all the patients and/or raise safety concerns. Base editing (BE) allows the introduction of point mutations (C>T by cytidine base editors, CBEs; A>G by adenine base editors, ABEs) without generating dangerous double strand breaks, thus allowing the multiplexed editing of several targets. Mutations leading to elevated fetal Hb (HbF) or reduced α-globin expression are associated with a better clinical phenotype in both β-thalassemia and SCD patients. Fetal β-like γ-globin compensates for adult β-globin deficiency and reduced α-globin levels impairs its precipitation and inhibit Hb polymerization in SCD. In this study, we have developed BE strategies aiming to (i) reactivate HbF by down-regulating BCL11A, a master repressor of γ-globin expression though inactivation of its enhancers, and (ii) reduce the α-globin levels through targeting of the MCS-R2 enhancer of the α-globin genes (HBA1/2). First, we used CBEs and ABEs to target the GATA1 or ATF4 activator binding site (BS) within the erythroid-specific BCL11A enhancers (+58-kb and +55-kb regions) in SCD HSPCs through RNA delivery and achieved efficiencies of up to ~90%. A colony forming cell (CFC) assay showed no impact on progenitors' viability. RBCs derived from base-edited HSPCs exhibited high HbF expression levels that were sufficient to ameliorate the sickling phenotype. Samples carrying different mutations within the GATA1 or ATF4 BS were associated with different HbF levels, implying that some nucleotides are more essential for GATA1 and ATF4 binding and the consequent BCL11A gene activation. In parallel, we used CBEs and ABEs to target the GATA1 or NF-E2 activator BS within the MCS-R2 enhancer of HBA1/2. We screened single guide RNAs (sgRNAs) in an erythroid cell line (K562) that allowed editing of different nucleotides within the GATA1 and NF-E2 BS and we identified the most efficient sgRNAs diminishing α-globin expression. Next, selected sgRNAs were delivered to SCD HSPCs, achieving high editing efficiency (~85%). A CFC assay showed a good progenitor viability. RT-qPCR and Western Blot analyses in the erythroid progeny of edited SCD HSPCs confirmed α-globin downregulation at RNA and protein levels. Lastly, we combined both strategies using ABEs to target simultaneously BS within the BCL11A and MCS-R2 enhancers in SCD HSPCs. The concomitant usage of two sgRNAs did not affect editing efficiency at the different targeted sites, which was ~80% at both BCL11A and MCS-R2 enhancers. The erythroid progeny of edited SCD HSPCs harboring mutations in both BCL11A and MCS-R2 enhancers showed downregulation of α-globin and up-regulation of γ-globin at RNA and protein expression level. We expect that this combined strategy will efficiently rescue the pathological phenotype of RBCs derived from both SCD and β-thalassemic HSPCs. In conclusion, our study identified the core nucleotides of both the BCL11A and HBA1/2 MCS-R2 enhancers, which are crucial for regulating the expression of these genes and represent potential targets for scarless editing through BE to finely modulate globin expression. We also developed efficient BE strategies to simultaneously reactivate HbF and downregulate the α-globin expression in patient HSPCs. Validation of the above-described results in HSPCs in vivo will provide sufficient proof of efficacy and safety to enable the clinical development of base-edited HSPCs for the therapy of β-hemoglobinopathies.

Use of a Foamy-Virus Vector System to Produce an 'Off-the-Shelf' Fcγ-CR-T Cell Product for the Treatment of Hematological and Solid Tumour Malignancies

Introduction Chimeric antigen receptor (CAR) T cells have transformed the field of cancer immunotherapy. Other approaches, such as the use of Fc gamma chimeric receptor (Fcγ-CR)-T cells have further expanded the applicability of such therapies in both solid and liquid tumours with the added benefit of tackling some of the hurdles associated with CAR-T therapies (such as loss or down-regulation of the target antigen). Previous studies have generated Fcγ-CR-T cells from autologous cells, which carry several limitations (prolonged production time, costly, manufacturing failure), whereas the use of Lentiviral vectors (LV) is endowed with extra limitations (packaging limits and mutagenesis risk). Our group has developed an in-house Fcγ-CR-T cell product, using a safer to LV, foamy virus (FV) vector and T cells from healthy donors. Materials We constructed FV vectors expressing the CD16 V158, which has higher Fc binding and is associated with higher tumour killing, the T cell stimulatory molecule CD3ζ and the costimulatory molecule CD28 and an EFP1a promoter. 2nd generation LV vector backbones were purchased from a commercial vendor. Peripheral blood from healthy individuals were used as T cell sources. T cells were activated by anti-CD3/CD28 beads and transduced with CD16-CR, LV or FV vectors. Transduction efficiency was assayed by flow cytometry (FCM) using an anti-CD16 antibody (Ab), on day 3. The human cell lines Raji, Panc01 and DLD-1 were used for functional assays, in the presence of the Abs Rituximab (anti-CD20) and Cetuximab (anti-EGFR), respectively, at a concentration of 0.1 ug/ml. The CR's Ab-binding capacity was assessed by incubating the CD16-CRs with the Abs for 30mins, followed by an anti-human Ig PE antibody and the related fluorescence intensity was assessed. To determine whether Ab binding to CD16-CR can promote aggregation of effector and target cells, CFSE-labelled target cells were mixed with Ab-coated CD16-CRs for 60 mins and the formation of CFSE-PE doublets was assessed by FCM. Their cytotoxic effect was evaluated against the CFSE-labelled Raji or DLD-1 or Panc01 cells at different ratios (5:1, 10:1) for 18 hours, in the presence of Rituximab (0.1ug/ml) or Cetuximab (0.1ug/ml), respectively. The % of live cells was assessed by flow cytometry and calculated as: [1-live targets (sample)/live targets (control)]x100. Results LV and FV vector titers were between 3-5x10^6 TU/ml and 4-5x10^6 TU/ml, respectively. Transduction efficiency ranged from 58.3-69.2% with FV vectors (MOI 3-5) and 85.2-85.9% with LV vectors (MOI 10-20). The Ab-binding capacity of FV-CD16-CRs was determined to be 68.7% (SEM 2.3) and 71.3% (SEM 3.1), (n=3) for Rituximab and Cetuximab, respectively, whereas that of LV-CD16-CRs was 72.1 (SEM 3.3) and 76.5 (SEM 2.8), (n=3), respectively. Cell aggregation of effector and target cells, assessed as doublets, was: (i) 32%, 39% and 36% for FV-CD16-CRs and (ii) 26%, 31% and 29% for LV-CD16-CRs, coated with Rituximab and Cetuximab, respectively, and it was specific for the Raji, DLD-1 and Panc01 cells, respectively. Next, we assessed whether CD16-CR T cells were able to kill target cells in the presence of specific antibodies. Results showed that the FV-CD16-CR-induced % cell lysis was: (i) 26.3% and 51.5% at 5:1 and 10:1 ratio, respectively, in the presence of Rituximab and Raji cells, (ii) 41.5% and 57.7% at 5:1 and 10:1 ratio, respectively, in the presence of Cetuximab and DLD1 cells and (iii) 39.4% and 57.3% at 5:1 and 10:1 ratio, respectively, in the presence of Cetuximab and Panc01 cells. For LV-CD16-CRs the respective % lysis was comparable. More importantly, this lysis was shown to be specific (no lysis noted with untransduced T cells) and significantly lower in the absence of the antibodies. Conclusion Our group has developed for the first time a FV vector for the generation of CD16-CR T cells, with an efficient gene transfer to human T cells and with potent in vitro cytotoxic properties, similar to their LV-derived counterpart. Overall, we provide a proof of concept that allogeneic, in-house Fcγ-CR-T cells derived from a non-pathogenic viral backbone such as the FV, could be a safe, efficient and affordable alternative to LV-derived vectors for immunotherapy. Our future aim is to further modify these Fcγ-CR-T cells, by interfering with immune checkpoint molecules, in order to achieve better tumour penetration and tackle the anergy induced by the tumour microenvironment.

Culture media selection and feeding strategy for high titer production of a lentiviral vector by stable producer clones cultivated at high cell density

The growing interest in the use of lentiviral vectors (LVs) for various applications has created a strong demand for large quantities of vectors. To meet the increased demand, we developed a high cell density culture process for production of LV using stable producer clones generated from HEK293 cells, and improved volumetric LV productivity by up to fivefold, reaching a high titer of 8.2 × 107 TU/mL. However, culture media selection and feeding strategy development were not straightforward. The stable producer clone either did not grow or grow to lower cell density in majority of six commercial HEK293 media selected from four manufacturers, although its parental cell line, HEK293 cell, grows robustly in these media. In addition, the LV productivity was only improved up to 53% by increasing cell density from 1 × 106 and 3.8 × 106 cells/mL at induction in batch cultures using two identified top performance media, even these two media supported the clone growth to 5.7 × 106 and 8.1 × 106 cells/mL, respectively. A combination of media and feed from different companies was required to provide diverse nutrients and generate synergetic effect, which supported the clone growing to a higher cell density of 11 × 106 cells/mL and also increasing LV productivity by up to fivefold. This study illustrates that culture media selection and feeding strategy development for a new clone or cell line can be a complex process, due to variable nutritional requirements of a new clone. A combination of diversified culture media and feed provides a broader nutrients and could be used as one fast approach to dramatically improve process performance.

Commercial-scale lentiviral vector manufacturing: is the myth busted?

Use of lentiviral vectors in clinical trials has increased through the last decade. Lentivirus based products have entered later trial phases, and cell and gene therapy products utilizing lentiviral vectors ex vivo have already been accepted on market by FDA and EMA. Thus, it is no wonder that there is a demand for commercial-scale lentiviral vector production. During the last ten years, large progress has fortunately occurred in both adherent and suspension production enabling manufacturing of large quantities of lentiviral vectors in relatively non-laborious manner. In this review, different scale-up options for lentiviral vector manufacturing are described, and aspects that should be taken into account while scaling up the process are covered.

Lentiviral Vectors Delivered with Biomaterials as Therapeutics for Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating trauma that can cause permanent disability, life-long chronic issues for sufferers and is a big socioeconomic burden. Regenerative medicine aims to overcome injury caused deficits and restore function after SCI through gene therapy and tissue engineering approaches. SCI has a multifaceted pathophysiology. Due to this, producing therapies that target multiple different cellular and molecular mechanisms might prove to be a superior approach in attempts at regeneration. Both biomaterials and nucleic acid delivery via lentiviral vectors (LVs) have proven to promote repair and restoration of function post SCI in animal models. Studies indicate that a combination of biomaterials and LVs is more effective than either approach alone. This review presents studies supporting the use of LVs and LVs delivered with biomaterials in therapies for SCI and summarises methods to combine LVs with biomaterials for SCI treatment. By summarising this knowledge this review aims to demonstrate how LV delivery with biomaterials can augment/compliment both LV and biomaterial therapeutic effects in SCI.

Designing Lentiviral Vectors for Gene Therapy of Genetic Diseases.

Lentiviral vectors are the most frequently used tool to stably transfer and express genes in the context of gene therapy for monogenic diseases. The vast majority of clinical applications involves an ex vivo modality whereby lentiviral vectors are used to transduce autologous somatic cells, obtained from patients and re-delivered to patients after transduction. Examples are hematopoietic stem cells used in gene therapy for hematological or neurometabolic diseases or T cells for immunotherapy of cancer. We review the design and use of lentiviral vectors in gene therapy of monogenic diseases, with a focus on controlling gene expression by transcriptional or post-transcriptional mechanisms in the context of vectors that have already entered a clinical development phase.

Single Transcription Factor-Based Differentiation Allowing Fast and Efficient Oligodendrocyte Generation via SOX10 Overexpression.

Oligodendrocytes are the main glial cell type in the central nervous system supporting the axonal part of neurons via myelin and lactate delivery. Both the conductive myelin formation and the energy support via lactate can be affected in diseases, such as multiple sclerosis and amyotrophic lateral sclerosis, respectively. Therefore, human disease modeling is needed to gain more mechanistic insights to drive drug discovery research. Here, patient-derived induced pluripotent stem cells (iPSCs) serve as a necessary tool providing an infinite cell source for patient-specific disease modeling, which allows investigation of oligodendrocyte involvement in human disease.Small molecule-based differentiation protocols to generate oligodendrocytes from pluripotent stem cells can last more than 90days. Here, we provide a transcription factor-based, fast and efficient protocol for generating O4+ oligodendrocytes in just 20-24days. After a neural induction phase of 8-12days, SOX10 is overexpressed either with the use of lentiviral vectors or via engineered iPSCs, which inducibly overexpress SOX10 after doxycycline addition. Using this last method, a pure O4+ cell population is achieved after keeping the SOX10-overexpressing neural stem cells in culture for an additional 10days. Furthermore, these O4+ cells can be co-cultured with iPSC-derived cortical neurons in 384-well format, allowing pro-myelinating drug screens. In conclusion, we provide a fast and efficient oligodendrocyte differentiation protocol allowing both in vitro human disease modeling and a high-throughput co-culture system for drug discovery.

Gene Therapy Applications of Non-Human Lentiviral Vectors.

Recent commercialization of lentiviral vector (LV)-based cell therapies and successful reports of clinical studies have demonstrated the untapped potential of LVs to treat diseases and benefit patients. LVs hold notable and inherent advantages over other gene transfer agents based on their ability to transduce non-dividing cells, permanently transform target cell genome, and allow stable, long-term transgene expression. LV systems based on non-human lentiviruses are attractive alternatives to conventional HIV-1-based LVs due to their lack of pathogenicity in humans. This article reviews non-human lentiviruses and highlights their unique characteristics regarding virology and molecular biology. The LV systems developed based on these lentiviruses, as well as their successes and shortcomings, are also discussed. As the field of gene therapy is advancing rapidly, the use of LVs uncovers further challenges and possibilities. Advances in virology and an improved understanding of lentiviral biology will aid in the creation of recombinant viral vector variants suitable for translational applications from a variety of lentiviruses.

Gene therapy for severe combined immunodeficiencies and beyond.

Ex vivo retrovirally mediated gene therapy has been shown within the last 20 yr to correct the T cell immunodeficiency caused by γc-deficiency (SCID X1) and adenosine deaminase (ADA) deficiency. The rationale was brought up by the observation of the revertant of SCIDX1 and ADA deficiency as a kind of natural gene therapy. Nevertheless, the first attempts of gene therapy for SCID X1 were associated with insertional mutagenesis causing leukemia, because the viral enhancer induced transactivation of oncogenes. Removal of this element and use of a promoter instead led to safer but still efficacious gene therapy. It was observed that a fully diversified T cell repertoire could be generated by a limited set (<1,000) of progenitor cells. Further advances in gene transfer technology, including the use of lentiviral vectors, has led to success in the treatment of Wiskott-Aldrich syndrome, while further applications are pending. Genome editing of the mutated gene may be envisaged as an alternative strategy to treat SCID diseases.

Optimized Production of Lentiviral Vectors for CAR-T Cell.

Advances in the use of lentiviral vectors for gene therapy applications have created a need for large-scale manufacture of clinical-grade viral vectors for transfer of genetic materials. Lentiviral vectors can transduce a wide range of cell types and integrate into the host genome of dividing and nondividing cells, resulting in long-term expression of the transgene both in vitro and in vivo. In this chapter, we present a method to transfect human cells, creating an easy platform to produce lentiviral vectors for CAR-T cell application.

Preclinical Proof-of-Concept, Analytical Development, and Commercial Scale Production of Lentiviral Vector in Adherent Cells

The therapeutic efficacy of a lentiviral vector (LV) expressing the herpes simplex virus thymidine kinase (HSV-TK) was studied in an immunocompetent rat glioblastoma model. Intraperitoneal ganciclovir injections (50 mg/kg/day) were administered for 14 consecutive days, resulting in reduced tumor volumes as monitored by MRI. Survival analyses revealed a significant improvement among the LV-expressing HSV-TK (LV-TK)/ganciclovir-treated animals when compared to non-treated control rats. However, a limiting factor in the use of LV has been the suboptimal small-scale production in flasks. Our aim during the translation phase, prior to entering the final pre-clinical and early clinical phases, was to develop a scalable, robust, and disposable manufacturing process for LV-TKs. We also aimed to minimize future process changes and enable production upscaling to make the process suitable for larger patient populations. The upstream process relies on fixed-bed iCELLis technology and transient plasmid transfection. This is the first time iCELLis 500 commercial-scale bioreactor was used for LV production. A testing strategy to determine the pharmacological activity of LV-TK drug product by measuring cell viability was developed, and the specificity of the potency assay was also proven. In this paper we focus on upstream process development while showing analytical development and the proof-of-concept of LV-TK functionality.