RESUMEN

Lentiviral vector (LVV)-mediated cell and gene therapies have the potential to cure diseases that currently require lifelong intervention. However, the requirement for plasmid transfection hinders large-scale LVV manufacture. Moreover, large-scale plasmid production, testing, and transfection contribute to operational risk and the high cost associated with this therapeutic modality. Thus, we developed LVV packaging and producer cell lines, which reduce or eliminate the need for plasmid transfection during LVV manufacture. To develop a packaging cell line, lentiviral packaging genes were stably integrated by random integration of linearized plasmid DNA. Then, to develop EGFP- and anti-CD19 chimeric antigen receptor-encoding producer cell lines, transfer plasmids were integrated by transposase-mediated integration. Single-cell isolation and testing were performed to isolate the top-performing clonal packaging and producer cell lines. Production of LVVs that encode various cargo genes revealed consistency in the production performance of the packaging and producer cell lines compared to the industry-standard four-plasmid transfection method. By reducing or eliminating the requirement for plasmid transfection, while achieving production performance consistent with the current industry standard, the packaging and producer cell lines developed here can reduce costs and operational risks of LVV manufacture, thus increasing patient access to LVV-mediated cell and gene therapies.

RESUMEN

Leukodystrophies are progressive single gene disorders affecting the white matter of the brain. Several gene therapy trials are in progress to address the urgent unmet need for this patient population. We performed a comprehensive literature review of all gene therapy clinical trials listed in www.clinicaltrials.gov through August 2024, and the relevant preclinical studies that enabled clinical translation. Of the approximately 50 leukodystrophies described to date, only eight have existing gene therapy clinical trials: metachromatic leukodystrophy, X-linked adrenoleukodystrophy, globoid cell leukodystrophy, Canavan disease, giant axonal neuropathy, GM2 gangliosidoses, Alexander disease and Pelizaeus-Merzbacher disease. What led to the emergence of gene therapy trials for these specific disorders? What preclinical data or disease context was enabling? For each of these eight disorders, we first describe its pathophysiology and clinical presentation. We discuss the impact of gene therapy delivery route, targeted cell type, delivery modality, dosage, and timing on therapeutic efficacy. We note that use of allogeneic hematopoietic stem cell transplantation in some leukodystrophies allowed for an accelerated path to clinic even in the absence of available animal models. In other leukodystrophies, small and large animal model studies enabled clinical translation of experimental gene therapies. Human clinical trials for the leukodystrophies include ex vivo lentiviral gene delivery, in vivo AAV-mediated gene delivery, and intrathecal antisense oligonucleotide approaches. We outline adverse events associated with each modality focusing specifically on genotoxicity and immunotoxicity. We review monitoring and management of events related to insertional mutagenesis and immune responses. The data presented in this review show that gene therapy, while promising, requires systematic monitoring to account for the precarious disease biology and the adverse events associated with new technology.

RESUMEN

BACKGROUND: For angioimmunoblastic T cell lymphoma (AITL), a rare cancer, no specific treatments are available and survival outcome is poor. We previously developed a murine model for AITL that mimics closely human disease and allows to evaluate new treatments. As in human AITL, the murine CD4+ follicular helper T (Tfh) cells are drivers of the malignancy. Therefore, chimeric antigen receptor (CAR) T cell therapy might represent a new therapeutic option. METHODS: To prevent fratricide among CAR T cells when delivering an CD4-specific CAR, we used a lentiviral vector (LV) encoding an anti-CD4 CAR, allowing exclusive entry into CD8 T cells. RESULTS: These anti-CD4CAR CD8-targeted LVs achieved in murine AITL biopsies high CAR-expression levels in CD8 T cells. Malignant CD4 Tfh cells were eliminated from the mAITL lymphoma, while the CAR + CD8 T cells expanded upon encounter with the CD4 receptor and were shaped into functional cytotoxic cells. Finally, in vivo injection of the CAR + CD8-LVs into our preclinical AITL mouse model carrying lymphomas, significantly prolonged mice survival. Moreover, the in vivo generated functional CAR + CD8 T cells efficiently reduced neoplastic T cell numbers in the mAITL tumors. CONCLUSION: This is the first description of in vivo generated CAR T cells for therapy of a T cell lymphoma. The strategy described offers a new therapeutic concept for patients suffering from CD4-driven T cell lymphomas.

Asunto(s)

Modelos Animales de Enfermedad , Inmunoterapia Adoptiva , Linfoma de Células T , Receptores Quiméricos de Antígenos , Animales , Ratones , Humanos , Receptores Quiméricos de Antígenos/inmunología , Inmunoterapia Adoptiva/métodos , Linfoma de Células T/terapia , Linfoma de Células T/inmunología , Linfoma de Células T/patología , Linfadenopatía Inmunoblástica/terapia , Linfadenopatía Inmunoblástica/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral

RESUMEN

Chronic kidney disease (CKD) is one of the leading causes of mortality worldwide because of kidney failure and the associated challenges of its treatment including dialysis and kidney transplantation. About one-third of CKD cases are linked to inherited monogenic factors, making them suitable for potential gene therapy interventions. However, the intricate anatomical structure of the kidney poses a challenge, limiting the effectiveness of targeted gene delivery to the renal system. In this review, we explore the progress made in the field of targeted gene therapy approaches and their implications for rare genetic kidney disorders, examining preclinical studies and prospects for clinical application. In vivo gene therapy is most commonly used for kidney-targeted gene delivery and involves administering viral and non-viral vectors through various routes such as systemic, renal vein and renal arterial injections. Small nucleic acids have also been used in preclinical and clinical studies for treating certain kidney disorders. Unexpectedly, hematopoietic stem and progenitor cells have been used as an ex vivo gene therapy vehicle for kidney gene delivery, highlighting their ability to differentiate into macrophages within the kidney, forming tunneling nanotubes that can deliver genetic material and organelles to adjacent kidney cells, even across the basement membrane to target the proximal tubular cells. As gene therapy technologies continue to advance and our understanding of kidney biology deepens, there is hope for patients with genetic kidney disorders to eventually avoid kidney transplantation.

RESUMEN

Lentiviral vectors are highly efficient gene delivery vehicles used extensively in the rapidly growing field of cell and gene therapy. Demand for efficient, large-scale, lentiviral vector bioprocessing is growing as more therapies reach late-stage clinical trials and are commercialized. However, despite substantial progress, several process inefficiencies remain. The unintended auto-transduction of viral vector-producing cells by newly synthesized lentiviral vector particles during manufacturing processes constitutes one such inefficiency which remains largely unaddressed. In this study, we determined that over 60% of functional lentiviral vector particles produced during an upstream production process were lost to auto-transduction, highlighting a major process inefficiency likely widespread within the industry. Auto-transduction of cells by particles pseudotyped with the widely used vesicular stomatitis virus G protein was inhibited via the adoption of a reduced extracellular pH during vector production, impairing the ability of the vector to interact with its target receptor. Employing a posttransfection pH shift to pH 6.7-6.8 resulted in a sevenfold reduction in vector genome integration events, arising from lentiviral vector-mediated transduction, within viral vector-producing cell populations and ultimately resulted in improved lentiviral vector production kinetics. The proposed strategy is scalable and cost-effective, providing an industrially relevant approach to improve lentiviral vector production efficiencies.

RESUMEN

Gene therapy has made considerable strides in recent years. More than 4000 protein-coding genes have been implicated in more than 6000 genetic diseases; next-generation sequencing has dramatically revolutionized the diagnosis of genetic diseases. Most genetic diseases are considered very rare or ultrarare, defined here as having fewer than 1:100,000 cases, but only one of the 12 approved gene therapies (excluding RNA therapies) targets an ultrarare disease. This article explores three gene supplementation therapy approaches suitable for various rare genetic diseases: lentiviral vector-modified autologous CD34+ hematopoietic stem cell transplantation, systemic delivery of adeno-associated virus (AAV) vectors to the liver, and local AAV delivery to the cerebrospinal fluid and brain. Together with RNA therapies, we propose a potential business model for these gene therapies.

Asunto(s)

Dependovirus , Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Terapia Genética/métodos , Humanos , Dependovirus/genética , Vectores Genéticos , Enfermedades Genéticas Congénitas/terapia , Enfermedades Genéticas Congénitas/genética , Enfermedades Raras/terapia , Enfermedades Raras/genética , Lentivirus/genética

RESUMEN

Transduction of producer cells during lentiviral vector (LVV) production causes the loss of 70-90% of viable particles. This process is called retro-transduction and it is a consequence of the interaction between the LVV envelope protein, VSV-G, and the LDL receptor located on the producer cell membrane, allowing lentiviral vector transduction. Avoiding retro-transduction in LVV manufacturing is crucial to improve net production and, therefore, the efficiency of the production process. Here, we describe a method for quantifying the transduction of producer cells and three different strategies that, focused on the interaction between VSV-G and the LDLR, aim to reduce retro-transduction.

Asunto(s)

Vectores Genéticos , Lentivirus , Receptores de LDL , Transducción Genética , Vectores Genéticos/genética , Lentivirus/genética , Humanos , Receptores de LDL/metabolismo , Receptores de LDL/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Células HEK293 , Glicoproteínas de Membrana

RESUMEN

Bringing effective cancer therapy in the form of chimeric antigen receptor technology to untapped markets faces numerous challenges, including a global shortage of therapeutic lentiviral or retroviral vectors on which all current clinical therapies using genetically modified T cells are based. Production of these lentiviral vectors in academic settings in principle opens the way to local production of therapeutic cells, which is the only economically viable approach to make this therapy available to patients in developing countries. The conditions for obtaining and concentrating lentiviral vectors have been optimized and described. The calcium phosphate precipitation method was found to be suitable for transfecting high cell-density cultures, a prerequisite for high titers. We describe protocols for gradually increasing production from 6-well plates to P100 plates, T-175 flasks, and 5-layer stacks while maintaining high titers, >108 transducing units. Concentration experiments using ultracentrifugation revealed the advantage of lower centrifugation speeds compared to competing protocols. The resulting batches of lentiviral vectors had a titer of 1010 infectious particles and were used to transduce primary human T lymphocytes generating chimeric antigen receptor T cells, the quality of which was checked and found potential applicability for treatment.

RESUMEN

The biological properties of human mesenchymal stromal cells (hMSCs) have been explored in over a thousand clinical trials in the last decade. Although hMSCs can be isolated from multiple sources, the degree of biological similarity between cell populations from these sources remains to be determined. A comparative study was performed investigating the growth kinetics and functionality of hMSCs isolated from adipose tissue (AT), bone marrow (BM) and umbilical cord tissue (UCT) expanded in monolayer over five passages. Adult hMSCs (AT, BM) had a slower proliferation ability than the UCT-hMSCs, with no apparent differences in their glucose consumption profile. BM-hMSCs produced higher concentrations of endogenous vascular endothelial growth factor (VEGF) compared to AT- and UCT-hMSCs. This study also revealed that UCT-hMSCs were more efficiently transduced by a lentiviral vector carrying a VEGF gene than their adult counterparts. Following cellular immunophenotypic characterization, no differences across the sources were found in the expression levels of the typical markers used to identify hMSCs. This work established a systematic approach for cell source selection depending on the hMSC's intended clinical application.

RESUMEN

Both cell surface and soluble extracellular glycosaminoglycans have been shown to interfere with the exogenous nucleic acid delivery efficiency of non-viral gene delivery, including lipoplex and polyplex-mediated transfection. Most gene therapy viral vectors used commercially and in clinical trials are currently manufactured using transient transfection-based bioprocesses. The growing demand for viral vector products, coupled with a global shortage in production capability, requires improved transfection technologies and processes to maximise process efficiency and productivity. Soluble extracellular glycosaminoglycans were found to accumulate in the conditioned cell culture medium of suspension adapted HEK293T cell cultures, compromising transfection performance and lentiviral vector production. The enzymatic degradation of specific, chondroitin sulphate-based, glycosaminoglycans with chondroitinase ABC was found to significantly enhance transfection performance. Additionally, we report significant improvements in functional lentiviral vector titre when cultivating cells at higher cell densities than those utilised in a control lentiviral vector bioprocess; an improvement that was further enhanced when cultures were supplemented with chondroitinase ABC prior to transfection. A 71.2% increase in functional lentiviral vector titre was calculated when doubling the cell density prior to transfection compared to the existing process and treatment of the high-density cell cultures with 0.1 U/mL chondroitinase ABC resulted in a further 18.6% increase in titre, presenting a method that can effectively enhance transfection performance.

RESUMEN

Hematopoietic stem cell gene therapy (HSCGT) is a promising therapeutic strategy for the treatment of neurodegenerative, metabolic disorders. The approach involves the ex vivo introduction of a missing gene into patients' own stem cells via lentiviral-mediated transduction (TD). Once transplanted back into a fully conditioned patient, these genetically modified HSCs can repopulate the blood system and produce the functional protein, previously absent or non-functional in the patient, which can then cross-correct other affected cells in somatic organs and the central nervous system. We previously developed an HSCGT approach for the treatment of Mucopolysaccharidosis type II (MPSII) (Hunter syndrome), a debilitating pediatric lysosomal disorder caused by mutations in the iduronate-2-sulphatase (IDS) gene, leading to the accumulation of heparan and dermatan sulfate, which causes severe neurodegeneration, skeletal abnormalities, and cardiorespiratory disease. In HSCGT proof-of-concept studies using lentiviral IDS fused to a brain-targeting peptide ApoEII (IDS.ApoEII), we were able to normalize brain pathology and behavior of MPSII mice. Here we present an optimized and validated good manufacturing practice hematopoietic stem cell TD protocol for MPSII in preparation for first-in-man studies. Inclusion of TEs LentiBOOST and protamine sulfate significantly improved TD efficiency by at least 3-fold without causing adverse toxicity, thereby reducing vector quantity required.

RESUMEN

BACKGROUND: Synaptic Ras GTPase activating protein 1 (SYNGAP1)-related non-specific intellectual disability is a neurodevelopmental disorder caused by an insufficient level of SynGAP1 resulting in a dysfunction of neuronal synapses and presenting with a wide array of clinical phenotypes. Hematopoietic stem cell gene therapy has the potential to deliver therapeutic levels of functional SynGAP1 to affected neurons upon transduction of hematopoietic stem and progenitor cells with a lentiviral vector. METHODS: As a novel approach toward the treatment of SYNGAP1, we have generated a lentiviral vector expressing a modified form of SynGAP1 for transduction of human CD34+ hematopoietic stem and progenitor cells. The gene-modified cells were then transplanted into adult immunodeficient SYNGAP1+/- heterozygous mice and evaluated for improvement of SYNGAP1-related clinical phenotypes. Expression of SynGAP1 was also evaluated in the brain tissue of transplanted mice. RESULTS: In our proof-of-concept study, we have demonstrated significant improvement of SYNGAP1-related phenotypes including an improvement in motor abilities observed in mice transplanted with the vector transduced cells because they displayed decreased hyperactivity in an open field assay and an increased latency to fall in a rotarod assay. An increased level of SynGAP1 was also detected in the brains of these mice. CONCLUSIONS: These early-stage results highlight the potential of this stem cell gene therapy approach as a treatment strategy for SYNGAP1.

Asunto(s)

Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Discapacidad Intelectual , Animales , Humanos , Ratones , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Terapia Genética/métodos , Vectores Genéticos/genética , Células Madre Hematopoyéticas/metabolismo , Discapacidad Intelectual/terapia , Discapacidad Intelectual/genética , Lentivirus/genética , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Transducción Genética

RESUMEN

Diamond-Blackfan anemia (DBA) is a rare genetic disorder affecting the bone marrow's ability to produce red blood cells, leading to severe anemia and various physical abnormalities. Approximately 75% of DBA cases involve heterozygous mutations in ribosomal protein (RP) genes, classifying it as a ribosomopathy, with RPS19 being the most frequently mutated gene. Non-RP mutations, such as in GATA1, have also been identified. Current treatments include glucocorticosteroids, blood transfusions, and hematopoietic stem cell transplantation (HSCT), with HSCT being the only curative option, albeit with challenges like donor availability and immunological complications. Gene therapy, particularly using lentiviral vectors and CRISPR/Cas9 technology, emerges as a promising alternative. This review explores the potential of gene therapy, focusing on lentiviral vectors and CRISPR/Cas9 technology in combination with non-integrating lentiviral vectors, as a curative solution for DBA. It highlights the transformative advancements in the treatment landscape of DBA, offering hope for individuals affected by this condition.

Asunto(s)

Anemia de Diamond-Blackfan , Terapia Genética , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/terapia , Terapia Genética/métodos , Humanos , Sistemas CRISPR-Cas/genética , Vectores Genéticos , Lentivirus/genética , Animales , Proteínas Ribosómicas/genética , Mutación/genética , Edición Génica/métodos

RESUMEN

Heart transplantation is associated with major hurdles, including the limited number of available organs for transplantation, the risk of rejection due to genetic discrepancies, and the burden of immunosuppression. In this study, we demonstrated the feasibility of permanent genetic engineering of the heart during ex vivo perfusion. Lentiviral vectors encoding for short hairpin RNAs targeting beta2-microglobulin (shß2m) and class II transactivator (shCIITA) were delivered to the graft during two hours of normothermic EVHP. Highly efficient genetic engineering was indicated by stable reporter gene expression in endothelial cells and cardiomyocytes. Remarkably, swine leucocyte antigen (SLA) class I and SLA class II expression levels were decreased by 66% and 76%, respectively, in the vascular endothelium. Evaluation of lactate, troponin T, and LDH levels in the perfusate and histological analysis showed no additional cell injury or tissue damage caused by lentiviral vectors. Moreover, cytokine secretion profiles (IL-6, IL-8, and TNF-α) of non-transduced and lentiviral vector-transduced hearts were comparable. This study demonstrated the ex vivo generation of genetically engineered hearts without compromising tissue integrity. Downregulation of SLA expression may contribute to reduce the immunogenicity of the heart and support graft survival after allogeneic or xenogeneic transplantation.

Asunto(s)

Vectores Genéticos , Trasplante de Corazón , Antígenos de Histocompatibilidad Clase I , Lentivirus , Animales , Lentivirus/genética , Trasplante de Corazón/métodos , Vectores Genéticos/genética , Porcinos , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Perfusión/métodos , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Microglobulina beta-2/genética , Citocinas/metabolismo , Ingeniería Genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/inmunología , Humanos , ARN Interferente Pequeño/genética , Supervivencia de Injerto/inmunología , Supervivencia de Injerto/genética , Células Endoteliales/metabolismo , Células Endoteliales/inmunología , Proteínas Nucleares , Transactivadores

RESUMEN

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 × 104 cells cm-2 providing similar specific productivities of infectious LV. Seeding at 3 × 104 cells cm-2 was selected, and the quasi-perfusion was modulated to minimize inhibitory metabolite accumulation and vector exposure at 37°C. Similar specific productivities of infectious LV and physical LV were achieved at 1, 2, and 3 vessel volumes per day (VVD), with 1 VVD selected to minimize downstream processing volumes. The optimized process was scaled 50-fold to 1,264 cm2 flasks, achieving similar LV titers. However, scaling up beyond this to a 6,320 cm2 multilayer flask reduced titers, 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 titers, respectively. The optimized flask process was successfully transferred to the iCELLis Nano (Cytiva) fixed-bed bioreactor, with quasi-perfusion at 1 VVD yielding 1.62 × 108 TU.

RESUMEN

A gene delivery system utilizing lentiviral vectors (LVs) requires high transduction efficiency for successful application in human gene therapy. Pseudotyping allows viral tropism to be expanded, widening the usage of LVs. While vesicular stomatitis virus G (VSV-G) single-pseudotyped LVs are commonly used, dual-pseudotyping is less frequently employed because of its increased complexity. In this study, we examined the potential of phenotypically mixed heterologous dual-pseudotyped LVs with VSV-G and Sendai virus hemagglutinin-neuraminidase (SeV-HN) glycoproteins, termed V/HN-LV. Our findings demonstrated the significantly improved transduction efficiency of V/HN-LV in various cell lines of mice, cynomolgus monkeys, and humans compared with LV pseudotyped with VSV-G alone. Notably, V/HN-LV showed higher transduction efficiency in human cells, including hematopoietic stem cells. The efficient incorporation of wild-type SeV-HN into V/HN-LV depended on VSV-G. SeV-HN removed sialic acid from VSV-G, and the desialylation of VSV-G increased V/HN-LV infectivity. Furthermore, V/HN-LV acquired the ability to recognize sialic acid, particularly N-acetylneuraminic acid on the host cell, enhancing LV infectivity. Overall, VSV-G and SeV-HN synergistically improve LV transduction efficiency and broaden its tropism, indicating their potential use in gene delivery.

Asunto(s)

Vectores Genéticos , Proteína HN , Lentivirus , Virus Sendai , Transducción Genética , Proteínas del Envoltorio Viral , Animales , Humanos , Vectores Genéticos/genética , Lentivirus/genética , Virus Sendai/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Ratones , Proteína HN/genética , Proteína HN/metabolismo , Línea Celular , Macaca fascicularis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Tropismo Viral , Células HEK293 , Técnicas de Transferencia de Gen , Terapia Genética/métodos

RESUMEN

Gene silencing without gene editing holds great potential for the development of safe therapeutic applications. Here, we describe a novel strategy to concomitantly repress multiple genes using zinc finger proteins fused to Krüppel-Associated Box repression domains (ZF-Rs). This was achieved via the optimization of a lentiviral system tailored for the delivery of ZF-Rs in hematopoietic cells. We showed that an optimal design of the lentiviral backbone is crucial to multiplex up to three ZF-Rs or two ZF-Rs and a chimeric antigen receptor. ZF-R expression had no impact on the integrity and functionality of transduced cells. Furthermore, gene repression in ZF-R-expressing T cells was highly efficient in vitro and in vivo during the entire monitoring period (up to 10 weeks), and it was accompanied by epigenetic remodeling events. Finally, we described an approach to improve ZF-R specificity to illustrate the path toward the generation of ZF-Rs with a safe clinical profile. In conclusion, we successfully developed an epigenetic-based cell engineering approach for concomitant modulation of multiple gene expressions that bypass the risks associated with DNA editing.

RESUMEN

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 to 4.64 × 107 TU mL-1 in 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 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L 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.

Asunto(s)

Reactores Biológicos , Vectores Genéticos , Lentivirus , Lentivirus/genética , Humanos , Vectores Genéticos/genética , Medio de Cultivo Libre de Suero , Línea Celular , Técnicas de Cultivo de Célula/métodos , Cultivo de Virus/métodos , Células HEK293 , Transfección/métodos

RESUMEN

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 âˆ¼ 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.

Asunto(s)

Vectores Genéticos , Lentivirus , Nanopartículas , Tamaño de la Partícula , Ultracentrifugación , Lentivirus/genética , Nanopartículas/química , Ultracentrifugación/métodos , Humanos , Receptores Quiméricos de Antígenos

RESUMEN

SCN5A mutations have been reported to cause various cardiomyopathies in humans. Most of the SCN5A mutations causes loss of function and thereby, alters the overall cellular function. Therefore, to understand the loss of SCN5A function in cardiomyocytes, we have knocked down the SCN5A gene (SCN5A-KD) in H9c2 cells and explored the cell phenotype and molecular behaviors in the presence and absence of isoproterenol (ISO), an adrenergic receptor agonist that induces cardiac hypertrophy. Expression of several genes related to hypertrophy, inflammation, fibrosis, and energy metabolism pathways were evaluated. It was found that the mRNA expression of hypertrophy-related gene, brain (B-type) natriuretic peptide (BNP) was significantly increased in SCN5A-KD cells as compared to 'control' H9c2 cells. There was a further increase in the mRNA expressions of BNP and ßMHC in SCN5A-KD cells after ISO treatment compared to their respective controls. Pro-inflammatory cytokine, tumor necrosis factor-alpha expression was significantly increased in 'SCN5A-KD' H9c2 cells. Further, metabolism-related genes like glucose transporter type 4, cluster of differentiation 36, peroxisome proliferator-activated receptor alpha, and peroxisome proliferator-activated receptor-gamma were significantly elevated in the SCN5A-KD cells as compared to the control cells. Upregulation of these metabolic genes is associated with increased ATP production. The study revealed that SCN5A knock-down causes alteration of gene expression related to cardiac hypertrophy, inflammation, and energy metabolism pathways, which may promote cardiac remodelling and cardiomyopathy.

Asunto(s)

Cardiomegalia , Isoproterenol , Canal de Sodio Activado por Voltaje NAV1.5 , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Cardiomegalia/genética , Cardiomegalia/metabolismo , Ratas , Línea Celular , Isoproterenol/farmacología , Miocitos Cardíacos/metabolismo , Péptido Natriurético Encefálico/genética , Péptido Natriurético Encefálico/metabolismo , Animales , Técnicas de Silenciamiento del Gen , Humanos , Mioblastos Cardíacos/metabolismo , Metabolismo Energético/genética , Regulación de la Expresión Génica/genética