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1.
PLoS One ; 19(6): e0297817, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38833479

RESUMO

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


Assuntos
Processamento Alternativo , Proliferação de Células , Vetores Genéticos , Indóis , Lentivirus , Transfecção , Humanos , Indóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Vetores Genéticos/genética , Lentivirus/genética , Transfecção/métodos , Células HEK293
2.
Cells ; 13(11)2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38891052

RESUMO

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.


Assuntos
Anemia de Diamond-Blackfan , Terapia Genética , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/terapia , Terapia Genética/métodos , Humanos , Sistemas CRISPR-Cas/genética , Vetores Genéticos , Lentivirus/genética , Animais , Proteínas Ribossômicas/genética , Mutação/genética , Edição de Genes/métodos
3.
J Nanobiotechnology ; 22(1): 329, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38858736

RESUMO

BACKGROUND: Cancer stem cells (CSCs) play a vital role in the occurrence, maintenance, and recurrence of solid tumors. Although, miR-145-5p can inhibit CSCs survival, poor understanding of the underlying mechanisms hamperes further therapeutic optimization for patients. Lentivirus with remarkable transduction efficiency is the most commonly used RNA carrier in research, but has shown limited tumor-targeting capability. METHODS: We have applied liposome to decorate lentivirus surface thereby yielding liposome-lentivirus hybrid-based carriers, termed miR-145-5p-lentivirus nanoliposome (MRL145), and systematically analyzed their potential therapeutic effects on liver CSCs (LCSCs). RESULTS: MRL145 exhibited high delivery efficiency and potent anti-tumor efficacy under in vitro and in vivo. Mechanistically, the overexpressed miR-145-5p can significantly suppress the self-renewal, migration, and invasion abilities of LCSCs by targeting Collagen Type IV Alpha 3 Chain (COL4A3). Importantly, COL4A3 can promote phosphorylating GSK-3ß at ser 9 (p-GSK-3ß S9) to inactivate GSK3ß, and facilitate translocation of ß-catenin into the nucleus to activate the Wnt/ß-catenin pathway, thereby promoting self-renewal, migration, and invasion of LCSCs. Interestingly, COL4A3 could attenuate the cellular autophagy through modulating GSK3ß/Gli3/VMP1 axis to promote self-renewal, migration, and invasion of LCSCs. CONCLUSIONS: These findings provide new insights in mode of action of miR-145-5p in LCSCs therapy and indicates that liposome-virus hybrid carriers hold great promise in miRNA delivery.


Assuntos
Lentivirus , Lipossomos , MicroRNAs , Células-Tronco Neoplásicas , MicroRNAs/genética , MicroRNAs/metabolismo , Lipossomos/química , Humanos , Animais , Camundongos , Lentivirus/genética , Linhagem Celular Tumoral , Células-Tronco Neoplásicas/metabolismo , Camundongos Nus , Neoplasias Hepáticas/terapia , Camundongos Endogâmicos BALB C , Movimento Celular , Glicogênio Sintase Quinase 3 beta/metabolismo , beta Catenina/metabolismo , Via de Sinalização Wnt
4.
Artigo em Inglês | MEDLINE | ID: mdl-38821669

RESUMO

Gene therapies have emerged as promising treatments for various conditions including inherited diseases as well as cancer. Ensuring their safe clinical application requires the development of appropriate safety testing strategies. Several guidelines have been provided by health authorities to address these concerns. These guidelines state that non-clinical testing should be carried out on a case-by-case basis depending on the modality. This review focuses on the genome safety assessment of frequently used gene therapy modalities, namely Adeno Associated Viruses (AAVs), Lentiviruses, designer nucleases and mRNAs. Important safety considerations for these modalities, amongst others, are vector integrations into the patient genome (insertional mutagenesis) and off-target editing. Taking into account the constraints of in vivo studies, health authorities endorse the development of novel approach methodologies (NAMs), which are innovative in vitro strategies for genotoxicity testing. This review provides an overview of NAMs applied to viral and CRISPR/Cas9 safety, including next generation sequencing-based methods for integration site analysis and off-target editing. Additionally, NAMs to evaluate the oncogenicity risk arising from unwanted genomic modifications are discussed. Thus, a range of promising techniques are available to support the safe development of gene therapies. Thorough validation, comparisons and correlations with clinical outcomes are essential to identify the most reliable safety testing strategies. By providing a comprehensive overview of these NAMs, this review aims to contribute to a better understanding of the genome safety perspectives of gene therapies.


Assuntos
Edição de Genes , Terapia Genética , Terapia Genética/métodos , Terapia Genética/efeitos adversos , Humanos , Edição de Genes/métodos , Animais , Dependovirus/genética , Vetores Genéticos , Sistemas CRISPR-Cas , Lentivirus/genética , Endonucleases/genética , Endonucleases/metabolismo , Testes de Mutagenicidade/métodos , Nucleotídeos
5.
Biomed Phys Eng Express ; 10(4)2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38772344

RESUMO

Lentiviral transduction is widely used in research, has shown promise in clinical trials involving gene therapy and has been approved for CAR-T cell immunotherapy. However, most modifications are doneex vivoand rely on systemic administration of large numbers of transduced cells for clinical applications. A novel approach utilizingin situbiomaterial-based gene delivery can reduce off-target side effects while enhancing effectiveness of the manipulation process. In this study, poly(ethylene glycol) diacrylate (PEGDA)-based scaffolds were developed to enablein situlentivirus-mediated transduction. Compared to other widely popular biomaterials, PEGDA stands out due to its robustness and cost-effectiveness. These scaffolds, prepared via cryogelation, are capable of flowing through surgical needles in bothin vitroandin vivoconditions, and promptly regain their original shape. Modification with poly(L-lysine) (PLL) enables lentivirus immobilization while interconnected macroporous structure allows cell infiltration into these matrices, thereby facilitating cell-virus interaction over a large surface area for efficient transduction. Notably, these preformed injectable scaffolds demonstrate hemocompatibility, cell viability and minimally inflammatory response as shown by ourin vitroandin vivostudies involving histology and immunophenotyping of infiltrating cells. This study marks the first instance of using preformed injectable scaffolds for delivery of lentivectors, which offers a non-invasive and localized approach for delivery of factors enablingin situlentiviral transduction suitable for both tissue engineering and immunotherapeutic applications.


Assuntos
Criogéis , Técnicas de Transferência de Genes , Lentivirus , Polietilenoglicóis , Polietilenoglicóis/química , Criogéis/química , Humanos , Lentivirus/genética , Animais , Sobrevivência Celular/efeitos dos fármacos , Alicerces Teciduais/química , Transdução Genética , Camundongos , Materiais Biocompatíveis/química , Terapia Genética/métodos , Propriedades de Superfície , Injeções , Polilisina/química
6.
JCI Insight ; 9(10)2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38775150

RESUMO

This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19 (RPS19), showing evidence of a new safe and effective therapy. The data show that, unlike patients with Fanconi anemia (FA), the hematopoietic stem cell (HSC) reservoir of patients with DBA was not significantly reduced, suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapy. Subsequently, 2 clinically applicable lentiviral vectors were developed. In the former lentiviral vector, PGK.CoRPS19 LV, a codon-optimized version of RPS19 was driven by the phosphoglycerate kinase promoter (PGK) already used in different gene therapy trials, including FA gene therapy. In the latter one, EF1α.CoRPS19 LV, RPS19 expression was driven by the elongation factor alpha short promoter, EF1α(s). Preclinical experiments showed that transduction of DBA patient CD34+ cells with the PGK.CoRPS19 LV restored erythroid differentiation, and demonstrated the long-term repopulating properties of corrected DBA CD34+ cells, providing evidence of improved erythroid maturation. Concomitantly, long-term restoration of ribosomal biogenesis was verified using a potentially novel method applicable to patients' blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion site analyses showed that lentivirus-mediated gene therapy was nontoxic.


Assuntos
Anemia de Diamond-Blackfan , Terapia Genética , Vetores Genéticos , Células-Tronco Hematopoéticas , Lentivirus , Proteínas Ribossômicas , Anemia de Diamond-Blackfan/terapia , Anemia de Diamond-Blackfan/genética , Humanos , Terapia Genética/métodos , Lentivirus/genética , Proteínas Ribossômicas/genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Animais , Camundongos , Masculino , Feminino , Ribossomos/metabolismo , Ribossomos/genética , Regiões Promotoras Genéticas , Mutação , Transplante de Células-Tronco Hematopoéticas/métodos
7.
Anal Chem ; 96(23): 9593-9600, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38804040

RESUMO

The limited biomolecular and functional stability of lentiviral vectors (LVVs) for cell therapy poses the need for analytical tools that can monitor their titers and activity throughout the various steps of expression and purification. In this study, we describe a rapid (25 min) and reproducible (coefficient of variance ∼0.5-2%) method that leverages size exclusion chromatography coupled with multiangle light scattering detection (SEC-MALS) to determine size, purity, and particle count of LVVs purified from bioreactor harvests. The SEC-MALS data were corroborated by orthogonal methods, namely, dynamic light scattering (DLS) and transmission electron microscopy. The method was also evaluated for robustness in the range of 2.78 × 105-2.67 × 107 particles per sample. Notably, MALS-based particle counts correlated with the titer of infectious LVVs measured via transduction assays (R2 = 0.77). Using a combination of SEC-MALS and DLS, we discerned the effects of purification parameters on LVV quality, such as the separation between heterogeneous LV, which can facilitate critical decision-making in the biomanufacturing of gene and cell therapies.


Assuntos
Difusão Dinâmica da Luz , Lentivirus , Lentivirus/genética , Lentivirus/isolamento & purificação , Humanos , Cromatografia em Gel , Células HEK293 , Tamanho da Partícula , Vetores Genéticos/genética , Vetores Genéticos/isolamento & purificação
8.
Exp Mol Pathol ; 137: 104898, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38729059

RESUMO

INTRODUCTION: NK cells are an untapped resource for cancer therapy. Sarcomas transduced with lentiviruses to express human IL-12 are only cleared in mice bearing mature human NK cells. However, systemic inflammation limits IL-12 utilization. Fate control a.k.a. "suicide mechanisms" regulate unchecked systemic inflammation caused by cellular immunotherapies. Despite increasing utilization, there remains limited data on immune consequences or tumor-directed effects of fate control. OBJECTIVES: We sought to engage the mutant thymidylate kinase (mTMPK) metabolic fate control system to regulate systemic inflammation and assess the impact on NK cell effector functions. METHODS: Primary human sarcoma short-passage samples and cell lines were transduced with LV/hu-IL-12_mTMPK engineering expression of IL-12 and an AZT-associated fate control enzyme. We assessed transduced sarcoma responses to AZT engagement and subsequent modulation of NK cell functions as measured by inflammatory cytokine production and cytotoxicity. RESULTS: AZT administration to transduced (LV/hu-IL-12_mTMPK) short-passage primary human sarcomas and human Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines, abrogated the robust expression of human IL-12. Fate control activation elicited a specific dose-dependent cytotoxic effect measured by metabolic activity (WST-1) and cell death (Incucyte). NK effector functions of IFN-γ and cytotoxic granule release were significantly augmented despite IL-12 abrogation. This correlated with preferentially induced expression of NK cell activation ligands. CONCLUSIONS: mTMPK fate control engagement terminates transduced sarcoma IL-12 production and triggers cell death, but also augments an NK cell-mediated response coinciding with metabolic stress activating surface ligand induction. Fate control engagement could offer a novel immune activation method for NK cell-mediated cancer clearance.


Assuntos
Interleucina-12 , Células Matadoras Naturais , Lentivirus , Sarcoma , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Humanos , Interleucina-12/genética , Interleucina-12/metabolismo , Lentivirus/genética , Sarcoma/imunologia , Sarcoma/genética , Sarcoma/patologia , Linhagem Celular Tumoral , Transdução Genética , Animais , Camundongos
9.
Eur J Pharm Biopharm ; 200: 114340, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38797222

RESUMO

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.


Assuntos
Vetores Genéticos , Lentivirus , Nanopartículas , Tamanho da Partícula , Ultracentrifugação , Lentivirus/genética , Nanopartículas/química , Ultracentrifugação/métodos , Humanos , Receptores de Antígenos Quiméricos
10.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 40(4): 289-295, 2024 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-38710512

RESUMO

Objective To evaluate the toxicology of targeting human epidermal growth factor receptor-2 chimeric antigen receptor T (HER2-CAR-T) cells and to provide a safety basis for the clinical evaluation of HER2-CAR-T cell therapy. Methods The recombinant lentiviral vector was used to generate HER2-CAR-T cells. Soft agar colony formation assay was used to observe the colony formation of HER2-CAR-T cells, and the colony formation rate was statistically analyzed. The HER2-CAR-T cell suspension was co-incubated with rabbit red blood cell suspension, and the hemolysis of red blood cells was evaluated by direct observation and microplate reader detection. The HER2-CAR-T cell preparation was injected into the ear vein of male New Zealand rabbits, and the stimulating effect of HER2-CAR-T cells on the blood vessels of the animals was observed by staining of tissue sections. The vesicular stomatitis virus envelope glycoprotein (VSV-G) gene of pMD 2.G vector was used as the target sequence, and the safety of the lentiviral vector was verified by real-time fluorescence quantitative PCR. The heart, liver, lung, and kidney of mice receiving HER2-CAR-T cell infusion were collected, and the lesions were observed by HE staining. Results The HER2-CAR-T cells were successfully prepared. These cells did not exhibit soft agar colony formation ability in vitro, and the HER2-CAR-T cell preparation did not cause hemolysis in New Zealand rabbit red blood cells. After the infusion of HER2-CAR-T cells into the ear vein of New Zealand rabbits, no obvious vascular stimulation response was found, and no specific amplification of VSV-G was detected. No obvious lesions were found in the heart, liver, lung and kidney tissues of the treatment group. Conclusion The prepared HER2-CAR-T cells have reliable safety.


Assuntos
Receptor ErbB-2 , Receptores de Antígenos Quiméricos , Animais , Humanos , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Coelhos , Camundongos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Masculino , Imunoterapia Adotiva/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Vetores Genéticos/genética , Lentivirus/genética , Feminino
11.
BMC Vet Res ; 20(1): 195, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38741095

RESUMO

Small ruminant lentiviruses (SRLVs) are widespread and infect goats and sheep. Several reports also suggest that SRLVs can infect wild ruminants. The presence of specific antibodies against SRLVs has been identified in wild ruminants from Poland, but no studies have been conducted to detect proviral DNA of SRLVs in these animals. Therefore, the purpose of this study was to examine samples from Polish wild ruminants to determine whether these animals can serve as reservoirs of SRLVs under natural conditions. A total of 314 samples were tested from red deer (n = 255), roe deer (n = 52) and fallow deer (n = 7) using nested real-time PCR. DNA from positive real-time PCR samples was subsequently used to amplify a CA fragment (625 bp) of the gag gene, a 1.2 kb fragment of the pol gene and an LTR-gag fragment. Three samples (0.95%) were positive according to nested real-time PCR using primers and probe specific for CAEV (SRLV group B). All the samples were negative for the primers and probe specific for MVV (SRLV A group). Only SRLV LTR-gag sequences were obtained from two red deer. Phylogenetic analysis revealed that these sequences were more closely related to CAEV than to MVV. Our results revealed that deer can carry SRLV proviral sequences and therefore may play a role in the epidemiology of SRLVs. To our knowledge, this is the first study describing SRLV sequences from red deer.


Assuntos
DNA Viral , Cervos , Infecções por Lentivirus , Provírus , Animais , Cervos/virologia , Polônia/epidemiologia , Provírus/genética , Infecções por Lentivirus/veterinária , Infecções por Lentivirus/virologia , Infecções por Lentivirus/epidemiologia , DNA Viral/genética , Lentivirus/isolamento & purificação , Lentivirus/genética , Lentivirus/classificação , Filogenia , Reação em Cadeia da Polimerase em Tempo Real/veterinária
12.
BMC Mol Cell Biol ; 25(1): 15, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38741034

RESUMO

BACKGROUND: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells. METHODS: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells. RESULTS: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 104 Vero cells. CONCLUSION: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.


Assuntos
Eletroporação , Vetores Genéticos , Transfecção , Animais , Chlorocebus aethiops , Células Vero , Eletroporação/métodos , Transfecção/métodos , Vetores Genéticos/genética , Lentivirus/genética , Transdução Genética/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos
13.
Methods Mol Biol ; 2799: 29-46, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38727901

RESUMO

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.


Assuntos
Hipocampo , Neurônios , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Hipocampo/metabolismo , Hipocampo/citologia , Neurônios/metabolismo , Animais , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , Células Cultivadas , Ratos , Humanos , Lentivirus/genética , Cultura Primária de Células/métodos , Expressão Gênica
14.
Biotechnol J ; 19(5): e2400090, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38719592

RESUMO

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.


Assuntos
Reatores Biológicos , Vetores Genéticos , Lentivirus , Lentivirus/genética , Humanos , Vetores Genéticos/genética , Meios de Cultura Livres de Soro , Linhagem Celular , Técnicas de Cultura de Células/métodos , Cultura de Vírus/métodos , Células HEK293 , Transfecção/métodos
15.
Am J Vet Res ; 85(6)2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38626794

RESUMO

OBJECTIVE: The aim of this study was to assess the efficacy and safety of a third-generation lentivirus-based vector encoding the feline erythropoietin (EPO) (feEPO) gene in vitro and in rodent models in vivo. This vector incorporates a genetic mechanism to facilitate the termination of the therapeutic effect in the event of supraphysiologic polycythemia, the herpes simplex virus thymidine kinase (HSV-TK) "suicide gene." ANIMALS: CFRK cells and replication-defective lentiviral vectors encoding feEPO were used for in vitro experiments. Eight Fischer rats were enrolled in the pilot in vivo study, 24 EPO-deficient mice were used in the initial mouse study, and 15 EPO-deficient mice were enrolled in the final mouse study. METHODS: Efficacy of a third-generation lentivirus encoding feEPO was determined in vitro using western blot assays. Subsequently, in a series of rodent experiments, animals were administered the viral vector in progressively increasing inoculation doses with serial measurements of blood packed cell volume (PCV) over time. RESULTS: We documented production of feEPO protein in transduced CRFK cells with subsequent cessation of production when treated with the HSV-TK substrate ganciclovir. In vivo, we demonstrated variably persistent elevated PCV values in treated rats and mice with eventual return to baseline values over time. CLINICAL RELEVANCE: These results provide justification for a lentiviral gene therapy approach to the treatment of nonregenerative anemia associated with chronic renal disease in cats.


Assuntos
Anemia , Eritropoetina , Terapia Genética , Vetores Genéticos , Lentivirus , Ratos Endogâmicos F344 , Animais , Eritropoetina/genética , Terapia Genética/veterinária , Lentivirus/genética , Camundongos , Anemia/veterinária , Anemia/terapia , Gatos , Ratos , Insuficiência Renal Crônica/terapia , Insuficiência Renal Crônica/veterinária , Masculino , Feminino , Linhagem Celular
16.
J Immunother Cancer ; 12(4)2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38658032

RESUMO

BACKGROUND: While immunotherapy has been highly successful for the treatment of some cancers, for others, the immune response to tumor antigens is weak leading to treatment failure. The resistance of tumors to checkpoint inhibitor therapy may be caused by T cell exhaustion resulting from checkpoint activation. METHODS: In this study, lentiviral vectors that expressed T cell epitopes of an experimentally introduced tumor antigen, ovalbumin, or the endogenous tumor antigen, Trp1 were developed. The vectors coexpressed CD40 ligand (CD40L), which served to mature the dendritic cells (DCs), and a soluble programmed cell death protein 1 (PD-1) microbody to prevent checkpoint activation. Vaccination of mice bearing B16.OVA melanomas with vector-transduced DCs induced the proliferation and activation of functional, antigen-specific, cytolytic CD8 T cells. RESULTS: Vaccination induced the expansion of CD8 T cells that infiltrated the tumors to suppress tumor growth. Vector-encoded CD40L and PD-1 microbody increased the extent of tumor growth suppression. Adoptive transfer demonstrated that the effect was mediated by CD8 T cells. Direct injection of the vector, without the need for ex vivo transduction of DCs, was also effective. CONCLUSIONS: This study suggests that therapeutic vaccination that induces tumor antigen-specific CD8 T cells coupled with a vector-expressed checkpoint inhibitor can be an effective means to suppress the growth of tumors that are resistant to conventional immunotherapy.


Assuntos
Vacinas Anticâncer , Inibidores de Checkpoint Imunológico , Lentivirus , Animais , Camundongos , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Lentivirus/genética , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Humanos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Linfócitos T CD8-Positivos/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Feminino
17.
Int J Mol Sci ; 25(7)2024 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-38612914

RESUMO

Hematopoietic reconstitution after hematopoietic stem cell transplantation (HSCT) is influenced by the number of transplanted cells. However, under certain conditions donor cell counts are limited and impair clinical outcome. Hematopoietic stem and progenitor cell (HSPC) expansion prior to HSCT is a widely used method to achieve higher donor cell counts and minimize transplantation-related risks such as graft failure or delayed engraftment. Still, expansion in a non-physiological environment can trigger cell death mechanisms and hence counteract the desired effect. We have shown earlier that during HSCT a relevant amount of HSPCs were lost due to apoptosis and that cell death inhibition in donor HSPCs improved engraftment in xenotransplantation experiments. Here, we assessed the effect of combined ex vivo expansion and cell death inhibition on HSPC yield and their reconstitution potential in vivo. During expansion with cytokines and the small molecule inhibitor StemRegenin 1, concomitant lentiviral overexpression of antiapoptotic BCL-XL resulted in an increased yield of transduced HSPCs. Importantly, BCL-XL overexpression enhanced the reconstitution potential of HSPCs in xenotransplantation experiments in vivo. In contrast, treatment with caspase and necroptosis inhibitors had no favorable effects on HSPC yields nor on cell viability. We postulate that overexpression of antiapoptotic BCL-XL, both during ex vivo expansion and transplantation, is a promising approach to improve the outcome of HSCT in situations with limited donor cell numbers. However, such apoptosis inhibition needs to be transient to avoid long-term sequelae like leukemia.


Assuntos
Apoptose , Lentivirus , Transplante Heterólogo , Lentivirus/genética , Células-Tronco Hematopoéticas , Morte Celular
18.
Viruses ; 16(4)2024 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-38675890

RESUMO

The high genetic heterogeneity of small ruminant lentiviruses (SRLV) renders the genetic characterization of the circulating strains crucial for the epidemiological investigation and the designation of effective diagnostic tools. In Greece, research data regarding the genetic diversity of the circulating SRLV strains is scarce, hindering the implementation of efficient surveillance and control programs. The objective of the study was to genetically characterize SRLV strains isolated from intensive dairy sheep farms in Greece and evaluate the variability of the immunodominant regions of the capsid protein. For this reason, a total of 12 SRLV-infected animals from four intensive dairy sheep farms with purebred Chios and Lacaune ewes were used for the amplification and sequencing of an 800 bp gag-pol fragment. The phylogenetic analyses revealed a breed-related circulation of strains; Chios ewes were infected with strains belonging exclusively to a separate group of genotype A, whereas strains belonging to subtype B2 were isolated from Lacaune ewes. Immunodominant epitopes of capsid protein were quite conserved among the strains of the same genotype, except for the Major Homology Region which showed some unique mutations with potential effects on viral evolution. The present study contributes to the extension of the current knowledge regarding the genetic diversity of SRLV strains circulating in sheep in Greece. However, broader genetic characterization studies are warranted for the exploration of possible recombinant events and the more comprehensive classification of the circulating strains.


Assuntos
Variação Genética , Genótipo , Infecções por Lentivirus , Filogenia , Doenças dos Ovinos , Animais , Ovinos , Grécia , Doenças dos Ovinos/virologia , Infecções por Lentivirus/veterinária , Infecções por Lentivirus/virologia , Feminino , Proteínas do Capsídeo/genética , Lentivirus/genética , Lentivirus/isolamento & purificação , Lentivirus/classificação
19.
EMBO Mol Med ; 16(6): 1427-1450, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38684862

RESUMO

Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice.


Assuntos
Células Endoteliais , Fator VIII , Terapia Genética , Vetores Genéticos , Hemofilia A , Lentivirus , Fígado , Animais , Hemofilia A/terapia , Hemofilia A/genética , Vetores Genéticos/genética , Células Endoteliais/metabolismo , Camundongos , Lentivirus/genética , Terapia Genética/métodos , Fígado/metabolismo , Fator VIII/genética , Fator VIII/metabolismo , Humanos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Transdução Genética/métodos , Hepatócitos/metabolismo , Hepatócitos/virologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo
20.
Sci Rep ; 14(1): 6958, 2024 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-38521856

RESUMO

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.


Assuntos
Proteínas do Citoesqueleto , Glaucoma de Ângulo Aberto , Glicoproteínas , Animais , Camundongos , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/terapia , Glaucoma de Ângulo Aberto/metabolismo , Pressão Intraocular/genética , Lentivirus/genética , Malha Trabecular/metabolismo
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