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1.
J Vis Exp ; (204)2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38436380

RESUMO

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.


Assuntos
MicroRNAs , Humanos , Animais , Camundongos , MicroRNAs/genética , Cavidade Peritoneal , Lentivirus/genética , Células HEK293 , Macrófagos , Modelos Animais de Doenças
2.
Biotechnol J ; 19(3): e2300348, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38472091

RESUMO

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


Assuntos
Lentivirus , Vírus , Lentivirus/genética , Vírus/genética , Filtração/métodos , Terapia Genética
3.
Curr Protoc ; 4(3): e1003, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38483112

RESUMO

The human lymphoblastoid cell line TK6 stands out as the most widely employed human cell line in genotoxicity testing, as recommended by various testing guidelines for in vitro assessments. Nevertheless, like many testing cell lines, TK6 lacks functional phase I drug-metabolizing enzymes crucial for chemical genotoxicity evaluations. This protocol introduces a lentivirus-based methodology for establishing a panel of TK6-derived cell lines, each expressing one of 14 cytochrome P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7). The utilization of a lentiviral expression system ensures stable transduction, offering notable advantages such as sustained transgene expression, high transduction efficiency, positive selection feasibility, and user-friendly application. Additionally, we present a detailed procedure for validating the enhanced expression of each CYP in the established cell lines through real-time PCR, western blotting, and mass spectrometry analysis. Lastly, we exemplify the application of these CYP-expressing TK6 cell lines in genotoxicity testing, employing a flow-cytometry-based in vitro micronucleus test. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Lentivirus production and transduction for TK6 cells Support Protocol: Selecting a single clone of CYP-expressing TK6 cells Basic Protocol 2: Validation of CYP expression in TK6 cell lines Basic Protocol 3: Application of transduced cell lines in flow-cytometry-based micronucleus assay.


Assuntos
Sistema Enzimático do Citocromo P-450 , Lentivirus , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2E1/genética , Linhagem Celular
4.
PLoS One ; 19(3): e0298542, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38457474

RESUMO

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies.


Assuntos
Infecções por HIV , HIV-1 , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Recombinases/metabolismo , HIV-1/fisiologia , Provírus/genética , Repetição Terminal Longa de HIV/genética , Infecções por HIV/terapia , Vetores Genéticos/genética
5.
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 , Glaucoma , Glicoproteínas , Camundongos , Animais , Pressão Intraocular/genética , Sistemas CRISPR-Cas/genética , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/terapia , Glaucoma de Ângulo Aberto/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , RNA Guia de Sistemas CRISPR-Cas , Glaucoma/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Malha Trabecular/metabolismo , Modelos Animais de Doenças
6.
Methods Mol Biol ; 2754: 533-549, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38512688

RESUMO

Tau pathology is a major hallmark of many neurodegenerative diseases summarized under the term tauopathies. In most of these disorders,  such as Alzheimer's disease, the neuronal axonal microtubule-binding Tau protein becomes mislocalized to the somatodendritic compartment. In human disease, this missorting of Tau is accompanied by an abnormally high phosphorylation state of the Tau protein, and several downstream pathological consequences (e.g., loss of microtubules, degradation of postsynaptic spines, impaired synaptic transmission, neuronal death). While some mechanisms of Tau sorting, missorting, and associated pathologies have been addressed in rodent models, few studies have addressed human Tau in physiological disease-relevant human neurons. Thus, suitable human-derived in vitro models are necessary. This protocol provides a simple step-by-step protocol for generating homogeneous cultures of cortical glutamatergic neurons using an engineered Ngn2 transgene-carrying WTC11 iPSC line. We further demonstrate strategies to improve neuronal maturity, that is, synapse formation, Tau isoform expression, and neuronal activity by co-culturing hiPSC-derived glutamatergic neurons with mouse-derived astrocytes. Finally, we describe a simple protocol for high-efficiency lentiviral transduction of hiPSC-derived neurons at almost all stages of differentiation.


Assuntos
Células-Tronco Pluripotentes Induzidas , Proteínas tau , Camundongos , Animais , Humanos , Proteínas tau/genética , Proteínas tau/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Neurônios/metabolismo , Axônios/metabolismo , Diferenciação Celular , Células Cultivadas
7.
Sci Rep ; 14(1): 3636, 2024 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-38351130

RESUMO

Small ruminant lentiviruses (SRLVs), are grouped in Retroviridae family, remain a significant loss in the small ruminant husbandry. As a result of unavailability of vaccine and effective treatment, the diagnosis plays a crucial role for the control of SRLV infection. However, the major challenge of diagnosis of SRLV infection is the genetic and antigenic variability of the viruses that can lead to a failure in serological detection. This study investigated the circulating strains of the viruses in goats in Thailand and an in-house ELISA was developed. The coding sequences for gag protein were optimized, synthesized, and expressed in Escherichia coli for increasing the sensitivity of ELISA test. A total of 365 serum samples were examined against the recombinant protein in an in-house ELISA. The results showed that the recombinant gag achieves 96.67% sensitivity and 93.18% specificity as compared with the commercially available ELISA test kit.


Assuntos
Doenças das Cabras , Infecções por Lentivirus , Doenças dos Ovinos , Ovinos , Animais , Lentivirus/genética , Cabras , Tailândia , Doenças das Cabras/diagnóstico , Ruminantes , Produtos do Gene gag/genética , Ensaio de Imunoadsorção Enzimática , Filogenia
8.
Mol Ther ; 32(3): 619-636, 2024 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-38310355

RESUMO

Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a rare X-linked recessive lysosomal storage disorder due to a mutation in the lysosomal enzyme iduronate-2-sulfatase (IDS) gene. IDS deficiency leads to a progressive, multisystem accumulation of glycosaminoglycans (GAGs) and results in central nervous system (CNS) manifestations in the severe form. We developed up to clinical readiness a new hematopoietic stem cell (HSC) gene therapy approach for MPS II that benefits from a novel highly effective transduction protocol. We first provided proof of concept of efficacy of our approach aimed at enhanced IDS enzyme delivery to the CNS in a murine study of immediate translational value, employing a lentiviral vector (LV) encoding a codon-optimized human IDS cDNA. Then the therapeutic LV was tested for its ability to efficiently and safely transduce bona fide human HSCs in clinically relevant conditions according to a standard vs. a novel protocol that demonstrated superior ability to transduce bona fide long-term repopulating HSCs. Overall, these results provide strong proof of concept for the clinical translation of this approach for the treatment of Hunter syndrome.


Assuntos
Iduronato Sulfatase , Mucopolissacaridose II , Humanos , Animais , Camundongos , Mucopolissacaridose II/terapia , Mucopolissacaridose II/tratamento farmacológico , Iduronato Sulfatase/genética , Iduronato Sulfatase/metabolismo , Terapia Genética , Sistema Nervoso Central/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Células-Tronco Hematopoéticas/metabolismo
9.
J Biosci ; 492024.
Artigo em Inglês | MEDLINE | ID: mdl-38384245

RESUMO

Inherited genetic disorders are progressive in nature and lead to organ dysfunction or death in severe cases. At present, there are no permanent treatment options for >95% of inherited disorders. Different modes of inheritance, type of gene(s) involved, and population-based variations add further complexity to finding suitable cures for approximately 400 million patients worldwide. Gene therapy is a very promising molecular technique for the treatment of rare genetic disorders. Gene therapy functions on the basis of restoration, replacement, inhibition, and, most recently, editing of gene(s) to rescue the disease phenotype. Recent reports show that increasing numbers of gene therapy clinical trials are using viral vectors (64.2%) when compared with non-viral vectors. Rapid development of efficient viral vector systems like the adeno-associated virus (AAV) and lentivirus has significantly contributed to this progress. Notably, AAV-mediated gene therapy has shown high potential for genetic disease treatment as evident from recent clinical trials for the eye (NCT00999609), blood (NCT00979238), and neuro-muscular systems (NCT02122952). Safety and efficacy are the two most critical features required for vector(s) to qualify for pre-clinical and clinical trial approval. The process of clinical-grade vector production, evaluation, and approvals for gene therapy products requires significant technological development, knowledge enhancement, and large financial investments. Additionally, trained manpower is required to meet the demands for constant technical innovation. These factors together contribute towards exorbitant prices for every dose of a gene therapy product and thus pose a challenge for the gene therapy field. The Indian subcontinent has traditionally lagged behind North America, Europe, Japan, and others in gene therapy clinical trials due to factors like inadequate industrial-scientific infrastructure, lack of accessible and organized patient databases, low financial investments, etc. However, over the last decade, increasing awareness of rare diseases, and international approvals of gene therapies such as Luxturna, Zolgensma, Hemgenix, etc., have spurred gene therapy development in India as well. In view of these advances, this article outlines gene therapy research, regulatory processes, and the launch of gene therapy clinical trials in India in the context of major developments worldwide. We briefly describe ongoing gene therapy research across Indian organizations and the nascent gene therapy product manufacturing. Further, we highlight the various initiatives from the medical and patient community to avail rehabilitation and gene therapy options. We briefly discuss the roles of regulatory agencies and guidelines for gene therapy clinical trials in India. We anticipate that this concise review will highlight the promise of gene therapy for the large population of rare disease patients in India.


Assuntos
Ensaios Clínicos como Assunto , Terapia Genética , Humanos , Terapia Genética/efeitos adversos , Vetores Genéticos/genética , Índia , Lentivirus/genética
10.
Sheng Wu Gong Cheng Xue Bao ; 40(2): 458-472, 2024 Feb 25.
Artigo em Chinês | MEDLINE | ID: mdl-38369833

RESUMO

Solid tumors lack well-defined targets for chimeric antigen receptor T-cell (CAR-T) therapy. Therefore, introducing a known target molecule, CD19, into solid tumor cell lines via lentiviral transduction to investigate the cytotoxicity of CD19 CAR-T cells can potentially support CAR-T cell therapy against solid tumors. In this study, a stable colon cancer CT26 cell line, CT26-CD19-FLUC-GFP, expressing CD19, firefly luciferase (FLUC), and green fluorescent protein (GFP), was constructed using a triple-plasmid lentiviral system. The growth characteristics of this cell line were consistent with those of the CT26 cell line. Subsequent flow cytometry analysis confirmed stable expression of CD19 and GFP in CT26-CD19-FLUC-GFP cells after serial passaging up to the 5th, 10th, and 22nd generations. Further validation revealed significantly higher levels of CD19 mRNA and FLUC expression in CT26-CD19-FLUC-GFP cells continuously passaged up to the 22nd generation compared to the control CT26 cells. In comparison to T cells, CD19 CAR-T cells demonstrated substantial cytotoxicity against CT26-CD19-FLUC-GFP cells and MC38-CD19 cells. One week after intraperitoneal implantation of CT26-CD19-FLUC-GFP cells into mice, FLUC expression in the peritoneal region could be detected. These results indicate the successful establishment of a stable CT26 cell line expressing CD19-FLUC-GFP, which can be specifically targeted by CD19 CAR-T cells.


Assuntos
Receptores de Antígenos Quiméricos , Camundongos , Animais , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Proteínas de Fluorescência Verde/genética , Luciferases de Vaga-Lume , Linfócitos T/metabolismo , Lentivirus/genética , Linhagem Celular Tumoral
11.
Sheng Wu Gong Cheng Xue Bao ; 40(2): 573-584, 2024 Feb 25.
Artigo em Chinês | MEDLINE | ID: mdl-38369842

RESUMO

Signal peptides (SP) are involved in regulating the secretion level and transmembrane translocation of chimeric antigen receptors (CAR), which is crucial for CAR-T cells. This study aimed to optimize the SP sequence by site-directed mutagenesis and investigate its impact on the killing function of CD19-CAR-T. Firstly, CAR vectors targeting CD19 containing wild-type SP (SP-wtY) or two mutant SP (SP-muK or SP-muR) were constructed using gene synthesis and molecular cloning techniques. The successfully constructed vector was packaged with lentivirus, and T cells were infected. The transfection efficiency of T cells was detected by flow cytometry, while the killing effect on target cells was assessed using the calcein release method. The secretion levels of cytokines interferon-γ (IFN-γ) and interferon-α (TNF-α) were measured using enzyme linked immunosorbent assay (ELISA). The results showed that successful construction of recombinant lentivirus plasmids with wild type and signal peptide mutation. After the transferring the lentivirus into T cells, the transfection efficiency of CD19-CAR carrying three signal peptides (SP-wtY, SP-muK, or SP-muR) were 33.9%, 35.5%, and 36.8%, respectively. Further killing assay showed that the tumor-killing effect of SP-muR cells was significantly higher than that of SP-muK and SP-wtY cells. When the ratio of effector to target was 10:1, the secretion levels of cytokines IFN-γ and TNF-α of CAR-T cells of the SP-muR group were significantly higher than those in SP-muK and SP-wtY groups. In summary, this study revealed that increasing the N-terminal positive charge of the signal peptide can improve the expression efficiency of CAR and promote the killing of CD19+ target cells. These findings provide a scientific basis the optimization and clinical application of CAR structure.


Assuntos
Receptores de Antígenos Quiméricos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Sinais Direcionadores de Proteínas/genética , Linfócitos T/metabolismo , Lentivirus/genética , Citocinas/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Mutagênese Sítio-Dirigida
12.
Acta Biomater ; 177: 157-164, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38364929

RESUMO

Efficient T cell engineering is central to the success of CAR T cell therapy but involves multiple time-consuming manipulations, including T cell isolation, activation, and transduction. These steps add complexity and delay CAR T cell manufacturing, which takes a mean time of 4 weeks. To streamline T cell engineering, we strategically combine two critical engineering solutions - T cell-specific lentiviral vectors and macroporous scaffolds - that enable T cell activation and transduction in a simple, single step. The T cell-specific lentiviral vectors (referred to as STAT virus) target T cells through the display of an anti-CD3 antibody and the CD80 extracellular domain on their surface and provide robust T cell activation. Biocompatible macroporous scaffolds (referred to as Drydux) mediate robust transduction by providing effective interaction between naïve T cells and viral vectors. We show that when unstimulated peripheral blood mononuclear cells (PBMCs) are seeded together with STAT lentivirus on Drydux scaffolds, T cells are activated, selectively transduced, and reprogrammed in a single step. Further, we show that the Drydux platform seeded with PBMCs and STAT lentivirus generates tumor-specific functional CAR T cells. This potent combination of engineered lentivirus and biomaterial scaffold holds promise for an effective, simple, and safe avenue for in vitro and in vivo T cell engineering. STATEMENT OF SIGNIFICANCE: Manufacturing T cell therapies involves lengthy and labor-intensive steps, including T cell selection, activation, and transduction. These steps add complexity to current CAR T cell manufacturing protocols and limit widespread patient access to this revolutionary therapy. In this work, we demonstrate the combination of engineered virus and biomaterial platform that, together, enables selective T cell activation and transduction in a single step, eliminating multistep T cell engineering protocols and significantly simplifying the manufacturing process.


Assuntos
Leucócitos Mononucleares , Linfócitos T , Humanos , Transdução Genética , Terapia Genética , Imunoterapia Adotiva/métodos , Lentivirus/genética , Vetores Genéticos
13.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 32(1): 274-281, 2024 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-38387934

RESUMO

OBJECTIVE: To construct recombinant lentivirus and adenovirus which regulate the expression of c-Cbl gene and evaluate their efficacy. METHODS: The interference lentivirus and overexpressed adenovirus targeting human c-Cbl gene were constructed by gene recombination technology. Quantitative PCR and western blotting were used to detect the expression changes in c-Cbl gene and its transcription after leukemia cells (HL60,THP1) were infected by virus. RESULTS: Three recombinant interfering lentiviral vectors targeting human c-Cbl genes to successfully constructed and were identified by DNA sequencing, and the titers of the packaged viruses were all greater than 1×108 TU/ml. Among them, shRNA-2 lentivirus had the highest interference efficiency, and the expression of c-Cbl gene and CBL protein were decreased about 95% and 60% respectively after leukemia cells were infected with shRNA-2; In addition, the recombinant overexpression adenovirus targeting human c-Cbl gene was packaged successfully with the virus titer greater than 1×109 TU/ml. When leukemia cells were infected with adenovirus, the expression of c-Cbl gene and CBL protein were up-regulated about 10 times and 1.5 times respectively. CONCLUSION: Both recombinant interfering lentivirus and overexpression adenovirus can efficiently infect leukemia cells and affect the expressions of c-Cbl gene and CBL protein. It will lay a preliminary foundation for the subsequent study on the function of c-Cbl gene in tumor cells.


Assuntos
Vetores Genéticos , Leucemia , Humanos , Adenoviridae/genética , Lentivirus/genética , RNA Interferente Pequeno/genética
14.
J Proteomics ; 291: 105037, 2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-38288553

RESUMO

Pompe disease is a lysosomal storage disorder caused by deficiency of acid alpha-glucosidase (GAA), resulting in glycogen accumulation with profound pathology in skeletal muscle. We recently developed an optimized form of lentiviral gene therapy for Pompe disease in which a codon-optimized version of the GAA transgene (LV-GAAco) was fused to an insulin-like growth factor 2 (IGF2) peptide (LV-IGF2.GAAco), to promote cellular uptake via the cation-independent mannose-6-phosphate/IGF2 receptor. Lentiviral gene therapy with LV-IGF2.GAAco showed superior efficacy in heart, skeletal muscle, and brain of Gaa -/- mice compared to gene therapy with untagged LV-GAAco. Here, we used quantitative mass spectrometry using TMT labeling to analyze the muscle proteome and the response to gene therapy in Gaa -/- mice. We found that muscle of Gaa -/- mice displayed altered levels of proteins including those with functions in the CLEAR signaling pathway, autophagy, cytoplasmic glycogen metabolism, calcium homeostasis, redox signaling, mitochondrial function, fatty acid transport, muscle contraction, cytoskeletal organization, phagosome maturation, and inflammation. Gene therapy with LV-GAAco resulted in partial correction of the muscle proteome, while gene therapy with LV-IGF2.GAAco resulted in a near-complete restoration to wild type levels without inducing extra proteomic changes, supporting clinical development of lentiviral gene therapy for Pompe disease. SIGNIFICANCE: Lysosomal glycogen accumulation is the primary cause of Pompe disease, and leads to a cascade of pathological events in cardiac and skeletal muscle and in the central nervous system. In this study, we identified the proteomic changes that are caused by Pompe disease in skeletal muscle of a mouse model. We showed that lentiviral gene therapy with LV-IGF2.GAAco nearly completely corrects disease-associated proteomic changes. This study supports the future clinical development of lentiviral gene therapy with LV-IGF2.GAAco as a new treatment option for Pompe disease.


Assuntos
Doença de Depósito de Glicogênio Tipo II , Animais , Camundongos , Terapia Genética/métodos , Glicogênio/metabolismo , Doença de Depósito de Glicogênio Tipo II/genética , Doença de Depósito de Glicogênio Tipo II/terapia , Doença de Depósito de Glicogênio Tipo II/patologia , Lentivirus/genética , Lentivirus/metabolismo , Lisossomos/metabolismo , Camundongos Knockout , Músculo Esquelético/metabolismo , Proteoma/metabolismo , Proteômica
15.
Cell Rep ; 43(2): 113697, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38294901

RESUMO

The pandemic HIV-1, HIV-1 group M, emerged from a single spillover event of its ancestral lentivirus from a chimpanzee. During human-to-human spread worldwide, HIV-1 diversified into multiple subtypes. Here, our interdisciplinary investigation mainly sheds light on the evolutionary scenario of the viral budding system of HIV-1 subtype C (HIV-1C), a most successfully spread subtype. Of the two amino acid motifs for HIV-1 budding, the P(T/S)AP and YPxL motifs, HIV-1C loses the YPxL motif. Our data imply that HIV-1C might lose this motif to evade immune pressure. Additionally, the P(T/S)AP motif is duplicated dependently of the level of HIV-1 spread in the human population, and >20% of HIV-1C harbored the duplicated P(T/S)AP motif. We further show that the duplication of the P(T/S)AP motif is caused by the expansion of the CTG triplet repeat. Altogether, our results suggest that HIV-1 has experienced a two-step evolution of the viral budding process during human-to-human spread worldwide.


Assuntos
Soropositividade para HIV , HIV-1 , Humanos , Animais , HIV-1/genética , Pandemias , Lentivirus , Divisão Celular , Pan troglodytes
16.
J Virol Methods ; 325: 114884, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38218417

RESUMO

HIV-1 based lentiviral viruses are considered powerful and versatile gene therapy vectors to deliver therapeutic genes to patients with hereditary or acquired diseases. These vectors can efficiently transduce a variety of cell types when dividing or non-dividing to provide permanent delivery and long-term gene expression. Demand for scalable manufacturing protocols able to generate enough high titre vector for widespread use of this technology is increasing and considerable efforts to improve vector production cost-effectively, is ongoing. Current methods for LV production mainly use transient transfection of producer cell lines. Cells can be grown at scale, either in 2D relying on culturing producer cells in multi-tray flask cell culture factories or in roller bottles or can be adapted to grow in 3D suspensions in large batch fermenters. This suits rapid production and testing of new vector constructs pre-clinically for their efficacy, particle titre and safety. In this study, we sought to improve lentiviral titre over time by testing two alternative commercially available transfection reagents Fugene® 6 and Genejuice® with the commonly used polycation, polyethyleneimine. Our aim was to identify less cytotoxic transfection reagents that could be used to generate LV particles at high titre past the often used 72 h period when vector is usually collected before producer cell death is caused due to post transfection cytotoxicity. We show that LV could be produced in extended culture using Genejuice® and even by transfected cells in glass flasks in suspension. Because this delivery agent is less toxic to 293 T producer cells, following optimisation of transfection we found that LV can be harvested for more than 10 days at high titre. Using our protocol, titres of 109 TU/ml and 108 TU/ml were routinely reached via traditional monolayer conditions or suspension cultures, respectively. We propose, this simple change in vector production enables large volumes of high titre vector to be produced, cost effectively for non-clinical in vivo and in vitro applications or for more stringent downstream clinical grade vector purification.


Assuntos
Vetores Genéticos , Lentivirus , Humanos , Lentivirus/genética , Células HEK293 , Transfecção , Técnicas de Cultura de Células/métodos
17.
ACS Synth Biol ; 13(2): 466-473, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38266181

RESUMO

We engineered HEK293T cells with a transgene encoding tetracycline-inducible expression of a Staphylococcus aureus nuclease incorporating a translocation signal. We adapted the unmodified and nuclease-engineered cell lines to grow in suspension in serum-free media, generating the HEK293TS and NuPro-2S cell lines, respectively. Transient transfection yielded 1.19 × 106 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 106 TU/mL from HEK293TS cells. DNA ladder disappearance revealed medium-resident nuclease activity arising from NuPro-2S cells in a tetracycline-inducible manner. DNA impurity levels in lentiviral material arising from NuPro-2S and HEK293TS cells were undetectable by SYBR Safe agarose gel staining. Direct measurement by PicoGreen reagent revealed DNA to be present at 636 ng/mL in lentiviral material from HEK293TS cells, an impurity level reduced by 89% to 70 ng/mL in lentiviral material from NuPro-2S cells. This reduction was comparable to the 23 ng/mL achieved by treating HEK293TS-derived lentiviral material with 50 units/mL Benzonase.


Assuntos
Fluoreto de Fosfato Acidulado , Vetores Genéticos , Lentivirus , Animais , Humanos , Lentivirus/genética , Vetores Genéticos/genética , Células HEK293 , Transfecção , DNA/genética , Tetraciclina , Mamíferos/genética
18.
STAR Protoc ; 5(1): 102854, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38294912

RESUMO

Modifying gene expression in lymphocytes is essential for immunological research; however, gene transfection methods for group 2 innate lymphoid cells (ILC2s) are not well established. Here, we present a protocol utilizing lentiviral vectors to introduce genes into human ILC2s. We detail steps for lentiviral solution preparation, transfection, and selection of transfected cells. This protocol allows overexpression or suppression of specific genes and evaluation of the changes in ILC2 biology. For complete details on the use and execution of this protocol, please refer to Irie et al. (2023).1.


Assuntos
Imunidade Inata , Lentivirus , Humanos , Imunidade Inata/genética , Lentivirus/genética , Linfócitos/metabolismo , Transfecção , Células Cultivadas
19.
J Med Virol ; 96(1): e29425, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38258313

RESUMO

The emergence of rapid and continuous mutations of severe acute respiratory syndrome 2 (SARS-CoV-2) spike glycoprotein that increased with the Omicron variant points out the necessity to anticipate such mutations for conceiving specific and adaptable therapies to avoid another pandemic. The crucial target for the antibody treatment and vaccine design is the receptor binding domain (RBD) of the SARS-CoV-2 spike. It is also the site where the virus has shown its high ability to mutate and consequently escape immune response. We developed a robust and simple method for generating a large number of functional SARS-CoV-2 spike RBD mutants by error-prone PCR and a novel nonreplicative lentivirus-based system. We prepared anti-RBD wild type (WT) polyclonal antibodies and used them to screen and select for mutant libraries that escape inhibition of virion entry into recipient cells expressing human angiotensin-converting enzyme 2 and transmembrane serine protease 2. We isolated, cloned, and sequenced six mutants totally bearing nine mutation sites. Eight mutations were found in successive WT variants, including Omicron and other recombinants, whereas one is novel. These results, together with the detailed functional analyses of two mutants provided the proof of concept for our approach.


Assuntos
COVID-19 , Lentivirus , Humanos , Lentivirus/genética , SARS-CoV-2/genética , Mutação
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