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1.
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
2.
Methods Mol Biol ; 2807: 287-298, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38743236

RESUMO

The inability of people living with HIV (PLWH) to eradicate human immunodeficiency virus (HIV) infection is due in part to the inadequate HIV-specific cellular immune response. The antiviral function of cytotoxic CD8+ T cells, which are crucial for HIV control, is impaired during chronic viral infection because of viral escape mutations, immune exhaustion, HIV antigen downregulation, inflammation, and apoptosis. In addition, some HIV-infected cells either localize to tissue sanctuaries inaccessible to CD8+ T cells or are intrinsically resistant to CD8+ T cell killing. The novel design of synthetic chimeric antigen receptors (CARs) that enable T cells to target specific antigens has led to the development of potent and effective CAR-T cell therapies. While initial clinical trials using anti-HIV CAR-T cells performed over 20 years ago showed limited anti-HIV effects, the improved CAR-T cell design, which enabled its success in treating cancer, has reinstated CAR-T cell therapy as a strategy for HIV cure with notable progress being made in the recent decade.Effective CAR-T cell therapy against HIV infection requires the generation of anti-HIV CAR-T cells with potent in vivo activity against HIV-infected cells. Preclinical evaluation of anti-HIV efficacy of CAR-T cells and their safety is fundamental for supporting the initiation of subsequent clinical trials in PLWH. For these preclinical studies, we developed a novel humanized mouse model supporting in vivo HIV infection, the development of viremia, and the evaluation of novel HIV therapeutics. Preclinical assessment of anti-HIV CAR-T cells using this mouse model involves a multistep process including peripheral blood mononuclear cells (PBMCs) harvested from human donors, T cell purification, ex vivo T cell activation, transduction with lentiviral vectors encoding an anti-HIV CAR, CAR-T cell expansion and infusion in mice intrasplenically injected with autologous PBMCs followed by the determination of CAR-T cell capacity for HIV suppression. Each of the steps described in the following protocol were optimized in the lab to maximize the quantity and quality of the final anti-HIV CAR-T cell products.


Assuntos
Infecções por HIV , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Humanos , Animais , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Camundongos , Infecções por HIV/imunologia , Infecções por HIV/terapia , Infecções por HIV/virologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T CD8-Positivos/imunologia , HIV-1/imunologia , Linfócitos T/imunologia , Transdução Genética
3.
Skelet Muscle ; 14(1): 9, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38702726

RESUMO

BACKGROUND: Adeno-associated virus (AAV)-based gene therapy is a promising strategy to treat muscle diseases. However, this strategy is currently confronted with challenges, including a lack of transduction efficiency across the entire muscular system and toxicity resulting from off-target tissue effects. Recently, novel myotropic AAVs named MyoAAVs and AAVMYOs have been discovered using a directed evolution approach, all separately demonstrating enhanced muscle transduction efficiency and liver de-targeting effects. However, these newly discovered AAV variants have not yet been compared. METHODS: In this study, we performed a comparative analysis of these various AAV9-derived vectors under the same experimental conditions following different injection time points in two distinct mouse strains. RESULTS: We highlight differences in transduction efficiency between AAV9, AAVMYO, MyoAAV2A and MyoAAV4A that depend on age at injection, doses and mouse genetic background. In addition, specific AAV serotypes appeared more potent to transduce skeletal muscles including diaphragm and/or to de-target heart or liver. CONCLUSIONS: Our study provides guidance for researchers aiming to establish proof-of-concept approaches for preventive or curative perspectives in mouse models, to ultimately lead to future clinical trials for muscle disorders.


Assuntos
Dependovirus , Terapia Genética , Vetores Genéticos , Camundongos Endogâmicos C57BL , Músculo Esquelético , Transdução Genética , Animais , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Músculo Esquelético/metabolismo , Camundongos , Transdução Genética/métodos , Terapia Genética/métodos , Masculino , Fígado/metabolismo , Camundongos Endogâmicos mdx
4.
Nat Commun ; 15(1): 3780, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38710714

RESUMO

Recombinant adeno-associated viruses (rAAVs) have emerged as promising gene therapy vectors due to their proven efficacy and safety in clinical applications. In non-human primates (NHPs), rAAVs are administered via suprachoroidal injection at a higher dose. However, high doses of rAAVs tend to increase additional safety risks. Here, we present a novel AAV capsid (AAVv128), which exhibits significantly enhanced transduction efficiency for photoreceptors and retinal pigment epithelial (RPE) cells, along with a broader distribution across the layers of retinal tissues in different animal models (mice, rabbits, and NHPs) following intraocular injection. Notably, the suprachoroidal delivery of AAVv128-anti-VEGF vector completely suppresses the Grade IV lesions in a laser-induced choroidal neovascularization (CNV) NHP model for neovascular age-related macular degeneration (nAMD). Furthermore, cryo-EM analysis at 2.1 Å resolution reveals that the critical residues of AAVv128 exhibit a more robust advantage in AAV binding, the nuclear uptake and endosome escaping. Collectively, our findings highlight the potential of AAVv128 as a next generation ocular gene therapy vector, particularly using the suprachoroidal delivery route.


Assuntos
Neovascularização de Coroide , Dependovirus , Terapia Genética , Vetores Genéticos , Epitélio Pigmentado da Retina , Animais , Dependovirus/genética , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Terapia Genética/métodos , Camundongos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/virologia , Neovascularização de Coroide/terapia , Neovascularização de Coroide/genética , Coelhos , Humanos , Técnicas de Transferência de Genes , Degeneração Macular/terapia , Degeneração Macular/genética , Degeneração Macular/patologia , Modelos Animais de Doenças , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Transdução Genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Camundongos Endogâmicos C57BL , Retina/metabolismo , Retina/virologia , Masculino , Células HEK293
5.
Folia Neuropathol ; 62(1): 32-46, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38741435

RESUMO

Human induced pluripotent stem cells (hiPSCs) are a potential source of somatic cells for cell therapies due to their ability to self-renew and differentiate into various cells of the body. To date, the clinical application of hiPSCs has been limited due to safety issues. The present study aims to standardize the safety procedure of the derivation of GMP-compliant induced pluripotent stem cell (iPSC) lines from human fibroblasts. The hiPSC lines were generated using the nonintegrative Sendai virus method to incorporate Yamanaka reprogramming factors (OCT3/4, SOX2, KLF4 and c-MYC) into cells. A constant temperature was maintained during the cell culture, including all stages of the culture after transduction with Sendai virus. Pluripotency was proved in six independently generated hiPSC lines from adult female (47 years old) and male (57 years old) donors' derived fibroblasts via alkaline phosphatase live (ALP) staining, qPCR, and immunocytochemistry. The hiPSC lines showed a gradual decrease in the presence of the virus with each subsequent passage, and this reduction was specific to the hiPSC line. The frequency and probability of chromosomal aberrations in hiPSCs were dependent on both the iPSC clone identity and sex of the donor. In summary, the generation of hiPSC for clinical applications requires safety standards application (biosafety protocol, quality control of hiPSC lines, viral and genetic integrity screening) from the first stages of the clonal selection of hiPSC from the same donor.


Assuntos
Células-Tronco Pluripotentes Induzidas , Fator 4 Semelhante a Kruppel , Vírus Sendai , Humanos , Feminino , Masculino , Pessoa de Meia-Idade , Linhagem Celular , Fibroblastos , Diferenciação Celular/fisiologia , Transdução Genética/métodos , Fatores Sexuais
6.
ISME J ; 18(1)2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-38739683

RESUMO

Temperate phages can interact with bacterial hosts through lytic and lysogenic cycles via different mechanisms. Lysogeny has been identified as the major form of bacteria-phage interaction in the coral-associated microbiome. However, the lysogenic-to-lytic switch of temperate phages in ecologically important coral-associated bacteria and its ecological impact have not been extensively investigated. By studying the prophages in coral-associated Halomonas meridiana, we found that two prophages, Phm1 and Phm3, are inducible by the DNA-damaging agent mitomycin C and that Phm3 is spontaneously activated under normal cultivation conditions. Furthermore, Phm3 undergoes an atypical lytic pathway that can amplify and package adjacent host DNA, potentially resulting in lateral transduction. The induction of Phm3 triggered a process of cell lysis accompanied by the formation of outer membrane vesicles (OMVs) and Phm3 attached to OMVs. This unique cell-lysis process was controlled by a four-gene lytic module within Phm3. Further analysis of the Tara Ocean dataset revealed that Phm3 represents a new group of temperate phages that are widely distributed and transcriptionally active in the ocean. Therefore, the combination of lateral transduction mediated by temperate phages and OMV transmission offers a versatile strategy for host-phage coevolution in marine ecosystems.


Assuntos
Antozoários , Halomonas , Prófagos , Halomonas/virologia , Halomonas/genética , Antozoários/microbiologia , Antozoários/virologia , Prófagos/genética , Prófagos/fisiologia , Animais , Lisogenia , Transdução Genética , Mitomicina/farmacologia
7.
Biomolecules ; 14(4)2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38672522

RESUMO

In this study, we introduce electrospun polydioxanone (PDO) nonwoven fabrics as a platform for the delivery of adeno-associated virus (AAV) vectors for transduction and genome editing by adhering them to organ surfaces, including the heart. AAV vectors were loaded onto the PDO fabrics by soaking the fabrics in a solution containing AAV vectors. In vitro, the amount of AAV vectors loaded onto the fabrics could be adjusted by changing their concentration in the solution, and the number of cells expressing the green fluorescent protein (GFP) encoded by the AAV vectors increased in correlation with the increasing amount of loaded AAV vectors. In vivo, both transduction and genome editing resulted in the observation of GFP expression around AAV vector-loaded PDO fabrics attached to the surfaces of mouse hearts, indicating effective transduction and expression at the target site. These results demonstrate the great potential of electrospun PDO nonwoven fabrics carrying therapeutic AAV vectors for gene therapy.


Assuntos
Dependovirus , Edição de Genes , Vetores Genéticos , Polidioxanona , Dependovirus/genética , Animais , Vetores Genéticos/genética , Polidioxanona/química , Edição de Genes/métodos , Camundongos , Humanos , Transdução Genética/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Terapia Genética/métodos , Miocárdio/metabolismo
8.
PLoS One ; 19(4): e0298866, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38687720

RESUMO

We demonstrate that applying electric field pulses to hepatocytes, in vitro, in the presence of enhanced green fluorescent protein (EGFP)-expressing adeno-associated virus (AAV8) vectors reduces the viral dosage required for a given transduction level by more than 50-fold, compared to hepatocytes exposed to AAV8-EGFP vectors without electric field pulse exposure. We conducted 48 experimental observations across 8 exposure conditions in standard well plates. The electric pulse exposures involved single 80-ms pulses with 375 V/cm field intensity. Our study suggests that electric pulse exposure results in enhanced EGFP expression in cells, indicative of increased transduction efficiency. The enhanced transduction observed in our study, if translated successfully to an in vivo setting, would be a promising indication of potential reduction in the required dose of AAV vectors. Understanding the effects of electric field pulses on AAV transduction in vitro is an important preliminary step.


Assuntos
Dependovirus , Vetores Genéticos , Proteínas de Fluorescência Verde , Transdução Genética , Dependovirus/genética , Humanos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Vetores Genéticos/genética , Células Hep G2 , Hepatócitos/metabolismo , Eletricidade
9.
Viruses ; 16(4)2024 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-38675893

RESUMO

The administration route affects the biodistribution of a gene transfer vector and the expression of a transgene. A simian adenovirus 1 vector carrying firefly luciferase and GFP reporter genes (SAdV1-GFluc) were constructed, and its biodistribution was investigated in a mouse model by bioluminescence imaging and virus DNA tracking with real-time PCR. Luciferase activity and virus DNA were mainly found in the liver and spleen after the intravenous administration of SAdV1-GFluc. The results of flow cytometry illustrated that macrophages in the liver and spleen as well as hepatocytes were the target cells. Repeated inoculation was noneffective because of the stimulated serum neutralizing antibodies (NAbs) against SAdV-1. A transient, local expression of low-level luciferase was detected after intragastric administration, and the administration could be repeated without compromising the expression of the reporter gene. Intranasal administration led to a moderate, constant expression of a transgene in the whole respiratory tract and could be repeated one more time without a significant increase in the NAb titer. An immunohistochemistry assay showed that respiratory epithelial cells and macrophages in the lungs were transduced. High luciferase activity was restricted at the injection site and sustained for a week after intramuscular administration. A compromised transgene expression was observed after a repeated injection. When these mice were intramuscularly injected for a third time with the human adenovirus 5 (HAdV-5) vector carrying a luciferase gene, the luciferase activity recovered and reached the initial level, suggesting that the sequential use of SAdV-1 and HAdV-5 vectors was practicable. In short, the intranasal inoculation or intramuscular injection may be the preferred administration routes for the novel SAdV-1 vector in vaccine development.


Assuntos
Adenovirus dos Símios , Genes Reporter , Vetores Genéticos , Animais , Vetores Genéticos/genética , Camundongos , Adenovirus dos Símios/genética , Distribuição Tecidual , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Transgenes , Replicação Viral , Luciferases de Vaga-Lume/genética , Camundongos Endogâmicos BALB C , Feminino , Transdução Genética , Modelos Animais , Baço/metabolismo , Baço/virologia , Fígado/metabolismo , Fígado/virologia , Anticorpos Neutralizantes/imunologia , Expressão Gênica , Injeções Intramusculares , Administração Intranasal
10.
Mol Ther ; 32(5): 1407-1424, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38429927

RESUMO

Maintaining functional adipose innervation is critical for metabolic health. We found that subcutaneous white adipose tissue (scWAT) undergoes peripheral neuropathy (PN) with obesity, diabetes, and aging (reduced small-fiber innervation and nerve/synaptic/growth-cone/vesicle markers, altered nerve activity). Unlike with nerve injuries, peripheral nerves do not regenerate with PN, and therefore new therapies are needed for treatment of this condition affecting 20-30 million Americans. Here, we validated a gene therapy approach using an adipocyte-tropic adeno-associated virus (AAV; serotype Rec2) to deliver neurotrophic factors (brain-derived neurotrophic factor [BDNF] and nerve growth factor [NGF]) directly to scWAT to improve tissue-specific PN as a proof-of-concept approach. AAVRec2-BDNF intra-adipose delivery improved tissue innervation in obese/diabetic mice with PN, but after longer periods of dietary obesity there was reduced efficacy, revealing a key time window for therapies. AAVRec2-NGF also increased scWAT innervation in obese mice and was more effective than BDNF, likely because Rec2 targeted adipocytes, the tissue's endogenous NGF source. AAVRec2-NGF also worked well even after 25 weeks of dietary obesity, unlike BDNF, which likely needs a vector that targets its physiological cellular source (stromal vascular fraction cells). Given the differing effects of AAVs carrying NGF versus BDNF, a combined therapy may be ideal for PN.


Assuntos
Adipócitos , Fator Neurotrófico Derivado do Encéfalo , Dependovirus , Terapia Genética , Vetores Genéticos , Obesidade , Gordura Subcutânea , Animais , Dependovirus/genética , Obesidade/terapia , Obesidade/metabolismo , Camundongos , Terapia Genética/métodos , Adipócitos/metabolismo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Gordura Subcutânea/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Modelos Animais de Doenças , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/genética , Técnicas de Transferência de Genes , Humanos , Masculino , Doenças do Sistema Nervoso Periférico/terapia , Doenças do Sistema Nervoso Periférico/etiologia , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Transdução Genética
11.
Mol Ther ; 32(5): 1311-1327, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38449314

RESUMO

While studying transgene expression after systemic administration of lentiviral vectors, we found that splenic B cells are robustly transduced, regardless of the types of pseudotyped envelope proteins. However, the administration of two different pseudotypes resulted in transduction of two distinct B cell populations, suggesting that each pseudotype uses unique and specific receptors for its attachment and entry into splenic B cells. Single-cell RNA sequencing analysis of the transduced cells demonstrated that different pseudotypes transduce distinct B cell subpopulations characterized by specific B cell receptor (BCR) genotypes. Functional analysis of the BCRs of the transduced cells demonstrated that BCRs specific to the pseudotyping envelope proteins mediate viral entry, enabling the vectors to selectively transduce the B cell populations that are capable of producing antibodies specific to their envelope proteins. Lentiviral vector entry via the BCR activated the transduced B cells and induced proliferation and differentiation into mature effectors, such as memory B and plasma cells. BCR-mediated viral entry into clonally specific B cell subpopulations raises new concepts for understanding the biodistribution of transgene expression after systemic administration of lentiviral vectors and offers new opportunities for BCR-targeted gene delivery by pseudotyped lentiviral vectors.


Assuntos
Linfócitos B , Vetores Genéticos , Lentivirus , Receptores de Antígenos de Linfócitos B , Transdução Genética , Transgenes , Proteínas do Envelope Viral , Lentivirus/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Animais , Camundongos , Linfócitos B/metabolismo , Linfócitos B/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Tropismo Viral , Humanos , Internalização do Vírus
12.
J Virol Methods ; 327: 114921, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38552881

RESUMO

Dendritic cells (DCs) play a pivotal role in maintaining immune tolerance. Using recombinant adenovirus (rAd) to deliver vectors to immature dendritic cells (imDCs) is an important method for studying the tolerogenic function of DCs. We found that using RPMI medium and a higher MOI during transduction increased the expression of CD80, CD86, and MHC-II on the surface of imDCs. Our data reveal a significant increase in the secretion of the pro-inflammatory cytokine IL-6 in the group showing the most pronounced phenotypic changes. In the mouse heart transplant model, imDCs with unstable phenotype and function due to adenoviral transduction resulted in an increased proportion of Th1 and Th17 cells in recipients. However, these effects can be managed, and our proposed optimized transduction strategy significantly minimizes these adverse effects. Our study holds significant implications for the development and optimization of immunotherapy utilizing tolerogenic dendritic cells.


Assuntos
Adenoviridae , Células Dendríticas , Vetores Genéticos , Imunoterapia , Transdução Genética , Células Dendríticas/imunologia , Animais , Adenoviridae/genética , Camundongos , Imunoterapia/métodos , Vetores Genéticos/genética , Transplante de Coração , Camundongos Endogâmicos C57BL , Interleucina-6/metabolismo , Tolerância Imunológica , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Células Th1/imunologia , Células Th17/imunologia , Antígeno B7-2/metabolismo , Antígeno B7-2/genética
13.
Cytotherapy ; 26(6): 586-591, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38551525

RESUMO

BACKGROUND AIMS: Gene therapy using lentiviral vectors (LVs) that harbor a functional ß-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). METHODS: After HSPCs were transduced with an LV encoding the therapeutic ß-globin (ßA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10-3 ng and 5 × 10-6 ng of target copy per reaction. RESULTS: We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of ßA-T87Q protein detected by reverse-phase high-performance liquid chromatography. CONCLUSIONS: Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting.


Assuntos
Terapia Genética , Vetores Genéticos , Células-Tronco Hematopoéticas , Lentivirus , Transdução Genética , Globinas beta , Humanos , Lentivirus/genética , Células-Tronco Hematopoéticas/metabolismo , Vetores Genéticos/genética , Globinas beta/genética , Transdução Genética/métodos , Terapia Genética/métodos , Talassemia beta/terapia , Talassemia beta/genética , Reação em Cadeia da Polimerase/métodos , Dosagem de Genes/genética
14.
Brain Behav Immun ; 118: 368-379, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38471576

RESUMO

Microglia play a central role in the etiology of many neuropathologies. Transgenic tools are a powerful experiment approach to gain reliable and specific control over microglia function. Adeno-associated virus (AAVs) vectors are already an indispensable tool in neuroscience research. Despite ubiquitous use of AAVs and substantial interest in the role of microglia in the study of central nervous system (CNS) function and disease, transduction of microglia using AAVs is seldom reported. This review explores the challenges and advancements made in using AAVs for expressing transgenes in microglia. First, we will examine the functional anatomy of the AAV capsid, which will serve as a basis for subsequent discussions of studies exploring the relationship between capsid mutations and microglia transduction efficacy. After outlining the functional anatomy of AAVs, we will consider the experimental evidence demonstrating AAV-mediated transduction of microglia and microglia-like cell lines followed by an examination of the most promising experimental approaches identified in the literature. Finally, technical limitations will be considered in future applications of AAV experimental approaches.


Assuntos
Dependovirus , Microglia , Animais , Dependovirus/genética , Transdução Genética , Microglia/metabolismo , Animais Geneticamente Modificados , Transgenes , Vetores Genéticos
15.
Mol Ther ; 32(3): 818-836, 2024 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-38297833

RESUMO

Directed evolution of natural AAV9 using peptide display libraries have been widely used in the search for an optimal recombinant AAV (rAAV) for transgene delivery across the blood-brain barrier (BBB) to the CNS following intravenous ( IV) injection. In this study, we used a different approach by creating a shuffled rAAV capsid library based on parental AAV serotypes 1 through 12. Following selection in mice, 3 novel variants closely related to AAV1, AAV-BBB6, AAV-BBB28, and AAV-BBB31, emerged as top candidates. In direct comparisons with AAV9, our novel variants demonstrated an over 270-fold improvement in CNS transduction and exhibited a clear bias toward neuronal cells. Intriguingly, our AAV-BBB variants relied on the LY6A cellular receptor for CNS entry, similar to AAV9 peptide variants AAV-PHP.eB and AAV.CAP-B10, despite the different bioengineering methods used and parental backgrounds. The variants also showed reduced transduction of both mouse liver and human primary hepatocytes in vivo. To increase clinical translatability, we enhanced the immune escape properties of our new variants by introducing additional modifications based on rational design. Overall, our study highlights the potential of AAV1-like vectors for efficient CNS transduction with reduced liver tropism, offering promising prospects for CNS gene therapies.


Assuntos
Barreira Hematoencefálica , Terapia Genética , Humanos , Animais , Camundongos , Terapia Genética/métodos , Capsídeo , Fígado , Peptídeos/genética , Dependovirus , Vetores Genéticos/genética , Transdução Genética
16.
J Integr Med ; 22(1): 72-82, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38307819

RESUMO

OBJECTIVE: Melittin and its derivative have been developed to support effective gene delivery systems. Their ability to facilitate endosomal release enhances the delivery of nanoparticle-based gene therapy. Nevertheless, its potential application in the context of viral vectors has not received much attention. Therefore, we would like to optimize the rAAV vector by Melittin analog to improve the transduction efficiency of rAAV in liver cancer cells and explore the mechanism of Melittin analog on rAAV. METHODS: Various melittin-derived peptides were inserted into loop VIII of the capsid protein in recombinant adeno-associated virus vectors. These vectors carrying either gfp or fluc genes were subjected to quantitative polymerase chain reaction assays and transduction assays in human embryonic kidney 293 (HEK293T) cells to investigate the efficiency of vector production and gene delivery. In addition, the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice. Finally, the intricate details of the vector-mediated transduction mechanisms were explored by using pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle. RESULTS: A total of 76 melittin-related peptides were identified from existing literature. Among them, CMA-3, p5RHH and aAR3 were found to significantly inhibit transduction of rAAV2 vector crude lysate. The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV. Mechanistically, bafilomycin A1, a vacuolar endosome acidification inhibitor, completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors. Most importantly, p5RHH-rAAV8 vectors also increased hepatic transduction in vivo in C57BL/6 mice. CONCLUSION: The incorporation of melittin analogs into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression. While further modifications remain an area of interest, our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery. Please cite this article as: Meng J, He Y, Yang H, Zhou L, Wang S, Feng X, Al-shargi OY, Yu X, Zhu L, Ling, C. Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency. J Integr Med. 2024; 22(1): 72-82.


Assuntos
Dependovirus , Meliteno , Camundongos , Masculino , Animais , Humanos , Dependovirus/genética , Meliteno/farmacologia , Meliteno/genética , Transdução Genética , Células HEK293 , Camundongos Endogâmicos C57BL , Vetores Genéticos
17.
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
18.
Gene Ther ; 31(5-6): 285-294, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38374348

RESUMO

Manufacturing of recombinant adeno-associated virus (AAV) vectors produces three types of capsids: full, intermediate, and empty. While there are different opinions about the impact of intermediate and empty capsids on safety and efficacy of AAV products, they are generally considered impurities because they are not the intended fully intact vector product. The presence of these impurities could impact product efficacy due to potential competition with fully packaged AAVs for cellular transduction, as well as have potential implications to patient safety due to increased capsid load during dosing. To determine the impact of intermediate capsids on potency, an AAV preparation was separated into fractions enriched for full, intermediate, or empty capsids. Using a matrix of in vitro (infectivity, gene expression, biological activity) and in vivo potency assays to determine potency as a function of capsid content, our results indicate that while intermediate capsids contribute to the vector genome titer of the product and are equally as infectious as full capsids, they do not contribute to the potency of the AAV product. This study confirms the criticality of reducing and controlling the level of intermediate capsids to ensure a more efficacious AAV product.


Assuntos
Capsídeo , Dependovirus , Vetores Genéticos , Dependovirus/genética , Capsídeo/metabolismo , Vetores Genéticos/genética , Humanos , Animais , Camundongos , Transdução Genética/métodos , Células HEK293 , Terapia Genética/métodos
19.
Biomed Pharmacother ; 171: 116148, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38232661

RESUMO

Decades of biological and clinical research have led to important advances in recombinant adeno-associated viruses rAAV-based gene therapy gene therapy. However, several challenges must be overcome to fully exploit the potential of rAAV vectors. Innovative approaches to modify viral genome and capsid elements have been used to overcome issues such as unwanted immune responses and off-targeting. While often successful, genetic modification of capsids can drastically reduce vector yield and often fails to produce vectors with properties that translate across different animal species, such as rodents, non-human primates, and humans. Here, we describe a chemical bioconjugation strategy to modify tyrosine residues on AAV capsids using specific ligands, thereby circumventing the need to genetically engineer the capsid sequence. Aromatic electrophilic substitution of the phenol ring of tyrosine residues on AAV capsids improved the in vivo transduction efficiency of rAAV2 vectors in both liver and retinal targets. This tyrosine bioconjugation strategy represents an innovative technology for the engineering of rAAV vectors for human gene therapy.


Assuntos
Dependovirus , Terapia Genética , Animais , Transdução Genética , Tirosina/genética , Fígado , Retina , Proteínas do Capsídeo/genética , Vetores Genéticos , Técnicas de Transferência de Genes
20.
Curr Gene Ther ; 24(4): 265-277, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38284735

RESUMO

Gene therapy for hemophilia has advanced tremendously after thirty years of continual study and development. Advancements in medical science have facilitated attaining normal levels of Factor VIII (FVIII) or Factor IX (FIX) in individuals with haemophilia, thereby offering the potential for their complete recovery. Despite the notable advancements in various countries, there is significant scope for further enhancement in haemophilia gene therapy. Adeno-associated virus (AAV) currently serves as the primary vehicle for gene therapy in clinical trials targeting haemophilia. Subsequent investigations will prioritize enhancing viral capsid structures, transgene compositions, and promoters to achieve heightened transduction efficacy, diminished immunogenicity, and more predictable therapeutic results. The present study indicates that whereas animal models have transduction efficiency that is over 100% high, human hepatocytes are unable to express clotting factors and transduction efficiency to comparable levels. According to the current study, achieving high transduction efficiency and high levels of clotting factor expression in human hepatocytes is still insufficient. It is also crucial to reduce the risk of cellular stress caused by protein overload. Despite encountering various hurdles, the field of haemophilia gene therapy holds promise for the future. As technology continues to advance and mature, it is anticipated that a personalized therapeutic approach will be developed to cure haemophilia effectively.


Assuntos
Dependovirus , Fator IX , Terapia Genética , Vetores Genéticos , Hemofilia A , Humanos , Hemofilia A/terapia , Hemofilia A/genética , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Animais , Fator IX/genética , Fator VIII/genética , Hepatócitos/metabolismo , Transdução Genética
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