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1.
J Pharm Biomed Anal ; 207: 114427, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34757284

RESUMO

Adeno-associated virus (AAV) represent a widely used delivery mechanism for gene therapy treatments currently being developed. The size and complexity of these molecules requires the development of sensitive analytical methods for detailed product characterization. Among the quality attributes that need to be monitored, characterization of the AAV capsid protein amino acid sequences and any associated post translational modifications (PTM) present, should be performed. As commonly used for recombinant protein analysis, LC-MS based peptide mapping can provide sequence coverage and PTM information to improve product understanding and the development and deployment of the associated manufacturing processes. In the current study, we report a fast and efficient method to digest AAV5 capsid proteins in only 30 min prior to peptide mapping analysis. The performance of different proteases in digesting AAV5 was compared and the benefits of using nanoflow liquid chromatography for separation prior to high resolution mass spectrometry to obtain 100% sequence coverage are highlighted. Characterization and quantitation of PTMs on AAV5 capsid proteins when using pepsin as a single protease is reported, thereby demonstrating the potential of this method to aid with complete characterization of AAV serotypes in gene therapy development laboratories.


Assuntos
Proteínas do Capsídeo , Dependovirus , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Cromatografia Líquida , Dependovirus/genética , Dependovirus/metabolismo , Digestão , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem
2.
BMC Genomics ; 22(1): 814, 2021 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-34763675

RESUMO

BACKGROUND: Engineered versions of adeno-associated virus (AAV) are commonly used in gene therapy but evidence revealing a potential oncogenic role of natural AAV in hepatocellular carcinoma (HCC) has raised concerns. The frequency of potentially oncogenic integrations has been reported in only a few populations. AAV infection and host genome integration in another type of liver cancer, cholangiocarcinoma (CCA), has been studied only in one cohort. All reported oncogenic AAV integrations in HCC come from strains resembling the fully sequenced AAV2 and partly sequenced AAV13. When AAV integration occurs, only a fragment of the AAV genome is detectable in later DNA or RNA sequencing. The integrated fragment is typically from the 3' end of the AAV genome, and this positional bias has been only partly explained. Three research groups searched for evidence of AAV integration in HCC RNAseq samples in the Cancer Genome Atlas (TCGA) but reported conflicting results. RESULTS: We collected and analyzed whole transcriptome and viral capture DNA sequencing in paired tumor and non-tumor samples from two liver cancer Asian cohorts from Thailand (N = 147, 47 HCC and 100 intrahepatic cholangiocarcinoma (iCCA)) and Mongolia (N = 70, all HCC). We found only one HCC patient with a potentially oncogenic integration of AAV, in contrast to higher frequency reported in European patients. There were no oncogenic AAV integrations in iCCA patients. AAV genomic segments are present preferentially in the non-tumor samples of Thai patients. By analyzing the AAV genome positions of oncogenic and non-oncogenic integrated fragments, we found that almost all the putative oncogenic integrations overlap the X gene, which is present and functional only in the strain AAV2 among all fully sequenced strains. This gene content difference could explain why putative oncogenic integrations from other AAV strains have not been reported. We resolved the discrepancies in previous analyses of AAV presence in TCGA HCC samples and extended it to CCA. There are 12 TCGA samples with an AAV segment and none are in Asian patients. AAV segments are present in preferentially in TCGA non-tumor samples, like what we observed in the Thai patients. CONCLUSIONS: Our findings suggest a minimal AAV risk of hepatocarcinogenesis in Asian liver cancer patients. The partial genome presence and positional bias of AAV integrations into the human genome has complicated analysis of possible roles of AAV in liver cancer.


Assuntos
Neoplasias dos Ductos Biliares , Carcinoma Hepatocelular , Neoplasias Hepáticas , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos , Carcinogênese , Carcinoma Hepatocelular/genética , Dependovirus/genética , Vírus da Hepatite B , Humanos , Neoplasias Hepáticas/genética , Tailândia , Integração Viral/genética
3.
Blood Adv ; 5(20): 4313, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34698767

RESUMO

The prospect of a clinical strategy using an adeno-associated virus (AAV) vector for expression of therapeutic levels of factor VIII (FVIII) has been highly desirable. This was initially anticipated by promising data from clinical studies on AAV5-FVIII in men with severe hemophilia A. However, long-term follow-up showed a unique efficacy concern on the sustainability and durability derived from a continuous decline in the FVIII transgene levels starting 1 year after vector injection through year 5. Additional follow-up of early-phase studies and outcomes of an ongoing phase 3 study will likely provide evidence on the feasibility of this approach. Here, the potential underlying mechanisms of the FVIII declining levels, together with the revision of several unique early and late onset findings, are discussed. The lack of long-term preclinical studies in large animal models prevents the firm conclusion that FVIII levels decline was unexpected. It is possible that the combination of vector manufacturing platform and dose, accompanied with ectopic expression of supraphysiologic levels of FVIII at short-term follow-up, may all contribute to the sustainability and durability of the transgene levels. Notably, vector readministration to further improve the FVIII levels is not feasible at this time. Thus, the need of a one-and-done AAV strategy to achieve sustain FVIII levels of expression is sine qua non to impact favorably the disease phenotype.


Assuntos
Dependovirus , Fator VIII , Animais , Dependovirus/genética , Fator VIII/genética , Terapia Genética , Vetores Genéticos , Humanos , Masculino , Estados Unidos , United States Food and Drug Administration
4.
Am J Sports Med ; 49(13): 3696-3707, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34643471

RESUMO

BACKGROUND: Gene transfer of the transcription factor SOX9 with clinically adapted recombinant adeno-associated virus (rAAV) vectors offers a powerful tool to durably enhance the repair process at sites of osteochondral injuries and counteract the development of perifocal osteoarthritis (OA) in the adjacent articular cartilage. PURPOSE: To examine the ability of an rAAV sox9 construct to improve the repair of focal osteochondral defects and oppose perifocal OA development over time in a large translational model relative to control gene transfer. STUDY DESIGN: Controlled laboratory study. METHODS: Standardized osteochondral defects created in the knee joints of adult sheep were treated with rAAV-FLAG-hsox9 relative to control (reporter) rAAV-lacZ gene transfer. Osteochondral repair and degenerative changes in the adjacent cartilage were monitored using macroscopic, histological, immunohistological, and biochemical evaluations after 6 months. The microarchitecture of the subchondral bone was assessed by micro-computed tomography. RESULTS: Effective, prolonged sox9 overexpression via rAAV was significantly achieved in the defects after 6 months versus rAAV-lacZ treatment. The application of rAAV-FLAG-hsox9 improved the individual parameters of defect filling, matrix staining, cellular morphology, defect architecture, surface architecture, subchondral bone, and tidemark as well as the overall score of cartilage repair in the defects compared with rAAV-lacZ. The overexpression of sox9 led to higher levels of proteoglycan production, stronger type II collagen deposition, and reduced type I collagen immunoreactivity in the sox9- versus lacZ-treated defects, together with decreased cell densities and DNA content. rAAV-FLAG-hsox9 enhanced semiquantitative histological subchondral bone repair, while the microstructure of the incompletely restored subchondral bone in the sox9 defects was not different from that in the lacZ defects. The articular cartilage adjacent to the sox9-treated defects showed reduced histological signs of perifocal OA changes versus rAAV-lacZ. CONCLUSION: rAAV-mediated sox9 gene transfer enhanced osteochondral repair in sheep after 6 months and reduced perifocal OA changes. These results underline the potential of rAAV-FLAG-hsox9 as a therapeutic tool to treat cartilage defects and afford protection against OA. CLINICAL RELEVANCE: The delivery of therapeutic rAAV sox9 to sites of focal injuries may offer a novel, convenient tool to enhance the repair of osteochondral defects involving both the articular cartilage and the underlying subchondral bone and provide a protective role by reducing the extent of perifocal OA.


Assuntos
Cartilagem Articular , Dependovirus , Animais , Cartilagem Articular/cirurgia , Dependovirus/genética , Vetores Genéticos , Modelos Animais , Ovinos , Microtomografia por Raio-X
5.
Mater Sci Eng C Mater Biol Appl ; 129: 112348, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34579876

RESUMO

The use of viral vectors for in vivo gene therapy can be severely limited by their immunogenicity. Non-viral vectors may represent an alternative, however, reports analyzing their immunogenicity are still lacking. Here, we studied the humoral immune response in a murine model triggered by artificial virus-like particles (AVLPs) carrying plasmid or antisense DNA. The AVLPs were assembled using a family of modular proteins based on bioinspired collagen-like and silk-like sequences that produce virus-like particles. We compared our AVLPs against an Adeno Associated Virus 1 (AAV), a widely used viral vector for in vivo gene delivery that has been approved by the FDA and EMA for gene therapy. We found that a 1000-fold higher mass of AVLPs than AAV are necessary to obtain similar specific antibody titters. Furthermore, we studied the stability of AVLPs against relevant biological reagents such as heparin and fetal bovine serum to ensure nucleic acid protection in biological media. Our study demonstrates that the AVLPs are stable in physiological conditions and can overcome safety limitations such as immunogenicity. The scarce humoral immunogenicity and high stability found with AVLPs suggest that they have potential to be used as stealth non-viral gene delivery systems for in vivo studies or gene therapy.


Assuntos
Dependovirus , Imunidade Humoral , Animais , Dependovirus/genética , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Camundongos
6.
Nat Commun ; 12(1): 5311, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493724

RESUMO

Although some effective therapies have been available for cancer, it still poses a great threat to human health and life due to its drug resistance and low response in patients. Here, we develop a ferroptosis-based therapy by combining iron nanoparticles and cancer-specific gene interference. The expression of two iron metabolic genes (FPN and LCN2) was selectively knocked down in cancer cells by Cas13a or microRNA controlled by a NF-κB-specific promoter. Cells were simultaneously treated by iron nanoparticles. As a result, a significant ferroptosis was induced in a wide variety of cancer cells. However, the same treatment had little effect on normal cells. By transferring genes with adeno-associated virus and iron nanoparticles, the significant tumor growth inhibition and durable cure were obtained in mice with the therapy. In this work, we thus show a cancer therapy based on gene interference-enhanced ferroptosis.


Assuntos
Proteínas de Transporte de Cátions/antagonistas & inibidores , Ferroptose/genética , Ferro/metabolismo , Lipocalina-2/antagonistas & inibidores , Neoplasias/terapia , Interferência de RNA , Espécies Reativas de Oxigênio/agonistas , Animais , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Dependovirus/genética , Dependovirus/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Lipocalina-2/genética , Lipocalina-2/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Nanopartículas/administração & dosagem , Nanopartículas/química , Neoplasias/genética , Neoplasias/mortalidade , Neoplasias/patologia , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Baço/metabolismo , Baço/patologia , Análise de Sobrevida , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Anal Chem ; 93(38): 12817-12821, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34519199

RESUMO

Adeno-associated virus (AAV)-based gene therapy is a rapidly developing field, requiring analytical methods for detailed product characterization. One important quality attribute of AAV products that requires monitoring is the amount of residual empty capsids following downstream processing. Traditionally, empty and full particles are quantified via analytical ultracentrifugation as well as anion exchange chromatography using ultraviolet or fluorescence detection. Here, we present a native mass spectrometry-based approach to assess the ratio of empty to full AAV-capsids without the need for excessive sample preparation. We report the rapid determination of the relative amount of empty capsids in AAV5 and AAV8 samples. The results correlate well with more conventional analysis strategies, demonstrating the potential of native mass spectrometry for the characterization of viral particles.


Assuntos
Capsídeo , Dependovirus , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Espectrometria de Massas
8.
J Integr Med ; 19(6): 515-525, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34538767

RESUMO

OBJECTIVE: Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells. METHODS: A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined. RESULTS: The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells. CONCLUSION: HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.


Assuntos
MicroRNAs , Tricosantina , Animais , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Células HEK293 , Humanos , Camundongos , MicroRNAs/genética
9.
J Control Release ; 338: 610-622, 2021 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-34481025

RESUMO

Ischemic stroke is still the major cause of disability worldwide. Although vascular endothelial growth factor (VEGF) is able to promote both angiogenesis and functional recovery, its use is limited by needle-induced injury, nonhomogenous VEGF distribution, and limited VEGF retention in the brain after intracranial or intravenous injection. Here, we first present a gelatin methacryloyl (GelMA) microneedle (MN)-based platform for the sustained and controlled local delivery of an adeno-associated virus (AAV) expressing human VEGF (AAV-VEGF) that achieves homogenous distribution and high transfection efficiency in ischemic brains. An ischemic stroke model was established in adult rats, and MNs loaded with AAV-VEGF were epicortically inserted into both the ischemic core and penumbra of these rats one day after the onset of ischemia. One week later, the inflammatory response and microneedle biocompatibility were assessed by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. Eight weeks later, angiogenesis and neural stem cell proliferation and migration were assessed. GelMA MN implantation did not elicit an obvious inflammatory response and had good biocompatibility in the brain. AAV-green fluorescent protein (GFP)-loaded MNs could achieve successful transfection and homogeneous distribution in the brain cortex three weeks postoperatively. MNs loaded with AAV-VEGF increased VEGF expression and enhanced functional angiogenesis and neurogenesis. In summary, MNs might emerge as a promising platform for delivering various therapeutics to treat ischemic stroke and repair other neurologically diseased tissues.


Assuntos
Isquemia Encefálica , Acidente Vascular Cerebral , Animais , Isquemia Encefálica/terapia , Dependovirus/genética , Neovascularização Fisiológica , Ratos , Recuperação de Função Fisiológica , Acidente Vascular Cerebral/terapia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
10.
Haemophilia ; 27(6): 967-973, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34553460

RESUMO

INTRODUCTION: Adeno-associated virus (AAV)-based gene therapy for haemophilia presents a challenge to the existing structure of haemophilia centres and requires a rethink of current collaboration and information exchange with the aim of ensuring a system that is fit-for-purpose for advanced therapies to maximise benefits and minimise risks. In Europe, a certification process based on the number of patients and facilities is offered to the haemophilia centres by European Haemophilia Network (EUHANET). AIM AND METHODS: This joint European Association for Haemophilia and Allied Disorders (EAHAD) and European Haemophilia Consortium (EHC) publication describes criteria for centres participating in gene therapy care that require a reassessment of the infrastructure of comprehensive care and provides an outlook on how these criteria can be implemented in the future work of haemophilia centres. RESULTS: The core definition of a haemophilia treatment centre remains, but additional roles could be implemented. A modifiable 'hub-and-spoke' model addresses all aspects associated with gene therapy, including preparation and administration of the gene therapy product, determination of coagulation and immunological parameters, joint score and function, and liver health. This will also include the strategy on how to follow-up patients for a long-term safety and efficacy surveillance. CONCLUSION: We propose a modifiable, networked 'hub and spoke' model with a long term safety and efficacy surveillance system. This approach will be progressively developed with the goal of making haemophilia centres better qualified to deliver gene therapy and to make gene therapy accessible to all persons with haemophilia, irrespective of their country or centre of origin.


Assuntos
Dependovirus , Hemofilia A , Certificação , Assistência Integral à Saúde , Dependovirus/genética , Terapia Genética , Hemofilia A/genética , Hemofilia A/terapia , Humanos
11.
Transl Vis Sci Technol ; 10(11): 15, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34520511

RESUMO

Purpose: Retinopathies display complex pathologies, including vasculopathies, inflammation, and fibrosis, leading ultimately to visual impairment. However, animal models accurately reflecting these pathologies are lacking. In this study, we evaluate the suitability of using Adeno-associated virus (AAV)-mediated long-term expression of cytokines to establish retinal pathology in the murine retina. Methods: We administered recombinant, Müller-glia targeted AAV-ShH10 into the mouse vitreous to induce retinal expression of either human vascular endothelial growth factor (VEGF)-A165, tumor necrosis factor alpha (TNF-α), or interleukin-6 (IL-6) and evaluated consequent effects by optical coherence tomography, fluorescein angiography, and histology. Results: Intravitreal injection of AAVs resulted in rapid and stable expression of the transgenes within 1 to 6 weeks. Akin to the role of VEGF-A in wet age-related macular degeneration, expression of VEGF-A led to several vasculopathies in mice, including neovascularization and vascular leakage. In contrast, the expression of the proinflammatory cytokines TNF-α or IL-6 induced retinal inflammation, as indicated by microglial activation. Furthermore, the expression of TNF-α, but not of IL-6, induced immune cell infiltration into the vitreous as well as vasculitis, and subsequently induced the development of fibrosis and epiretinal membranes. Conclusions: In summary, the long-term expression of human VEGF-A165, TNF-α, or IL-6 in the mouse eye induced specific pathologies within 6 weeks that mimic different aspects of human retinopathies. Translational Relevance: AAV-mediated expression of human genes in mice is an attractive approach to provide valuable insights into the underlying molecular mechanisms causing retinopathies and is easily adaptable to other genes and preclinical species supporting drug discovery for retinal diseases.


Assuntos
Fator de Necrose Tumoral alfa , Fator A de Crescimento do Endotélio Vascular , Animais , Dependovirus/genética , Humanos , Interleucina-6/genética , Camundongos , Retina , Fator de Necrose Tumoral alfa/genética , Fator A de Crescimento do Endotélio Vascular/genética
12.
Nat Commun ; 12(1): 5343, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504088

RESUMO

Mucopolysaccharidosis type IVA (MPSIVA) or Morquio A disease, a lysosomal storage disorder, is caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, resulting in keratan sulfate (KS) and chondroitin-6-sulfate accumulation. Patients develop severe skeletal dysplasia, early cartilage deterioration and life-threatening heart and tracheal complications. There is no cure and enzyme replacement therapy cannot correct skeletal abnormalities. Here, using CRISPR/Cas9 technology, we generate the first MPSIVA rat model recapitulating all skeletal and non-skeletal alterations experienced by patients. Treatment of MPSIVA rats with adeno-associated viral vector serotype 9 encoding Galns (AAV9-Galns) results in widespread transduction of bones, cartilage and peripheral tissues. This led to long-term (1 year) increase of GALNS activity and whole-body correction of KS levels, thus preventing body size reduction and severe alterations of bones, teeth, joints, trachea and heart. This study demonstrates the potential of AAV9-Galns gene therapy to correct the disabling MPSIVA pathology, providing strong rationale for future clinical translation to MPSIVA patients.


Assuntos
Condroitina Sulfatases/genética , Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética/métodos , Mucopolissacaridose IV/terapia , Sistema Musculoesquelético/metabolismo , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Cartilagem Articular/ultraestrutura , Condroitina Sulfatases/deficiência , Condroitina Sulfatases/metabolismo , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos/genética , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Mucopolissacaridose IV/enzimologia , Mucopolissacaridose IV/genética , Sistema Musculoesquelético/patologia , Sistema Musculoesquelético/ultraestrutura , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resultado do Tratamento
13.
Cell Host Microbe ; 29(9): 1437-1453.e8, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34428428

RESUMO

The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and can be deployed easily. Here, AAVCOVID-1, an adeno-associated viral (AAV), spike-gene-based vaccine candidate demonstrates potent immunogenicity in mouse and non-human primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques. Peak neutralizing antibody titers are sustained at 1 year and complemented by functional memory T cell responses. The AAVCOVID vector has no relevant pre-existing immunity in humans and does not elicit cross-reactivity to common AAVs used in gene therapy. Vector genome persistence and expression wanes following injection. The single low-dose requirement, high-yield manufacturability, and 1-month stability for storage at room temperature may make this technology well suited to support effective immunization campaigns for emerging pathogens on a global scale.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/genética , Dependovirus/genética , Dependovirus/metabolismo , Feminino , Humanos , Imunogenicidade da Vacina/imunologia , Memória Imunológica/imunologia , Macaca fascicularis , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Transgenes/genética , Vacinação/métodos , Carga Viral/imunologia
14.
PLoS Pathog ; 17(8): e1009758, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34379705

RESUMO

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development.


Assuntos
COVID-19 , Doenças Transmissíveis Emergentes , Modelos Animais de Doenças , Células 3T3 , Enzima de Conversão de Angiotensina 2/genética , Animais , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , COVID-19/imunologia , COVID-19/patologia , COVID-19/fisiopatologia , Chlorocebus aethiops , Dependovirus/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução Genética , Células Vero
15.
Exp Eye Res ; 210: 108728, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34390734

RESUMO

PURPOSE: Activation of bone morphogenetic protein (BMP) 4 signaling promotes the survival of retinal ganglion cell (RGC) after acute injury. Chordin-like 1 (CHRDL1) is an endogenous BMP antagonist. In this study, we researched whether CHRDL1 was involved in BMP4 signaling and regulation of RGC degeneration in a mouse model of glaucoma. METHODS: Magnetic microbeads were intracameral injected to induce experimental glaucoma in a mouse model. A recombinant adeno-associated virus (rAAV) system was designed for overexpression of BMP4 or CHRDL1 in mouse retina. Immunohistochemistry and hematoxylin-eosin (HE) stains were performed to identify changes in retinal morphology. Electroretinogram (ERG) recordings were used to assess changes in visual function. RESULTS: The mRNA expression levels of Bmp4 and its downstream BMPRIa, small mothers against decapentaplegic 1 (Smad1), were significantly upregulated in retinas with glaucoma. RGC survival was significantly enhanced in the beads + AAV-BMP4 group and significantly reduced in the beads + AAV-CHRDL1 group, compared with the beads + AAV-EGFP group. Similar results were observed in retinal explant culture in vitro. Consistent with these findings, the photopic negative response (PhNR)responses in ERG, which indicate RGC function, were restored in mice overexpressing BMP4, whereas a-wave and b-wave responses were not. Activation of CHRLD1 inhibited Smad1/5/8 phosphorylation and exacerbated RGC damage. The expression of Glial fibrillary acidic protein (GFAP) was decreased significantly in beads + AAV-BMP4 group. CONCLUSIONS: BMP4 promoted RGC survival and visual function in an experimental glaucoma model. Activation of CHRDL1 exaggerated RGC degeneration by inhibiting the BMP4/Smad1/5/8 pathway. The mechanism of BMP4/Smad1/5/8 pathway may be related to the inhibition of glial cell activation. Our studies suggested that BMP4 and CHRLD1 might serve as therapeutic targets in glaucoma.


Assuntos
Proteína Morfogenética Óssea 4/genética , Modelos Animais de Doenças , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/fisiologia , Glaucoma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Ganglionares da Retina/fisiologia , Animais , Proteína Morfogenética Óssea 4/antagonistas & inibidores , Sobrevivência Celular , Dependovirus/genética , Eletrorretinografia , Vetores Genéticos , Glaucoma/fisiopatologia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Pressão Intraocular/fisiologia , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Retina/fisiopatologia
16.
Cancer Sci ; 112(11): 4570-4579, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34459070

RESUMO

Although the inhibition of acid ceramidase (AC) is known to induce antitumor effects in various cancers, there are few reports in pancreatic cancer, and the underlying mechanisms remain unclear. Moreover, there is currently no safe administration method of AC inhibitor. Here the effects of gene therapy using siRNA and shRNA for AC inhibition with its mechanisms for pancreatic cancer were investigated. The inhibition of AC by siRNA and shRNA using an adeno-associated virus 8 (AAV8) vector had antiproliferative effects by inducing apoptosis in pancreatic cancer cells and xenograft mouse model. Acid ceramidase inhibition elicits mitochondrial dysfunction, reactive oxygen species accumulation, and manganese superoxide dismutase suppression, resulting in apoptosis of pancreatic cancer cells accompanied by ceramide accumulation. These results elucidated the mechanisms underlying the antitumor effect of AC inhibition in pancreatic cancer cells and suggest the potential of the AAV8 vector to inhibit AC as a therapeutic strategy.


Assuntos
Ceramidase Ácida/antagonistas & inibidores , Terapia Genética/métodos , Doenças Mitocondriais/etiologia , Estresse Oxidativo , Neoplasias Pancreáticas/terapia , RNA Interferente Pequeno/uso terapêutico , Ceramidase Ácida/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Ceramidas/metabolismo , Dependovirus , Vetores Genéticos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Distribuição Aleatória , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Vis Exp ; (174)2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34424236

RESUMO

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of optogenetics, however, requires that safety and efficacy be demonstrated in animal models, ideally in non-human primates (NHPs), because of their neurological similarity to humans. The number of candidate vectors that are potentially useful for neuroscience and medicine is vast, and no high-throughput means to test these vectors yet exists. Thus, there is a need for techniques to make multiple spatially and volumetrically accurate injections of viral vectors into NHP brain that can be identified unambiguously through postmortem histology. Described herein is such a method. Injection cannulas are constructed from coupled polytetrafluoroethylene and stainless-steel tubes. These cannulas are autoclavable, disposable, and have low minimal-loading volumes, making them ideal for the injection of expensive, highly concentrated viral vector solutions. An inert, red-dyed mineral oil fills the dead space and forms a visible meniscus with the vector solution, allowing instantaneous and accurate measurement of injection rates and volumes. The oil is loaded into the rear of the cannula, reducing the risk of co-injection with the vector. Cannulas can be loaded in 10 min, and injections can be made in 20 min. This procedure is well suited for injections into awake or anesthetized animals. When used to deliver high-quality viral vectors, this procedure can produce robust expression of optogenetic proteins, allowing optical control of neural activity and behavior in NHPs.


Assuntos
Optogenética , Vigília , Animais , Encéfalo , Dependovirus/genética , Vetores Genéticos/genética , Primatas
18.
Sci Rep ; 11(1): 15694, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344952

RESUMO

Respiratory syncytial virus (RSV) infection is a common cause of hospitalisation in infants and the elderly. Palivizumab prophylaxis is the only approved treatment modality but is costly and only offered to select vulnerable populations. Here, we investigated gene delivery approaches via recombinant adeno-associated virus (rAAV2/8) and simian immunodeficiency virus (rSIV.F/HN) vectors to achieve sustained in vivo production of palivizumab in a murine model. Delivery of palivizumab-expressing vectors 28 days prior to RSV challenge resulted in complete protection from RSV-induced weight loss. This approach offers prophylaxis against RSV infection, allowing for wider use and reduction in treatment costs in vulnerable populations.


Assuntos
Anticorpos Monoclonais Humanizados/genética , Expressão Gênica , Terapia Genética , Palivizumab/genética , Infecções por Vírus Respiratório Sincicial/terapia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios , Animais , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Engenharia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Injeções Intramusculares , Lentivirus/genética , Camundongos , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Transdução Genética , Resultado do Tratamento
19.
Zhonghua Xue Ye Xue Za Zhi ; 42(6): 452-458, 2021 Jun 14.
Artigo em Chinês | MEDLINE | ID: mdl-34384150

RESUMO

Objectives: To verify the effects and mechanisms of natural MSC-exosome in treating acute GVHD in mice, explore and establish a method for targeted modification of MSC-exosome, and verify the functions of the modified MSC-exosome. Methods: In different doses of MSC-exosome groups and MSC group, weight loss in acute GVHD mice was observed; then the proliferation levels of activated T cells were measured through T cell activation experiment in vitro and OVA antigen-specific T cell activation experiment in vivo. AAV2YF3 mutants carrying PD-L1 and PD-L1-ITGB1 were obtained after the construction of recombinant expression vectors and were then applied to infect human MSC to modify their exosome. The immunoregulatory functions of the modified MSC-exosome were measured with the abovementioned methods. Results: ①Mouse MSC-exosome (300 µg×3 times) and MSC (1×10(6)×3 times) effectively alleviated the weight loss in acute GVHD mice. ②Compared with IL-2, 10, 25 and 50 µg human MSC-exosome inhibited the proliferation of activated T cells in vitro, respectively, 86.0% (IL-2) , 40.0%, 39.6%, and 42.8%; compared with PBS, 50, 100 and 200 µg mouse MSC-exosome inhibited the proliferation of antigen-specific activated OT-1 cells in vivo, respectively, 42.6%, 33.1%, 14.2%, and 10.6%. ③After the infection of AAV2YF3 mutant carrying PD-L1 or PD-L1-ITGB1, the positive proportion of MSC-exosome exceeds 40% and 60%, respectively. ④Compared with the natural state, MSC-exosome modified by PD-L1 or PD-L1-ITGB1 showed better proliferation inhibitory effect in vivo and increased the proportion of Treg cells in vitro. Conclusions: MSC-exosome exhibited similar immunomodulatory effects with MSC. MSC-exosome after PD-L1 and PD-L1-ITGB1-targeted modification effectively inhibited the proliferation of activated T cells and increased the proportion of Treg cells.


Assuntos
Exossomos , Doença Enxerto-Hospedeiro , Células-Tronco Mesenquimais , Animais , Dependovirus/genética , Humanos , Camundongos , Linfócitos T Reguladores
20.
Nat Commun ; 12(1): 4722, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354059

RESUMO

Mutations in the LaminA gene are a common cause of monogenic dilated cardiomyopathy. Here we show that mice with a cardiomyocyte-specific Lmna deletion develop cardiac failure and die within 3-4 weeks after inducing the mutation. When the same Lmna mutations are induced in mice genetically deficient in the LINC complex protein SUN1, life is extended to more than one year. Disruption of SUN1's function is also accomplished by transducing and expressing a dominant-negative SUN1 miniprotein in Lmna deficient cardiomyocytes, using the cardiotrophic Adeno Associated Viral Vector 9. The SUN1 miniprotein disrupts binding between the endogenous LINC complex SUN and KASH domains, displacing the cardiomyocyte KASH complexes from the nuclear periphery, resulting in at least a fivefold extension in lifespan. Cardiomyocyte-specific expression of the SUN1 miniprotein prevents cardiomyopathy progression, potentially avoiding the necessity of developing a specific therapeutic tailored to treating each different LMNA cardiomyopathy-inducing mutation of which there are more than 450.


Assuntos
Cardiomiopatia Dilatada/genética , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Dependovirus/genética , Feminino , Humanos , Lamina Tipo A/deficiência , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução Genética
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