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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.
Nat Commun ; 12(1): 6239, 2021 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-34716331

RESUMO

Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some AAV serotypes are secreted in a pre-lytic manner as free or extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a frameshifted open reading frame in the AAV cap gene, is a novel viral egress factor. MAAP contains a highly conserved, cationic amphipathic domain critical for AAV secretion. Wild type or recombinant AAV with a mutated MAAP start site (MAAPΔ) show markedly attenuated secretion and correspondingly, increased intracellular retention. Trans-complementation with MAAP restored secretion of multiple AAV/MAAPΔ serotypes. Further, multiple processing and analytical methods corroborate that one plausible mechanism by which MAAP promotes viral egress is through AAV/EV association. In addition to characterizing a novel viral egress factor, we highlight a prospective engineering platform to modulate secretion of AAV vectors or other EV-associated cargo.


Assuntos
Dependovirus/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Virais/metabolismo , Liberação de Vírus , Membrana Celular/química , Dependovirus/patogenicidade , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microrganismos Geneticamente Modificados/metabolismo , Domínios Proteicos , Proteínas Virais/química , Proteínas Virais/genética
6.
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
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.
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
9.
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
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.
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
12.
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
13.
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
14.
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
15.
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
16.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361120

RESUMO

A major limiting factor for systemically delivered gene therapies is the lack of novel tissue specific AAV (Adeno-associated virus) derived vectors. Bispecific antibodies can be used to redirect AAVs to specific target receptors. Here, we demonstrate that the insertion of a short linear epitope "2E3" derived from human proprotein-convertase subtilisin/kexin type 9 (PCSK9) into different surface loops of the VP capsid proteins can be used for AAV de-targeting from its natural receptor(s), combined with a bispecific antibody-mediated retargeting. We chose to target a set of distinct disease relevant membrane proteins-fibroblast activation protein (FAP), which is upregulated on activated fibroblasts within the tumor stroma and in fibrotic tissues, as well as programmed death-ligand 1 (PD-L1), which is strongly upregulated in many cancers. Upon incubation with a bispecific antibody recognizing the 2E3 epitope and FAP or PD-L1, the bispecific antibody/rAAV complex was able to selectively transduce receptor positive cells. In summary, we developed a novel, rationally designed vector retargeting platform that can target AAVs to a new set of cellular receptors in a modular fashion. This versatile platform may serve as a valuable tool to investigate the role of disease relevant cell types and basis for novel gene therapy approaches.


Assuntos
Anticorpos Biespecíficos/imunologia , Proteínas do Capsídeo/imunologia , Capsídeo/imunologia , Dependovirus/genética , Endopeptidases/imunologia , Epitopos/imunologia , Vetores Genéticos/administração & dosagem , Proteínas de Membrana/imunologia , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/imunologia , Pró-Proteína Convertase 9/metabolismo , Transdução Genética
17.
Nat Commun ; 12(1): 4934, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400638

RESUMO

Rhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a RhoP23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo.


Assuntos
Terapia Genética , RNA/genética , Retinite Pigmentosa/genética , Retinite Pigmentosa/terapia , Animais , Dependovirus/genética , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Vetores Genéticos , Células HEK293 , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA/metabolismo , Interferência de RNA , Splicing de RNA , Retina , Degeneração Retiniana , Rodopsina/genética , Rodopsina/metabolismo
18.
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
19.
Gene ; 803: 145889, 2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34371094

RESUMO

Although seen as a revolution in modern science, gene therapy has been plagued by failed clinical trials and controversial ethics in the last thirty years. Moreover, there is no comprehensive, in-depth, high-quality analysis of global gene therapy patents. This paper proposes a method to correctly retrieve patents to address the issue and use it for the patent landscape. The results show the global patent landscape of gene therapy, with the United States dominating the field, while China has emerged as a leader in recent years. For various reasons, the EU, Korea, and Japan lag in the development of patented technologies. China has edged closer to the US in both live and indefinite patents, with the Chinese Academy of Military Medical Sciences and the Chinese Academy of Sciences leading the way, surpassing primary applicants such as the US Department of Health and Human Services, the University of California, and the University of Pennsylvania. The study also reveals four broad categories of technologies that have been extensively studied in gene therapy: basic biology of the gene and diseases, diseases being treated, gene delivery methods, and potential adverse events. What is more, Adeno-Associated Virus, Retrovirus, and Lentivirus are the most prevalent gene therapy delivery vectors after 2014. The industrial development trend revealed in this paper can provide an evidence-based basis for scientific research management and decision-making.


Assuntos
Terapia Genética , Vetores Genéticos/classificação , Patentes como Assunto , China , Dependovirus/genética , União Europeia , Humanos , Japão , Lentivirus/genética , República da Coreia , Retroviridae/genética , Estados Unidos
20.
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
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