RESUMO
Adeno-associated viruses 2 (AAV2) are minute viruses renowned for their capacity to infect human cells and akin organisms. They have recently emerged as prominent candidates in the field of gene therapy, primarily attributed to their inherent non-pathogenic nature in humans and the safety associated with their manipulation. The efficacy of AAV2 as gene therapy vectors hinges on their ability to infiltrate host cells, a phenomenon reliant on their competence to construct a capsid capable of breaching the nucleus of the target cell. To enhance their infection potential, researchers have extensively scrutinized various combinatorial libraries by introducing mutations into the capsid, aiming to boost their effectiveness. The emergence of high-throughput experimental techniques, like deep mutational scanning (DMS), has made it feasible to experimentally assess the fitness of these libraries for their intended purpose. Notably, machine learning is starting to demonstrate its potential in addressing predictions within the mutational landscape from sequence data. In this context, we introduce a biophysically-inspired model designed to predict the viability of genetic variants in DMS experiments. This model is tailored to a specific segment of the CAP region within AAV2's capsid protein. To evaluate its effectiveness, we conduct model training with diverse datasets, each tailored to explore different aspects of the mutational landscape influenced by the selection process. Our assessment of the biophysical model centers on two primary objectives: (i) providing quantitative forecasts for the log-selectivity of variants and (ii) deploying it as a binary classifier to categorize sequences into viable and non-viable classes.
Assuntos
Mutação , Humanos , Proteínas do Capsídeo/genética , Dependovirus/genética , Parvovirinae/genéticaRESUMO
We determined the transcription profile of adeno-associated virus type 2 (AAV2)-infected primary human fibroblasts. Subsequent analysis revealed that cells respond to AAV infection through changes in several significantly affected pathways, including cell cycle regulation, chromatin modulation, and innate immune responses. Various assays were performed to validate selected differentially expressed genes and to confirm not only the quality but also the robustness of the raw data. One of the genes upregulated in AAV2-infected cells was interferon-γ inducible factor 16 (IFI16). IFI16 is known as a multifunctional cytosolic and nuclear innate immune sensor for double-stranded as well as single-stranded DNA, exerting its effects through various mechanisms, such as interferon response, epigenetic modifications, or transcriptional regulation. IFI16 thereby constitutes a restriction factor for many different viruses among them, as shown here, AAV2 and thereof derived vectors. Indeed, the post-transcriptional silencing of IFI16 significantly increased AAV2 transduction efficiency, independent of the structure of the virus/vector genome. We also show that IFI16 exerts its inhibitory effect on AAV2 transduction in an immune-modulatory independent way by interfering with Sp1-dependent transactivation of wild-type AAV2 and AAV2 vector promoters. IMPORTANCE: Adeno-associated virus (AAV) vectors are among the most frequently used viral vectors for gene therapy. The lack of pathogenicity of the parental virus, the long-term persistence as episomes in non-proliferating cells, and the availability of a variety of AAV serotypes differing in their cellular tropism are advantageous features of this biological nanoparticle. To deepen our understanding of virus-host interactions, especially in terms of antiviral responses, we present here the first transcriptome analysis of AAV serotype 2 (AAV2)-infected human primary fibroblasts. Our findings indicate that interferon-γ inducible factor 16 acts as an antiviral factor in AAV2 infection and AAV2 vector-mediated cell transduction in an immune-modulatory independent way by interrupting the Sp1-dependent gene expression from viral or vector genomes.
Assuntos
Dependovirus , Fibroblastos , Proteínas Nucleares , Fosfoproteínas , Transdução Genética , Humanos , Dependovirus/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fibroblastos/virologia , Fibroblastos/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Imunidade Inata , Vetores Genéticos/genética , Parvovirinae/genética , Células CultivadasRESUMO
Recombinant Muscovy duck parvovirus (rMDPV) is a product of genetic recombination between classical Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV). The recombination event took place within a 1.1-kb DNA segment located in the middle of the VP3 gene, and a 187-bp sequence extending from the P9 promoter to the 5' initiation region of the Rep1 ORF. This resulted in the alteration of five amino acids within VP3. Despite these genetic changes, the precise influence of recombination and amino acid mutations on the pathogenicity of rMDPV remains ambiguous. In this study, based on the rMDPV strain ZW and the classical MDPV strain YY, three chimeric viruses (rZW-mP9, rZW-mPR187, and rYY-rVP3) and the five amino acid mutations-introduced mutants (rZW-g5aa and rYY-5aa(ZW)) were generated using reverse genetic technology. When compared to the parental virus rZW, rZW-g5aa exhibited a prolonged mean death time (MDT) and a decreased median lethal dose (ELD50) in embryonated duck eggs. In contrast, rYY-5aa(ZW) did not display significant differences in MDT and ELD50 compared to rYY. In 2-day-old Muscovy ducklings, infection with rZW-g5aa and rYY-5aa(ZW) resulted in mortality rates of only 20% and 10%, respectively, while infections with the three chimeric viruses (rZW-mP9, rZW-mPR187, rYY-rVP3) and rZW still led to 100% mortality. Notably, rYY-rVP3, containing the VP3 region from strain ZW, exhibited 50% mortality in 6-day-old Muscovy ducklings and demonstrated significant horizontal transmission. Collectively, our findings indicate that recombination and consequent amino acid changes in VP3 have a synergistic impact on the heightened virulence of rMDPV in Muscovy ducklings.
Assuntos
Proteínas do Capsídeo , Patos , Infecções por Parvoviridae , Mutação Puntual , Doenças das Aves Domésticas , Recombinação Genética , Animais , Virulência , Infecções por Parvoviridae/virologia , Infecções por Parvoviridae/veterinária , Doenças das Aves Domésticas/virologia , Proteínas do Capsídeo/genética , Parvovirinae/genética , Parvovirinae/patogenicidadeRESUMO
Tetraparvovirus is an emerging parvovirus infecting a variety of mammals and humans, and associated with human diseases including severe acute respiratory infection and acute encephalitis syndrome. In the present study, a Tetraparvovirus ungulate 1 (formerly known as bovine hokovirus) strain HNU-CBY-2023 was identified and characterized from diseased Chinese Simmental from Hunan province, China. The nearly complete genome of HNU-CBY-2023 is 5346 nt in size and showed genomic identities of 85-95.5% to the known Tetraparvovirus ungulate 1 strains from GenBank, indicating a rather genetic variation. Phylogenetic and genetic divergence analyses indicated that Tetraparvovirus ungulate 1 could be divided into two genotypes (I and II), and HNU-CBY-2023 was clustered into genotype II. This study, for the first time, identified Tetraparvovirus ungulate 1 from domestic cattle from mainland China, which will be helpful to understand the prevalence and genetic diversity of Tetraparvovirus ungulate 1.
Assuntos
Doenças dos Bovinos , Variação Genética , Genoma Viral , Genótipo , Infecções por Parvoviridae , Filogenia , Animais , Bovinos , China , Doenças dos Bovinos/virologia , Doenças dos Bovinos/epidemiologia , Infecções por Parvoviridae/veterinária , Infecções por Parvoviridae/virologia , Infecções por Parvoviridae/epidemiologia , Genoma Viral/genética , Parvovirinae/genética , Parvovirinae/isolamento & purificação , Parvovirinae/classificação , Análise de Sequência de DNA , DNA Viral/genética , População do Leste AsiáticoRESUMO
Adeno-associated Virus (AAV) is a promising vector for gene therapy. However, few studies have focused on producing virus-like particles (VLPs) of AAV in cells, especially in E. coli. In this study, we describe a method to produce empty VP3-only VLPs of AAV2 in E. coli by co-expressing VP3 and assembly-activating protein (AAP) of AAV2. Although the yields of VLPs produced with our method were low, the VLPs were able to self-assemble in E. coli without the need of in vitro capsid assembly. The produced VLPs were characterized by immunological detection and transmission electron microscopy (TEM). In conclusion, this study demonstrated that capsid assembly of AAV2 is possible in E. coli, and E. coli may be a candidate system for production of VLPs of AAV.
Assuntos
Proteínas do Capsídeo , Dependovirus , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Dependovirus/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/biossíntese , Vírion/genética , Vírion/metabolismo , Montagem de Vírus , Vetores Genéticos/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/química , Parvovirinae/genética , HumanosRESUMO
Short-beak and dwarf syndrome (SBDS) is caused by infection with novel goose parvovirus (NGPV), which leads to intestinal dysbiosis, developmental delay, short beak, lameness, and paralysis in ducks and is the cause of skeletal health problems. NGPV infection can cause intestinal microbial disturbances, but it is still unclear whether the intestinal microbiota affects the pathogenicity of NGPV. Here, the effects of intestinal microbiota on NGPV-induced SBDS in Cherry Valley ducks were assessed by establishing a duck model for gut microflora depletion/reestablishment through antibiotics (ABX) treatment/fecal microbiota transplanted (FMT). By measuring body weight, beak length, beak width and tarsal length, we found that SBDS clinical symptoms were alleviated in ducks treated with ABX, but not in FMT ducks. Next, we conducted a comprehensive analysis of bone metabolism, gut barrier integrity, and inflammation levels using quantitative real-time PCR (qPCR), enzyme linked immunosorbent assay (ELISA), biochemical analysis and histological analysis. The results showed that ABX treatment improved bone quality reduced bone resorption, mitigated tissue lesions, protected intestinal barrier integrity, and inhibited systemic inflammation in NGPV-infected ducks. Moreover, cecal microflora composition and short-chain fatty acids (SCFAs) production were examined by bacterial 16S rRNA sequencing and gas chromatography. The results revealed that ABX treatment mitigated the decreased abundance of Firmicutes and Bacteroidota in NGPV-infected ducks, as well as increased SCFAs production. Furthermore, ABX treatment reduced the mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) and nuclear factor κB (NF-κB) expression, which are correlated with systemic inflammation in SBDS ducks. These findings suggested that intestinal microflora depletion alleviated NGPV-induced SBDS by maintaining intestinal homeostasis, inhibiting inflammatory response and alleviating bone resorption. These results provide evidence for the pivotal role of intestinal microbiota in the process of SBDS and contribute a theoretical basis for the feasibility of microecological preparation as a method to control SBDS.
Assuntos
Patos , Microbioma Gastrointestinal , Infecções por Parvoviridae , Parvovirinae , Doenças das Aves Domésticas , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/microbiologia , Infecções por Parvoviridae/veterinária , Infecções por Parvoviridae/virologia , Parvovirinae/genética , Parvovirinae/fisiologia , Antibacterianos/farmacologia , Antibacterianos/administração & dosagem , Transplante de Microbiota Fecal/veterináriaRESUMO
The recombinant adeno-associated virus (rAAV) vectors used in gene therapy are usually produced by transfecting three different plasmids (Adenoviral helper plasmid (pHelper), AAV rep/cap plasmids (pRepCap), and Transgene plasmid (pAAV-GOI)) into human embryonic kidney 293 (HEK293) cells. However, the high proportion of unwanted empty capsids generated during rAAV production is problematic. To simultaneously enhance the genome titer and full capsid ratio, the ratio of the three plasmids transfected into HEK293 cells was optimized using design-of-experiment (DoE). AAV2 and AAV9, which have different production kinetics, were selected as cell-associated and secreted model AAVs, respectively. In 125 mL Erlenmeyer flasks, the genome titers of rAAV2 and rAAV9 at DoE-optimized plasmid weight ratios (pHelper:pRep2Cap2:pAAV-GOI = 1:3.52:0.50 for rAAV2 and pHelper:pRep2Cap9:pAAV-GOI = 1:1.44:0.27 for rAAV9) were 2.23-fold and 2.26-fold higher than those in the widely used plasmid weight ratio (1:1:1), respectively. In addition, compared with the plasmid ratio of 1:1:1, the relative VP3 band intensities of rAAV2 and rAAV9, which represent the relative empty capsid ratios, were reduced by 26% and 25%, respectively, at the DoE-optimized plasmid ratio. Reduced empty capsid ratios in the DoE-optimized plasmid ratios were also confirmed using transmission electron microscopy (TEM). Taken together, regardless of the AAV serotype, DoE-aided optimization of the triple plasmid ratio was found to be an efficient means of improving the production of rAAV with a high full capsid ratio.
Assuntos
Capsídeo , Parvovirinae , Humanos , Células HEK293 , Vetores Genéticos/genética , Dependovirus/genética , Plasmídeos/genética , Proteínas do Capsídeo/genética , Parvovirinae/genéticaRESUMO
Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show that when injected intravitreally, AAV2 engineered with a reporter gene driven by cytomegalovirus (CMV) enhancer and chicken ß-actin (CBA) promoters, displays ubiquitous and high RGC expression, similar to its synthetic derivative AAV8BP2. A novel AAV2 vector combining the promoter of the human RGC-selective γ-synuclein (hSNCG) gene and woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted upstream and downstream of a reporter gene, respectively, induces widespread transduction and strong transgene expression in RGCs. High transduction efficiency and selectivity to RGCs is further achieved by incorporating in the vector backbone a leading CMV enhancer and an SV40 intron at the 5' and 3' ends, respectively, of the reporter gene. As a delivery vehicle of hSIRT1, a 2.2-kb therapeutic gene with anti-apoptotic, anti-inflammatory and anti-oxidative stress properties, this recombinant vector displayed improved transduction efficiency, a strong, widespread and selective RGC expression of hSIRT1, and increased RGC survival following optic nerve crush. Thus, AAV2 vector carrying hSNCG promoter with additional regulatory sequences may offer strong potential for enhanced effects of candidate gene therapies targeting RGCs.
Assuntos
Infecções por Citomegalovirus , Parvovirinae , Humanos , Células Ganglionares da Retina/metabolismo , Terapia Genética , Transgenes , Nervo Óptico , Dependovirus/genética , Parvovirinae/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Vetores Genéticos/genéticaRESUMO
Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector to manipulate activity of neurons and their circuitry in the primate brain. In the present study, we created a novel vector of which capsid was composed of capsid proteins derived from both of the AAV serotypes 1 and 2 (AAV1 and AAV2). Following the injection into the frontal cortex of macaque monkeys, this mosaic vector, termed AAV2.1 vector, was found to exhibit the excellence in transgene expression (for AAV1 vector) and neuron specificity (for AAV2 vector) simultaneously. To explore its applicability to chemogenetic manipulation and in vivo calcium imaging, the AAV2.1 vector expressing excitatory DREADDs or GCaMP was injected into the striatum or the visual cortex of macaque monkeys, respectively. Our results have defined that such vectors secure intense and stable expression of the target proteins and yield conspicuous modulation and imaging of neuronal activity.
Assuntos
Dependovirus , Parvovirinae , Animais , Dependovirus/metabolismo , Transdução Genética , Vetores Genéticos/genética , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Transgenes , Primatas/genética , Parvovirinae/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Neurônios/metabolismoRESUMO
Adeno-associated virus serotype 2 (AAV2) is a viral vector that can be used to deliver therapeutic genes to diseased cells in the retina. One strategy for altering AAV2 vectors involves the mutation of phosphodegron residues, which are thought to be phosphorylated/ubiquitinated in the cytosol, facilitating degradation of the vector and the inhibition of transduction. As such, mutation of phosphodegron residues have been correlated with increased transduction of target cells, however, an assessment of the immunobiology of wild-type and phosphodegron mutant AAV2 vectors following intravitreal (IVT) delivery to immunocompetent animals is lacking in the current literature. In this study, we show that IVT of a triple phosphodegron mutant AAV2 capsid is associated with higher levels of humoral immune activation, infiltration of CD4 and CD8 T-cells into the retina, generation of splenic germinal centre reactions, activation of conventional dendritic cell subsets, and elevated retinal gliosis compared to wild-type AAV2 capsids. However, we did not detect significant changes in electroretinography arising after vector administration. We also demonstrate that the triple AAV2 mutant capsid is less susceptible to neutralisation by soluble heparan sulphate and anti-AAV2 neutralising antibodies, highlighting a possible utility for the vector in terms of circumventing pre-existing humoral immunity. In summary, the present study highlights novel aspects of rationally-designed vector immunobiology, which may be relevant to their application in preclinical and clinical settings.
Assuntos
Capsídeo , Parvovirinae , Camundongos , Animais , Capsídeo/metabolismo , Sorogrupo , Transdução Genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Parvovirinae/genética , Dependovirus/metabolismo , Vetores Genéticos/genéticaRESUMO
Adeno-associated virus (AAV) is a nonenveloped single-stranded DNA (ssDNA) icosahedral T=1 virus being developed as a vector for clinical gene delivery systems. Currently, there are approximately 160 AAV clinical trials, with AAV2 being the most widely studied serotype. To further understand the AAV gene delivery system, this study investigates the role of viral protein (VP) symmetry interactions on capsid assembly, genome packaging, stability, and infectivity. A total of 25 (seven 2-fold, nine 3-fold, and nine 5-fold symmetry interface) AAV2 VP variants were studied. Six 2-fold and two 5-fold variants did not assemble capsids based on native immunoblots and anti-AAV2 enzyme-linked immunosorbent assays (ELISAs). Seven of the 3-fold and seven of the 5-fold variants that assembled capsids were less stable, while the only 2-fold variant that assembled had ~2°C higher thermal stability (Tm) than recombinant wild-type AAV2 (wtAAV2). Three of the 3-fold variants (AAV2-R432A, AAV2-L510A, and N511R) had an approximately 3-log defect in genome packaging. Consistent with previous reports of the 5-fold axes, the region of the capsid is important for VP1u externalization and genome ejection, and one 5-fold variant (R404A) had a significant defect in viral infectivity. The structures of wtAAV2 packaged with a transgene (AAV2-full) and without a transgene (AAV2-empty) and one 5-fold variant (AAV2-R404A) were determined by cryo-electron microscopy and three dimensional (3D)-image reconstruction to 2.8, 2.9, and 3.6 Å resolution, respectively. These structures revealed the role of stabilizing interactions on the assembly, stability, packaging, and infectivity of the virus capsid. This study provides insight into the structural characterization and functional implications of the rational design of AAV vectors. IMPORTANCE Adeno-associated viruses (AAVs) have been shown to be useful vectors for gene therapy applications. Consequently, AAV has been approved as a biologic for the treatment of several monogenic disorders, and many additional clinical trials are ongoing. These successes have generated significant interest in all aspects of the basic biology of AAV. However, to date, there are limited data available on the importance of the capsid viral protein (VP) symmetry-related interactions required to assemble and maintain the stability of the AAV capsids and the infectivity of the AAV capsids. Characterizing the residue type and interactions at these symmetry-driven assembly interfaces of AAV2 has provided the foundation for understanding their role in AAV vectors (serotypes and engineered chimeras) and has determined the residues or regions of the capsid that can or cannot tolerate alterations.
Assuntos
Capsídeo , Parvovirinae , Capsídeo/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Sorogrupo , Microscopia Crioeletrônica , Proteínas do Capsídeo/metabolismo , Parvovirinae/genética , Parvovirinae/metabolismo , Proteínas Virais/metabolismo , Vetores Genéticos , Montagem de VírusRESUMO
Birds carry a large number of viruses that may cause diseases in animals or humans. At present, information about the virome of zoo birds is limited. In this study, using viral metagenomics, we investigated the fecal virome of zoo birds collected from a zoo in Nanjing, Jiangsu Province, China. Three novel parvoviruses were obtained and characterized. The genomes of the three viruses are 5,909, 4,411, and 4,233 nt in length, respectively, and contain four or five ORFs. Phylogenetic analysis showed that these three novel parvoviruses clustered with other strains and formed three different clades. Pairwise comparison of NS1 amino acid sequences showed that Bir-01-1 shared 44.30-74.92% aa sequence identity with other parvoviruses belonging to the genus Aveparvovirus, while Bir-03-1 and Bir-04-1 shared less than 66.87% and 53.09% aa sequence identity, respectively, with other parvoviruses belonging to the genus Chaphamaparvovirus. Each of these three viruses was identified as a member of a novel species based on the species demarcation criteria for parvoviruses. These findings broaden our knowledge of the genetic diversity of parvoviruses and provide epidemiological data regarding potential outbreaks of parvovirus disease in birds.
Assuntos
Infecções por Parvoviridae , Parvovirinae , Parvovirus , Vírus , Animais , Humanos , Filogenia , Parvovirus/genética , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/veterinária , Aves , Parvovirinae/genéticaRESUMO
Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken ß-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken ß-actin/short ß-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.
Assuntos
Infecções por Citomegalovirus , Parvovirinae , Camundongos , Animais , Humanos , Células Ganglionares da Retina/metabolismo , Actinas/genética , Actinas/metabolismo , Transdução Genética , Camundongos Endogâmicos C57BL , Transgenes , Dependovirus/genética , Dependovirus/metabolismo , Parvovirinae/genética , Proteínas de Fluorescência Verde/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Vetores Genéticos/genéticaRESUMO
PURPOSE: To evaluate the safety profile of lenadogene nolparvovec (Lumevoq) in patients with Leber hereditary optic neuropathy. DESIGN: Pooled analysis of safety data from 5 clinical studies. METHODS: A total of 189 patients received single unilateral or bilateral intravitreal injections of a recombinant adeno-associated virus 2 (rAAV2/2) vector encoding the human wild-type ND4 gene. Adverse events (AEs) were collected throughout the studies, up to 5 years. Intraocular inflammation and increased intraocular pressure (IOP) were ocular AEs of special interest. Other assessments included ocular examinations, vector bio-dissemination, and systemic immune responses against rAAV2/2. RESULTS: Almost all patients (95.2%) received 9 × 1010 viral genomes and 87.8% had at least 2 years of follow-up. Most patients (75.1%) experienced at least one systemic AE, but systemic treatment-related AEs occurred in 3 patients; none were serious. Intraocular inflammation was reported in 75.6% of lenadogene nolparvovec-treated eyes. Almost all intraocular inflammations occurred in the anterior chamber (58.8%) or in the vitreous (40.3%), and were of mild (90.3%) or moderate (8.8%) intensity; most resolved with topical corticosteroids alone. All IOP increases were mild to moderate in intensity. No AE led to study discontinuation. Bio-dissemination of lenadogene nolparvovec and systemic immune response were limited. The safety profile was comparable for patients treated bilaterally and unilaterally. CONCLUSIONS: Lenadogene nolparvovec had a good overall safety profile with excellent systemic tolerability, consistent with limited bio-dissemination. The product was well tolerated, with mostly mild ocular side effects responsive to conventional ophthalmologic treatments.
Assuntos
Atrofia Óptica Hereditária de Leber , Parvovirinae , Humanos , Atrofia Óptica Hereditária de Leber/tratamento farmacológico , Atrofia Óptica Hereditária de Leber/genética , Vetores Genéticos , Parvovirinae/genética , Terapia Genética , Inflamação/etiologiaRESUMO
Waterfowl parvovirus (WPFs) has multiple effects on the intestinal tract, but the effects of recombinant Muscovy duck parvovirus (rMDPV) have not been elucidated. In this study, 48 one-day-old Muscovy ducklings were divided into an infected group and a control group. Plasma and ileal samples were collected from both groups at 2, 4, 6, and 8 days post-infection (dpi), both six ducklings at a time. Next, we analyzed the genomic sequence of the rMDPV strain. Results showed that the ileal villus structure was destroyed seriously at 4, 6, 8 dpi, and the expression of ZO-1, Occludin, and Claudin-1 decreased at 4, 6 dpi; 4, 6, 8 dpi; and 2, 6 dpi, respectively. Intestinal cytokines IFN-α, IL-1ß and IL-6 increased at 6 dpi; 8 dpi; and 6, 8 dpi, respectively, whereas IL-2 decreased at 6, 8 dpi. The diversity of ileal flora increased significantly at 4 dpi and decreased at 8 dpi. The bacteria Ochrobactrum and Enterococcus increased and decreased at 4, 8 dpi; 2, 4 dpi, respectively. Plasma MDA increased at 2 dpi, SOD, CAT, and T-AOC decreased at 2, 4, 8 dpi; 4, 8 dpi; and 4, 6, 8 dpi, respectively. These results suggest that rMDPV infection led to early intestinal barrier dysfunction, inflammation, ileac microbiota disruption, and oxidative stress.
Assuntos
Infecções por Parvoviridae , Parvovirinae , Parvovirus , Doenças das Aves Domésticas , Animais , Patos , Infecções por Parvoviridae/veterinária , Parvovirinae/genética , Parvovirus/genéticaRESUMO
Choroidal neovascularization (CNV) is a defining characteristic feature of neovascular age-related macular degeneration (nAMD) that frequently results in irreversible vision loss. The current strategies for the treatment of nAMD are mainly based on neutralizing vascular endothelial growth factor (VEGF). However, anti-VEGF therapies are often associated with subretinal fibrosis that eventually leads to damages in macula. In this study, we tested whether an anti-fibrotic and anti-angiogenic protein CCN5 can potentially be an effective and safe therapeutic modality in a mouse model of CNV. Laser photocoagulation was utilized to induce CNV, which was followed by intravitreal injection of recombinant adeno-associated virus serotype 2 encoding CCN5 (rAAV2-CCN5). Our data demonstrated that rAAV2-CCN5, but not a control viral vector, rAAV2-VLP, prominently attenuated both CNV lesions and angiogenesis. Aflibercept, which was utilized as a positive control, exhibited similar effects on CNV lesions and angiogenesis in our experimental settings. Upon laser photocoagulation, retinal pigmented epithelium (RPE) cells underwent significant morphological changes including cellular enlargement and loss of hexagonality. rAAV2-CCN5 significantly normalized these morphological defects. Laser photocoagulation also led to fibrotic deformation in RPE cells through inducing epithelial-mesenchymal transition (EMT), which was completely blocked by rAAV2-CCN5. In a striking contrast, aflibercept as well as rAAV2-VLP failed to exhibit any effects on EMT. Collectively, this study suggest that CCN5 might provide a potential novel strategy for the treatment of nAMD with a capability to inhibit CNV and fibrosis simaultaneously.
Assuntos
Neovascularização de Coroide , Parvovirinae , Animais , Neovascularização de Coroide/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Epitélio/metabolismo , Fibrose , Camundongos , Camundongos Endogâmicos C57BL , Parvovirinae/genética , Fator A de Crescimento do Endotélio VascularRESUMO
Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near-universal entry receptor, AAVR, and structures detected by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now ß-strand A is the minor conformer and, for the major conformer, partially ordered N termini near the 2-fold axis join the canonical capsid jellyroll fold at the ßA-ßB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCE With 150 clinical trials for 30 diseases under way, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.
Assuntos
Dependovirus , Parvovirinae , Dependovirus/metabolismo , Tomografia com Microscopia Eletrônica , Parvovirinae/química , Parvovirinae/genética , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
Since the outbreak of short beak and dwarfish syndrome (SBDS) in Cherry Valley Pekin ducks in China, novel goose parvovirus (NGPV) has been isolated. Till now, little is known about the NGPV pathogenesis toward Cherry Valley Pekin ducks. Besides, due to detection of duck circovirus co-infection in SBDS clinical cases, whether sole NGPV infection can reproduce all the typical symptoms of SBDS remains unclear. In this study, based on the NGPV isolate SDJN19, an infectious plasmid clone pJNm containing the entire SDJN19 genome was constructed. Transfection of pJNm in embryonated duck eggs resulted in generation of the infectious virus carrying the genetic marker, named rJNm. rJNm infection of 2-day-old Cherry Valley Pekin ducks reproduced all the typical signs of SBDS, including beak atrophy, tongue protrusion, and growth retardation. rJNm can infect Cherry Valley Pekin ducks through the horizontal transmission route, and the infected ducks exhibited the characteristic SBDS symptoms. A high level of serum precipitation antibodies (above 5log2) were induced in the surviving ducks, however, high viral loads were still detected in the duck organs, suggesting persistent NGPV infection in ducks. By incorporating the homologous Rep1 and VP1 gene from classical GPV, two chimeric viruses rJN-cVP1 and rJN-cRep1 were generated. Duck infection tests revealed that the non-structural protein Rep1 played a crucial role in the NGPV pathogenicity. The present result lays a solid foundation for further exploring how the Rep protein contributes to the NGPV pathogenesis.
Assuntos
Infecções por Parvoviridae , Parvovirinae , Doenças das Aves Domésticas , Animais , Bico/patologia , Infecções por Parvoviridae/veterinária , Parvovirinae/genética , ReproduçãoRESUMO
Intravenous (IV) administration of naturally occurring adeno-associated virus (AAV) vectors are liver tropic, with a significant proportion of the total vector dose mediating gene expression in liver hepatocytes. AAV capsids that are directed toward other organs such as lung may be useful for therapy of nonliver-based diseases. Based on the knowledge that the lung capillary endothelium is the first capillary bed encountered by an intravenously administered AAV vector, and that the lung endothelium glycocalyx is enriched in negatively charged sialic acid, we hypothesized that adding positively changed lysine residues to the AAV capsid would enhance AAV biodistribution to the lung after IV administration. Using site-directed mutagenesis, two lysine residues were inserted into variable loop VIII of the AAV serotype 5 capsid (AAV5-PK2). Organ distribution of AAV5-PK2 was compared with that of AAV5, AAV2, and AAV2-7m8 4 weeks after IV administration (1011 gc) to C57Bl/6 male mice. As predicted, after IV administration, AAV5-PK2 had the highest biodistribution in the lung (p < 0.02 compared with AAV5, AAV2, and AAV2-7m8). Furthermore, biodistribution to liver of AAV5-PK2 was 2 logs decreased compared with AAV5 (p < 10-4) with a ratio of AAV5-PK2 lung to liver of 62-fold compared with AAV5 of 0.2-fold (p < 0.0003). The AAV5-PK2 capsid represents a lung-tropic AAV vector that is also significantly detargeted from the liver, a property that may be useful in lung-directed gene therapies.
Assuntos
Capsídeo , Parvovirinae , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Feminino , Vetores Genéticos/genética , Fígado/metabolismo , Pulmão/metabolismo , Lisina/análise , Lisina/genética , Lisina/metabolismo , Masculino , Camundongos , Parvovirinae/genética , Distribuição Tecidual , Transdução GenéticaRESUMO
Dietary, environmental, and hereditary causes were reported as causative agents of angel wing syndrome in waterfowl. Since 2017, several Muscovy duck flocks at Behira governorate were found to exhibit this syndrome associated with the clinical symptoms of goose parvovirus (GPV) infection. Four strains of goose parvovirus named HS1-HS4 were isolated and identified from diseased ducks at some of these flocks. Phylogenetic analysis revealed clustering of these strains together and within a distinct monophyletic group in relation to GPV strains of Derzsy's disease and short beak and dwarfism syndrome (SBDS). Nucleotide identities with goose parvovirus strain B of Derzsy's disease were 95.7%-96.6%, and with the strain JS1603 of SBDS they were 96.8%-97.4%. However, nucleotide identities with Muscovy duck parvovirus strain FM were 74.1%-74.6%. The disease was reproduced experimentally via oral-route artificial infection with HS1 strain, and both clinical symptoms of goose parvovirus and angel wing syndrome were observed in the artificially infected Muscovy ducks, but with less severity in geese. This study demonstrated clear evidence for induction of angel wing syndrome, at least partially, with GPV infection in Muscovy duck. To the authors' knowledge, this is the first work to mention a viral cause of angel wing syndrome in waterfowl.
Participación del parvovirus del ganso en la inducción del síndrome de ala de ángel en patos reales. Se han reportado causas dietéticas, ambientales y hereditarias como agentes causales del síndrome de alas de ángel en aves acuáticas. Desde el año 2017, se descubrió que varias parvadas de patos reales en la gobernación de Behira presentaban este síndrome asociado con los síntomas clínicos de la infección por parvovirus del ganso (con las siglas en inglés GPV). Se aislaron e identificaron cuatro cepas de parvovirus de ganso denominadas HS1HS4 de patos enfermos en algunas de estas parvadas. El análisis filogenético reveló el agrupamiento de estas cepas juntas y dentro de un grupo monofilético distinto con relación con las cepas de parvovirus de ganso asociadas con la enfermedad de Derzsy y el síndrome de pico corto y enanismo (con las siglas en inglés SBDS). Las identidades de nucleótidos con la cepa B del parvovirus de la enfermedad de Derzsy fueron del 95.7 % al 96.6 %, y con la cepa JS1603 del síndrome de pico corto y enanismo fueron del 96.8 % al 97.4 %. Sin embargo, las identidades de nucleótidos con la cepa FM del parvovirus del pato Muscovy fueron del 74.1 % al 74.6 %. La enfermedad se reprodujo experimentalmente a través de una infección artificial por vía oral con la cepa HS1 y se observaron signos clínicos de parvovirus del ganso y del síndrome del ala de ángel en los patos reales infectados artificialmente, pero con menor gravedad en los gansos. Este estudio demostró una clara evidencia de la inducción del síndrome del ala de ángel, al menos parcialmente, con la infección por parvovirus del ganso en el pato real. Según el conocimiento de los autores, este es el primer trabajo que menciona una causa viral del síndrome del ala de ángel en aves acuáticas.