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1.
Int J Mol Sci ; 24(9)2023 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-37175977

RESUMO

CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.


Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Sistemas CRISPR-Cas/genética , Edição de Genes , Reparo do DNA por Junção de Extremidades , Genoma
2.
J Immunol ; 204(8): 2169-2176, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32161099

RESUMO

Currently 247 million people are living with chronic hepatitis B virus infection (CHB), and the development of novel curative treatments is urgently needed. Immunotherapy is an attractive approach to treat CHB, yet therapeutic approaches to augment the endogenous hepatitis B virus (HBV)-specific T cell response in CHB patients have demonstrated little success. In this study, we show that strain 68-1 rhesus macaque (RM) CMV vaccine vectors expressing HBV Ags engender HBV-specific CD8+ T cells unconventionally restricted by MHC class II and the nonclassical MHC-E molecule in RM. Surface staining of human donor and RM primary hepatocytes (PH) ex vivo revealed the majority of PH expressed MHC-E but not MHC class II. HBV-specific, MHC-E-restricted CD8+ T cells from RM vaccinated with RM CMV vaccine vectors expressing HBV Ags recognized HBV-infected PH from both human donor and RM. These results provide proof-of-concept that MHC-E-restricted CD8+ T cells could be harnessed for the treatment of CHB, either through therapeutic vaccination or adoptive immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Hepatócitos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Animais , Hepatite B Crônica/virologia , Hepatócitos/virologia , Macaca mulatta
3.
Viruses ; 16(2)2024 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-38400071

RESUMO

Metabolic-dysfunction-associated fatty liver disease (MAFLD) is a major cause of morbidity and mortality in HIV-infected individuals, even those receiving optimal antiretroviral therapy. Here, we utilized the SIV rhesus macaque model and advanced laparoscopic techniques for longitudinal collection of liver tissue to elucidate the timing of pathologic changes. The livers of both SIV-infected (N = 9) and SIV-naïve uninfected (N = 8) macaques were biopsied and evaluated at four time points (weeks -4, 2, 6, and 16-20 post-infection) and at necropsy (week 32). SIV DNA within the macaques' livers varied by over 4 logs at necropsy, and liver SIV DNA significantly correlated with SIV RNA in the plasma throughout the study. Acute phase liver pathology (2 weeks post-infection) was characterized by evidence for fat accumulation (microvesicular steatosis), a transient elevation in both AST and cholesterol levels within the serum, and increased hepatic expression of the PPARA gene associated with cholesterol metabolism and beta oxidation. By contrast, the chronic phase of the SIV infection (32 weeks post-infection) was associated with sinusoidal dilatation, while steatosis resolved and concentrations of AST and cholesterol remained similar to those in uninfected macaques. These findings suggest differential liver pathologies associated with the acute and chronic phases of infection and the possibility that therapeutic interventions targeting metabolic function may benefit liver health in people newly diagnosed with HIV.


Assuntos
Fígado Gorduroso , Infecções por HIV , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Humanos , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Macaca mulatta , Infecções por HIV/complicações , Hepatócitos/metabolismo , DNA , Colesterol
4.
J Nat Prod ; 76(4): 685-93, 2013 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-23517093

RESUMO

Mining the genome sequence of Burkholderia thailandensis MSMB43 revealed a cryptic biosynthetic gene cluster resembling that of FR901464 (4), a prototype spliceosome inhibitor produced by Pseudomonas sp. No. 2663. Transcriptional analysis revealed a cultivation condition in which a regulatory gene of the cryptic gene cluster is adequately expressed. Consequently, three new compounds, named thailanstatins A (1), B (2), and C (3), were isolated from the fermentation broth of B. thailandensis MSMB43. Thailanstatins are proposed to be biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase pathway. They differ from 4 by lacking an unstable hydroxyl group and by having an extra carboxyl moiety; those differences endow thailanstatins with a significantly greater stability than 4 as tested in phosphate buffer at pH 7.4. In vitro assays showed that thailanstatins inhibit pre-mRNA splicing as potently as 4, with half-maximal inhibitory concentrations in the single to sub-µM range. Cell culture assays indicated that thailanstatins also possess potent antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the single nM range. This work provides new chemical entities for research and development and new structure-activity information for chemical optimization of related spliceosome inhibitors.


Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Burkholderia/química , Piranos/isolamento & purificação , Piranos/farmacologia , Antineoplásicos/química , Ensaios de Seleção de Medicamentos Antitumorais , Genômica , Humanos , Família Multigênica , Pseudomonas/química , Piranos/química , Precursores de RNA/efeitos dos fármacos , Compostos de Espiro/química , Compostos de Espiro/isolamento & purificação , Relação Estrutura-Atividade
5.
Sci Transl Med ; 15(689): eade5795, 2023 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-36989376

RESUMO

Yellow fever virus (YFV) is a reemerging global health threat, driven by several factors, including increased spread of the mosquito vector and rapid urbanization. Although a prophylactic vaccine exists, vaccine hesitancy, supply deficits, and distribution difficulties leave specific populations at risk of severe YFV disease, as evidenced by recent outbreaks in South America. To establish a treatment for patients with severe YFV infection, we tested 37 YFV-specific monoclonal antibodies isolated from vaccinated humans and identified two capable of potently neutralizing multiple pathogenic primary YFV isolates. Using both hamster and nonhuman primate models of lethal YFV infection, we demonstrate that a single administration of either of these two potently neutralizing antibodies during acute infection fully controlled viremia and prevented severe disease and death in treated animals. Given the potential severity of YFV-induced disease, our results show that these antibodies could be effective in saving lives and fill a much-needed void in managing YFV cases during outbreaks.


Assuntos
Vacina contra Febre Amarela , Febre Amarela , Cricetinae , Animais , Humanos , Vírus da Febre Amarela , Anticorpos Neutralizantes/uso terapêutico , Vacina contra Febre Amarela/efeitos adversos , Febre Amarela/prevenção & controle , Anticorpos Antivirais/uso terapêutico , Anticorpos Monoclonais/uso terapêutico
6.
J Med Chem ; 65(4): 3388-3403, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35133171

RESUMO

Histone acetylation is a prominent epigenetic modification linked to the memory loss symptoms associated with neurodegenerative disease. The use of existing histone deacetylase inhibitor (HDACi) drugs for treatment is precluded by their weak blood-brain barrier (BBB) permeability and undesirable toxicity. Here, we address these shortcomings by developing a new class of disulfide-based compounds, inspired by the scaffold of the FDA-approved HDACi romidepsin (FK288). Our findings indicate that our novel compound MJM-1 increases the overall level of histone 3 (H3) acetylation in a prostate cancer cell line. In mice, MJM-1 injected intraperitoneally (i.p.) crossed the BBB and could be detected in the hippocampus, a brain region that mediates memory. Consistent with this finding, we found that the post-training i.p. administration of MJM-1 enhanced hippocampus-dependent spatial memory consolidation in male mice. Therefore, MJM-1 represents a potential lead for further optimization as a therapeutic strategy for ameliorating cognitive deficits in aging and neurodegenerative diseases.


Assuntos
Encéfalo/metabolismo , Inibidores de Histona Desacetilases/síntese química , Memória Espacial/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacocinética , Inibidores de Histona Desacetilases/farmacologia , Camundongos , Camundongos Endogâmicos BALB C
7.
Nat Commun ; 13(1): 2995, 2022 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-35637225

RESUMO

Hepatitis B virus has infected a third of the world's population, and 296 million people are living with chronic infection. Chronic infection leads to progressive liver disease, including hepatocellular carcinoma and liver failure, and there remains no reliable curative therapy. These gaps in our understanding are due, in large part, to a paucity of animal models of HBV infection. Here, we show that rhesus macaques regularly clear acute HBV infection, similar to adult humans, but can develop long-term infection if immunosuppressed. Similar to patients, we longitudinally detected HBV DNA, HBV surface antigen, and HBV e antigen in the serum of experimentally infected animals. In addition, we discovered hallmarks of HBV infection in the liver, including RNA transcription, HBV core and HBV surface antigen translation, and covalently closed circular DNA biogenesis. This pre-clinical animal model will serve to accelerate emerging HBV curative therapies into the clinic.


Assuntos
Hepatite B Crônica , Hepatite B , Neoplasias Hepáticas , Animais , Antígenos de Superfície , Vírus da Hepatite B/genética , Humanos , Macaca mulatta
8.
Synth Syst Biotechnol ; 1(1): 34-38, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29062925

RESUMO

Thailanstatin A (TST-A) is a potent antiproliferative natural product discovered by our group from Burkholderia thailandensis MSMB43 through a genome-guided approach. The limited supply of TST-A, due to its low titer in bacterial fermentation, modest stability and very low recovery rate during purification, has hindered the investigations of TST-A as an anticancer drug candidate. Here we report the significant yield improvement of TST-A and its direct precursor, thailanstatin D (TST-D), through metabolic engineering of the thailanstatin biosynthetic pathway in MSMB43. Deletion of tstP, which encodes a dioxygenase involved in converting TST-A to downstream products including FR901464 (FR), resulted in 58% increase of the TST-A titer to 144.7 ± 2.3 mg/L and 132% increase of the TST-D titer to 14.6 ± 0.5 mg/L in the fermentation broth, respectively. Deletion of tstR, which encodes a cytochrome P450 involved in converting TST-D to TST-A, resulted in more than 7-fold increase of the TST-D titer to 53.2 ± 12.1 mg/L in the fermentation broth. An execution of 90 L pilot-scale fed-batch fermentation of the tstP deletion mutant in a 120-L fermentor led to the preparation of 714 mg of TST-A with greater than 98.5% purity. The half-life of TST-D in a phosphate buffer was found to be at least 202 h, significantly longer than that of TST-A or FR, suggesting superior stability. However, the IC50 values of TST-D against representative human cancer cell lines were determined to be greater than those of TST-A, indicating weaker antiproliferative activity. This work enabled us to prepare sufficient quantities of TST-A and TST-D for our ongoing translational research.

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