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1.
bioRxiv ; 2023 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-38014175

RESUMO

Lipid nanoparticle (LNP) delivery of CRISPR ribonucleoproteins (RNPs) has the potential to enable high-efficiency in vivo genome editing with low toxicity and an easily manufactured technology, if RNP efficacy can be maintained during LNP production. In this study, we engineered a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) using directed evolution to generate iGeoCas9 evolved variants capable of robust genome editing of cells and organs. iGeoCas9s were significantly better at editing cells than wild-type GeoCas9, with genome editing levels >100X greater than those induced by the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP:LNP complexes edited a variety of cell lines and induced homology-directed repair (HDR) in cells receiving co-delivered single-stranded DNA (ssDNA) templates. Using tissue-selective LNP formulations, we observed genome editing of 35‒56% efficiency in the liver or lungs of mice that received intravenous injections of iGeoCas9 RNP:LNPs. In particular, iGeoCas9 complexed to acid-degradable LNPs edited lung tissue in vivo with an average of 35% efficiency, a significant improvement over editing efficiencies observed previously using viral or non-viral delivery strategies. These results show that thermostable Cas9 RNP:LNP complexes are a powerful alternative to mRNA:LNP delivery vehicles, expanding the therapeutic potential of genome editing.

2.
Biochemistry ; 62(24): 3533-3547, 2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-37729550

RESUMO

Neurological disorders are often debilitating conditions with no cure. The majority of current therapies are palliative rather than disease-modifying; therefore, new strategies for treating neurological disorders are greatly needed. mRNA-based therapeutics have great potential for treating such neurological disorders; however, challenges with delivery have limited their clinical potential. Lipid nanoparticles (LNPs) are a promising delivery vector for the brain, given their safer toxicity profile and higher efficacy. Despite this, very little is known about LNP-mediated delivery of mRNA into the brain. Here, we employ MC3-based LNPs and successfully deliver Cre mRNA and Cas9 mRNA/Ai9 sgRNA to the adult Ai9 mouse brain; greater than half of the entire striatum and hippocampus was found to be penetrated along the rostro-caudal axis by direct intracerebral injections of MC3 LNP mRNAs. MC3 LNP Cre mRNA successfully transfected cells in the striatum (∼52% efficiency) and hippocampus (∼49% efficiency). In addition, we demonstrate that MC3 LNP Cas9 mRNA/Ai9 sgRNA edited cells in the striatum (∼7% efficiency) and hippocampus (∼3% efficiency). Further analysis demonstrates that MC3 LNPs mediate mRNA delivery to multiple cell types including neurons, astrocytes, and microglia in the brain. Overall, LNP-based mRNA delivery is effective in brain tissue and shows great promise for treating complex neurological disorders.


Assuntos
Nanopartículas , Doenças do Sistema Nervoso , Animais , Camundongos , RNA Guia de Sistemas CRISPR-Cas , Encéfalo , RNA Mensageiro/genética , RNA Interferente Pequeno
3.
RSC Adv ; 13(16): 10636-10641, 2023 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-37025664

RESUMO

Covalent inhibitors of the papain-like protease (PLpro) from SARS-CoV-2 have great potential as antivirals, but their non-specific reactivity with thiols has limited their development. In this report, we performed an 8000 molecule electrophile screen against PLpro and identified an α-chloro amide fragment, termed compound 1, which inhibited SARS-CoV-2 replication in cells, and also had low non-specific reactivity with thiols. Compound 1 covalently reacts with the active site cysteine of PLpro, and had an IC50 of 18 µM for PLpro inhibition. Compound 1 also had low non-specific reactivity with thiols and reacted with glutathione 1-2 orders of magnitude slower than other commonly used electrophilic warheads. Finally, compound 1 had low toxicity in cells and mice and has a molecular weight of only 247 daltons and consequently has great potential for further optimization. Collectively, these results demonstrate that compound 1 is a promising lead fragment for future PLpro drug discovery campaigns.

4.
Bioact Mater ; 25: 387-398, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36844366

RESUMO

Nanoparticle-based drug delivery systems have the potential to revolutionize medicine, but their low vascular permeability and rapid clearance by phagocytic cells have limited their medical impact. Nanoparticles delivered at the in utero stage can overcome these key limitations due to the high rate of angiogenesis and cell division in fetal tissue and the under-developed immune system. However, very little is known about nanoparticle drug delivery at the fetal stage of development. In this report, using Ai9 CRE reporter mice, we demonstrate that lipid nanoparticle (LNP) mRNA complexes can deliver mRNA in utero, and can access and transfect major organs, such as the heart, the liver, kidneys, lungs and the gastrointestinal tract with remarkable efficiency and low toxicity. In addition, at 4 weeks after birth, we demonstrate that 50.99 ± 5.05%, 36.62 ± 3.42% and 23.7 ± 3.21% of myofiber in the diaphragm, heart and skeletal muscle, respectively, were transfected. Finally, we show here that Cas9 mRNA and sgRNA complexed to LNPs were able to edit the fetal organs in utero. These experiments demonstrate the possibility of non-viral delivery of mRNA to organs outside of the liver in utero, which provides a promising strategy for treating a wide variety of devastating diseases before birth.

5.
Chem Commun (Camb) ; 58(19): 3166-3169, 2022 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-35170593

RESUMO

This article reports the synthesis and characterization of a novel self-immolative linker, based on thiocarbonates, which releases a free thiol upon activation via enzymes. We demonstrate that thiocarbonate self-immolative linkers can be used to detect the enzymes penicillin G amidase (PGA) and nitroreductase (NTR) with high sensitivity using absorption spectroscopy. Paired with modern thiol amplification technology, the detection of PGA and NTR were achieved at concentrations of 160 nM and 52 nM respectively. In addition, the PGA probe was shown to be compatible with both biological thiols and enzymes present in cell lysates.


Assuntos
Nitrorredutases/análise , Penicilina Amidase/análise , Compostos de Sulfidrila/química , Estrutura Molecular , Nitrorredutases/metabolismo , Penicilina Amidase/metabolismo , Espectrometria de Fluorescência
6.
Adv Healthc Mater ; 11(9): e2102118, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34861744

RESUMO

Protein-based therapeutics have the potential to treat a variety of diseases, however, safe and effective methods for delivering them into cells need to be developed before their clinical potential can be realized. Peptide fusions have great potential for improving intracellular delivery of proteins. However, very few peptides have been identified that can increase the intracellular delivery of proteins, and new peptides that can enhance intracellular protein delivery are greatly needed. In this report, the authors demonstrate that the coiled-coil forming peptide (KVSALKE)5 (termed K5) can function as a cell penetrating peptide (CPP), and can also complex other proteins that contain its partner peptide E5. It is shown here that GFP and Cas9 fused to the K5 peptide has dramatically enhanced cell uptake in a variety of cell lines, and is able to edit neurons and astrocytes in the striatum and hippocampus of mice after a direct intracranial injection. Collectively, these studies demonstrate that the coiled-coil forming peptide (KVSALKE)5 is a new class of multifunctional CPPs that has great potential for improving the delivery of proteins into cells and in vivo.


Assuntos
Peptídeos Penetradores de Células , Animais , Transporte Biológico , Peptídeos Penetradores de Células/uso terapêutico , Camundongos , Proteínas/metabolismo
7.
Mol Pharm ; 19(1): 67-79, 2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34931518

RESUMO

The development of endosomal disruptive agents is a major challenge in the field of drug delivery and pharmaceutical chemistry. Current endosomal disruptive agents are composed of polymers, peptides, and nanoparticles and have had limited clinical impact. Alternatives to traditional endosomal disruptive agents are therefore greatly needed. In this report, we introduce a new class of low molecular weight endosomal disruptive agents, termed caged surfactants, that selectively disrupt endosomes via reversible PEGylation under acidic endosomal conditions. The caged surfactants have the potential to address several of the limitations hindering the development of current endosomal disruptive agents, such as high toxicity and low excretion, and are amenable to traditional medicinal chemistry approaches for optimization. In this report, we synthesized three generations of caged surfactants and demonstrated that they can enhance the ability of cationic lipids to deliver mRNA into primary cells. We also show that caged surfactants can deliver siRNA into cells when modified with the RNA-binding dye thiazole orange. We anticipate that the caged surfactants will have numerous applications in pharmaceutical chemistry and drug delivery given their versatility.


Assuntos
Sistemas de Liberação de Medicamentos , Ácidos Nucleicos/administração & dosagem , Tensoativos/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Endossomos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , RNA Mensageiro/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Relação Estrutura-Atividade , Tensoativos/administração & dosagem , Tensoativos/química
8.
Chem Commun (Camb) ; 56(91): 14207-14210, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33111731

RESUMO

There is great interest in developing strategies to deliver proteins into the cytoplasm of cells. We report here a PEG-poly-eosin block copolymer (PEG-pEosin) that can encapsulate proteins and release them in active form under mildly acidic conditions. A PEG-pEosin formulation composed of Cre and the endosomolytic protein LLO efficiently performed gene editing in cells and in the brains of mice after an intracranial injection.


Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Amarelo de Eosina-(YS)/farmacologia , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas Hemolisinas/antagonistas & inibidores , Polietilenoglicóis/farmacologia , Animais , Toxinas Bacterianas/genética , Sobrevivência Celular/efeitos dos fármacos , Amarelo de Eosina-(YS)/química , Eritrócitos/efeitos dos fármacos , Edição de Genes , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Concentração de Íons de Hidrogênio , Camundongos , Modelos Moleculares , Estrutura Molecular , Polietilenoglicóis/química
9.
Chem Sci ; 11(33): 8973-8980, 2020 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34123152

RESUMO

Reduction sensitive linkers (RSLs) have the potential to transform the field of drug delivery due to their ease of use and selective cleavage in intracellular environments. However, despite their compelling attributes, developing reduction sensitive self-immolative linkers for aliphatic amines has been challenging due to their poor leaving group ability and high pK a values. Here a traceless self-immolative linker composed of a dithiol-ethyl carbonate connected to a benzyl carbamate (DEC) is presented, which can modify aliphatic amines and release them rapidly and quantitatively after disulfide reduction. DEC was able to reversibly modify the lysine residues on CRISPR-Cas9 with either PEG, the cell penetrating peptide Arg10, or donor DNA, and generated Cas9 conjugates with significantly improved biological properties. In particular, Cas9-DEC-PEG was able to diffuse through brain tissue significantly better than unmodified Cas9, making it a more suitable candidate for genome editing in animals. Furthermore, conjugation of Arg10 to Cas9 with DEC was able to generate a self-delivering Cas9 RNP that could edit cells without transfection reagents. Finally, conjugation of donor DNA to Cas9 with DEC increased the homology directed DNA repair (HDR) rate of the Cas9 RNP by 50% in HEK 293T cell line. We anticipate that DEC will have numerous applications in biotechnology, given the ubiquitous presence of aliphatic amines on small molecule and protein therapeutics.

10.
Chem Commun (Camb) ; 55(31): 4562-4565, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30931453

RESUMO

In this report, we designed and synthesized a novel fluorescent single tailed surfactant (termed FEDS), which can disrupt endosomes, complex lipofectamine, and can also identify cells that have been transfected. FEDS was able to increase the gene editing efficiency of lipofectamine/Cas9 ribonucleoprotein by 300% via a combination of fluorescent based enrichment and endosomal disruption.


Assuntos
Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Lipídeos/química , Animais , Linhagem Celular , Endossomos/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Citometria de Fluxo , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Coelhos
11.
RSC Adv ; 8(9): 4900-4904, 2018 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-35539523

RESUMO

A micrometer-sized hybrid crystalline material was facilely fabricated from the synergistic coordination chemistry of two types of 2-hydroxyazobenzene building blocks and cobalt ions. This hybrid crystalline material functioned as both receptor and signal reporter in cyanide (CN-) sensing with ratiometric absorption responses at 456 nm and 537 nm. The mechanism of CN- sensing involves partial ligand dissociation accompanied by the formation of a new organic metal-CN- adduct. We believe that this protocol would be valuable in achieving the expected selectivities and sensitivities for a wide variety of analytes in many chemical and biological systems in the future.

12.
Mol Syst Des Eng ; 3(4): 599-603, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30740245

RESUMO

The development of antibiotics against Gram-negative bacteria is a central problem in drug discovery. In this report, we demonstrate that aromatic sulfonyl fluorides with a nitro group in their ortho position have remarkable antibacterial activity and are active against drug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug resistant Acinetobacter baumannii, and Pseudomonas aeruginosa.

13.
PLoS One ; 10(6): e0128967, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26047497

RESUMO

Melanin production is important to the pathogenicity and survival of some bacterial pathogens. In Bacillus anthracis, loss of hmgA, encoding homogentisate 1,2-dioxygenase, results in accumulation of a melanin-like pigment called pyomelanin. Pyomelanin is produced in the mutant as a byproduct of disrupted catabolism of L-tyrosine and L-phenylalanine. Accumulation of pyomelanin protects B. anthracis cells from UV damage but not from oxidative damage. Neither loss of hmgA nor accumulation of pyomelanin alter virulence gene expression, sporulation or germination. This is the first investigation of homogentisate 1,2-dioxygenase activity in the Gram-positive bacteria, and these results provide insight into a conserved aspect of bacterial physiology.


Assuntos
Bacillus anthracis/fisiologia , Homogentisato 1,2-Dioxigenase/metabolismo , Melaninas/metabolismo , Substâncias Protetoras/metabolismo , Antraz/microbiologia , Bacillus anthracis/genética , Bacillus anthracis/efeitos da radiação , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Homogentisato 1,2-Dioxigenase/genética , Humanos , Melaninas/genética , Mutagênese , Estresse Oxidativo , Fenilalanina/metabolismo , Tirosina/metabolismo , Raios Ultravioleta
14.
J Bacteriol ; 197(3): 626-35, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25422307

RESUMO

Loss of the cytochrome c maturation system in Bacillus cereus results in increased transcription of the major enterotoxin genes nhe, hbl, and cytK and the virulence regulator plcR. Increased virulence factor production occurs at 37°C under aerobic conditions, similar to previous findings in Bacillus anthracis. Unlike B. anthracis, much of the increased virulence gene expression can be attributed to loss of only c551, one of the two small c-type cytochromes. Additional virulence factor expression occurs with loss of resBC, encoding cytochrome c maturation proteins, independently of the presence of the c-type cytochrome genes. Hemolytic activity of strains missing either cccB or resBC is increased relative to that in the parental strain, while sporulation efficiency is unaffected in the mutants. Increased virulence gene expression in the ΔcccB and ΔresBC mutants occurs only in the presence of an intact plcR gene, indicating that this process is PlcR dependent. These findings suggest a new mode of regulation of B. cereus virulence and reveal intriguing similarities and differences in virulence regulation between B. cereus and B. anthracis.


Assuntos
Bacillus cereus/metabolismo , Citocromos c/metabolismo , Expressão Gênica , Processamento de Proteína Pós-Traducional , Fatores de Virulência/biossíntese , Enterotoxinas/biossíntese , Proteínas Hemolisinas/biossíntese , Temperatura
15.
J Bacteriol ; 195(23): 5242-9, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24056109

RESUMO

The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation, while the closely related organism Bacillus subtilis carries only one copy of ccdA. To investigate the roles of the two ccdA gene copies in B. anthracis, strains were constructed without each ccdA gene, and one strain was constructed without both copies simultaneously. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. Complementation and expression analyses indicate that ccdA2 encodes the primary CcdA in B. anthracis, active in all three pathways. While CcdA1 retains activity in cytochrome c maturation and virulence control, it has completely lost its activity in the sporulation pathway. In support of this finding, expression of ccdA1 is strongly reduced when cells are grown under sporulation-inducing conditions. When the activities of CcdA1 and CcdA2 were analyzed in B. subtilis, neither protein retained activity in cytochrome c maturation, but CcdA2 could still function in sporulation. These observations reveal the complexities of thiol-disulfide oxidoreductase function in pathways relevant to virulence and physiology.


Assuntos
Bacillus anthracis/metabolismo , Bacillus anthracis/patogenicidade , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Membrana/metabolismo , Esporos Bacterianos/fisiologia , Bacillus anthracis/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Citocromos c/genética , Citocromos c/metabolismo , Deleção de Genes , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Fatores de Tempo , Transcriptoma , Virulência
16.
Comput Math Methods Med ; 2013: 983829, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23861727

RESUMO

Metuximab is the generic name of Licartin, a new drug for radioimmunotherapy of hepatocellular carcinoma. Although it is known to be a mouse monoclonal antibody against CD147, the complete epitope mediating the binding of metuximab to CD147 remains unknown. We panned the Ph.D.-12 phage display peptide library against metuximab and got six mimotopes. The following bioinformatics analysis based on mimotopes suggested that metuximab recognizes a conformational epitope composed of more than 20 residues. The residues of its epitope may include T28, V30, K36, L38, K57, F74, D77, S78, D79, D80, Q81, G83, S86, N98, Q100, L101, H102, G103, P104, V131, P132, and K191. The homology modeling of metuximab and the docking of CD147 to metuximab were also performed. Based on the top one docking model, the epitope was predicted to contain 28 residues: AGTVFTTV (23-30), I37, D45, E84, V88, EPMGTANIQLH (92-102), VPP (131-133), Q164, and K191. Almost half of the residues predicted on the basis of mimotope analysis also appear in the docking result, indicating that both results are reliable. As the predicted epitopes of metuximab largely overlap with interfaces of CD147-CD147 interactions, a structural mechanism of metuximab is proposed as blocking the formation of CD147 dimer.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Basigina/química , Basigina/metabolismo , Mapeamento de Epitopos/métodos , Sequência de Aminoácidos , Animais , Basigina/genética , Sítios de Ligação , Biologia Computacional , Mapeamento de Epitopos/estatística & dados numéricos , Humanos , Camundongos , Modelos Moleculares , Biblioteca de Peptídeos , Multimerização Proteica , Homologia Estrutural de Proteína
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