RESUMEN
Gapmer antisense oligonucleotides (gapmers) sometimes cleave nontarget pre-mRNAs by recognizing target-like intronic/exonic portions. This off-target RNA cleavage could be a major cause of the hepatotoxicity that is induced by gapmers. In line with these findings, we hypothesized that gapmers with higher specificity have less hepatotoxicity, and that those with lower specificity have greater toxicity. To examine this concept, we investigated various Malat1-targeting gapmers with various computationally evaluated target specificities. We had expected that higher specificity gapmers would have lower hepatotoxicity, but these factors were not significantly related. In silico analysis of gapmer sequences does not always contribute to mitigating the risk of hepatotoxicity. Transcriptome analysis indicated that nontoxic gapmers do not cleave off-target RNAs, although they have many target-like RNA sequences. The present results shed light on the mechanism of the hepatotoxicity of gapmers.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Oligonucleótidos Antisentido/efectos adversos , Precursores del ARN/efectos adversos , ARN Largo no Codificante/genética , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Línea Celular Tumoral , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Biología Computacional , Simulación por Computador , Perfilación de la Expresión Génica/métodos , Humanos , Ratones , Oligonucleótidos Antisentido/administración & dosificación , Precursores del ARN/administración & dosificación , ARN Largo no Codificante/antagonistas & inhibidores , ARN Mensajero/genética , Ribonucleasa H/antagonistas & inhibidores , Ribonucleasa H/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
Cholesterol (Chol) conjugation to the 5' or 3' end of antisense oligonucleotide (ASO) enables delivery to the liver, and Chol conjugation at the gap region can also be expected to improve delivery to the liver. In this study, we synthesized ASOs bearing the Chol-conjugated thiono triester and evaluated their activity and hepatic accumulation. We found that Chol conjugations at the gap region improved in vitro activity and hepatic accumulation when compared to unconjugated ASOs. However, Chol conjugation with phosphorothioate linkage did not improve in vivo activity in the liver, suggesting the importance of cleaving the phosphodiester between ASO and Chol. These results offer useful information for tuning the oligonucleotide structure to improve pharmaceutical properties and designing ASOs for multiple ligand conjugations and combinations with end modification.
Asunto(s)
Ésteres del Colesterol/farmacocinética , Hígado/metabolismo , Oligonucleótidos Antisentido/farmacocinética , Animales , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Masculino , Ratones Endogámicos C57BL , Interferencia de ARN , Distribución TisularRESUMEN
Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei.
Asunto(s)
Silenciador del Gen , Hígado/metabolismo , Oligonucleótidos Antisentido/toxicidad , Oligonucleótidos/toxicidad , Ribonucleasa H/genética , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos/genética , Oligonucleótidos Antisentido/genética , Ribonucleasa H/metabolismoRESUMEN
TriantennaryN-acetyl galactosamine (GalNAc, GN3) and lipophilic ligands such as cholesterol andα-tocopherol conjugations dramatically improve the distribution and efficacy of second-generation antisense oligonucleotides (ASOs) in the whole liver. To characterize ligands for delivery to liver cells based on pharmacokinetics and efficacy, we used a locked nucleic acid gapmer of ASO targeting apolipoprotein B as a model compound and evaluated the amount of ASO and apolipoprotein B mRNA in the whole liver, hepatocytes, and nonparenchymal (NP) cells as well as plasma total cholesterol after administration of ASO conjugated with these ligands to mice. Compared with unconjugated ASO, GN3 conjugation increased the amount (7-fold) and efficacy (more than 10-fold) of ASO in hepatocytes only and showed higher efficacy than the increased rate of the amount of ASO. On the other hand, lipophilic ligand conjugations led to increased delivery (3- to 5-fold) and efficacy (5-fold) of ASO to both hepatocytes and NP cells. GN3 and lipophilic ligand conjugations increased the area under the curve of ASOs and the pharmacodynamic duration but did not change the half-life in hepatocytes and NP cells compared with unconjugated ASO. In the liver, the phosphodiester bond between ASO and these ligands was promptly cleaved to liberate unconjugated ASO. These ligand conjugations reduced plasma total cholesterol compared with unconjugated ASO, although these ASOs were well tolerated with no elevation in plasma transaminases. These findings could facilitate ligand selection tailored to liver cells expressed in disease-related genes and could contribute to the discovery and development of RNA interference-based therapy.
Asunto(s)
Acetilgalactosamina/química , Apolipoproteínas B/efectos de los fármacos , Hepatocitos/metabolismo , Lípidos/química , Hígado/metabolismo , Oligonucleótidos Antisentido/farmacocinética , ARN Mensajero/farmacocinética , Animales , Colesterol/sangre , Técnicas de Transferencia de Gen , Semivida , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química , Interferencia de ARN , ARN Mensajero/administración & dosificación , ARN Mensajero/química , Transaminasas/metabolismoRESUMEN
Oligonucleotides composed of natural nucleotides are inapplicable for biotechnical and therapeutic use due to its instability under biological conditions. Therminator DNA polymerases, mutant DNA polymerases of thermophilic marine archaea, show that they can efficiently synthesize fully 2'-fluoro-modified (2'F-) oligonucleotides. Furthermore, the sequence analysis reveals that the oligonucleotide sequence is highly accurate, especially the fidelity of a 2'F-oligonucleotide synthesized by Therminator II is more accurate than that of natural RNA synthesized by conventional RNA polymerase. These finding would be helpful for the synthesis of chemically modified oligonucleotides, for the use of biotechnical or medical applications.
Asunto(s)
Archaea/enzimología , ADN Polimerasa Dirigida por ADN/metabolismo , Oligonucleótidos/química , ARN/química , Secuencia de Bases , Halogenación , Modelos Moleculares , Datos de Secuencia Molecular , Oligonucleótidos/síntesis química , Oligonucleótidos/metabolismo , ARN/síntesis química , ARN/metabolismoRESUMEN
A bio-nanocapsule (BNC) is an ~50-nm hepatitis B virus (HBV) subviral particle comprising HBV envelope L proteins and a lipid bilayer, and is synthesized in recombinant Saccharomyces cerevisiae. When BNCs are administered intravenously in a mouse xenograft model, they can accumulate specifically in human liver-derived tissues and enter cells efficiently by the HBV-derived human liver-specific infection machinery, localized at the outer-membrane pre-S region of the L protein. BNC specificity for the human liver can be altered to other tissues by substituting the pre-S region using targeting molecules (e.g., antibodies, lectins, cytokines). BNCs can spontaneously form complexes with liposomes (LPs) by the membrane fusogenic activity of the pre-S region. LPs containing various therapeutic materials (e.g., chemicals, proteins, DNA, RNA) can therefore be covered with BNCs to form an ~150-nm BNC-LP conjugate. BNC-LP conjugates injected intravenously can deliver incorporated materials to target tissues specifically and efficiently by utilizing the HBV-derived infection machinery. The stability of BNC-LP conjugates in the blood circulation is similar to that of PEGylated LPs. In this chapter, we describe the preparation and in vivo application of BNC-LP conjugates, and the potential of BNC-LP conjugates as in vivo pinpoint drug delivery systems.
Asunto(s)
Sistemas de Liberación de Medicamentos , Terapia Genética , Liposomas/química , Biología Molecular/métodos , Nanocápsulas/química , Animales , Vectores Genéticos/química , Vectores Genéticos/genética , Virus de la Hepatitis B/química , Virus de la Hepatitis B/genética , Humanos , Ratones , Saccharomyces cerevisiae/genéticaRESUMEN
A wide variety of nanoparticles (NPs) that can deliver incorporated therapeutic materials such as compounds, proteins, genes and siRNAs to the human liver have been developed to treat liver-related diseases. This review describes NP-based drug and gene delivery systems such as liposomes (including lipoplex), polymer micelles, polymers (including polyplex) and viral vectors. It focuses upon the modification of these NPs to enhance liver specificity or delivery efficiency in vitro and in vivo. We discuss recent advances in drug and gene delivery systems specific to the human liver utilizing bio-nanocapsules comprising hepatitis B virus (HBV) envelope L protein, which has a pivotal role in HBV infection. These NP-based medicines may offer novel strategies for the treatment of liver-related diseases and contribute to the development of nanomedicines targeting other tissues.
Asunto(s)
Sistemas de Liberación de Medicamentos , Técnicas de Transferencia de Gen , Nanopartículas/administración & dosificación , Animales , Vectores Genéticos/administración & dosificación , Humanos , Liposomas , Hígado/metabolismo , Hepatopatías/terapia , Polímeros/química , Distribución TisularRESUMEN
We previously developed the bio-nanocapsule, which consists of hepatitis B virus envelope L proteins. The bio-nanocapsule can be used to deliver genes and drugs specifically to the human liver-derived tissues in xenograft models, presumably by utilizing the human liver-specific mechanism of hepatitis B virus infection. The hepatitis B virus tropism is highly restricted to humans and higher primates. Thus, to evaluate the in vivo therapeutic effects of forthcoming bio-nanocapsule-based medicines, it will be crucial to develop an animal model whose liver is susceptible to both bio-nanocapsule and hepatitis B virus. In the present study, we aimed to establish a bio-nanocapsule-susceptible animal model using transgenic rats expressing squamous cell carcinoma antigen-1 (SCCA1), which has been proposed to be a receptor for hepatitis B virus, interacting with the hepatitis B virus envelope protein and enhancing the cellular uptake of hepatitis B virus. We show that the recombinant SCCA1 protein interacts directly with bio-nanocapsule and inhibits its attachment to the cultured human liver-derived cells. Furthermore, we have established a transgenic rat that specifically expresses SCCA1 in the liver and also demonstrate that the amount of bio-nanocapsule accumulated in the liver is significantly increased by the SCCA1 expression. Histological analysis suggests that bio-nanocapsule is preferentially incorporated into the SCCA1-expressing hepatocytes but not into macrophages, such as Küppfer cells, nor into endothelial cells. Therefore, this animal model is expected to be useful for the development of bio-nanocapsule-based medicines.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Hígado/metabolismo , Nanocápsulas/uso terapéutico , Serpinas/metabolismo , Proteínas del Envoltorio Viral/farmacocinética , Animales , Virus de la Hepatitis B/química , Humanos , Nanocápsulas/virología , Ratas , Ratas Transgénicas , Distribución Tisular , Proteínas del Envoltorio Viral/uso terapéuticoRESUMEN
Metastasis is a key aspect of tumor malignancy, and several malignant tumors show expression of various mature N-type glycans. In particular, beta1-6 branching N-acetylglucosamine (GlcNAc) is abundantly expressed as a part of high-mannose glycans in various highly metastatic cancers. Phaseolus vulgaris agglutinin-L(4) isolectin (L(4)-PHA), which adheres to beta1-6 GlcNAc specifically, has been used for in situ cancer diagnosis. Bionanocapsules (BNCs), hollow particles with a diameter of approximately 80 nm and composed of hepatitis B surface antigen (HBsAg) and a lipid bilayer, have been developed as human liver-specific nanocapsules for in vivo drug delivery system. In this study, we have generated L(4)-PHA-displaying BNCs (PHA-BNCs) and examined whether L(4)-PHA could retarget the BNCs to malignant tumors as a "biosensor" distinguishing tumor metastaticity. Fluorescence-labeled PHA-BNCs injected systemically into a mouse xenograft model were found to accumulate in beta1-6 GlcNAc-expressing malignant tumors. The PHA-BNCs were able to deliver DNA to the malignant cancer cells. These results open up the possibility of using L(4)-PHA lectin as a targeting molecule in a drug delivery system, and of using PHA-BNCs as a novel nanodevice for malignant tumor-specific bioimaging and drug delivery.
Asunto(s)
Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Fitohemaglutininas/administración & dosificación , Animales , Línea Celular Tumoral , ADN Recombinante/administración & dosificación , ADN Recombinante/genética , Sistemas de Liberación de Medicamentos , Terapia Genética/métodos , Humanos , Neoplasias Hepáticas Experimentales/diagnóstico , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/secundario , Masculino , Ratones , Ratones Desnudos , N-Acetilglucosaminiltransferasas/metabolismo , Nanocápsulas , Trasplante de Neoplasias , Neoplasias/enzimología , Phaseolus , Fitoterapia , Trasplante HeterólogoRESUMEN
Human hepatocyte-specific delivery of green fluorescent protein was succeeded in the mouse xenograft model by fusion with hepatitis B virus surface antigen pre-S regions (pre-S(1+2)), not with each pre-S region. The entire pre-S region would be useful for human liver-specific delivery of therapeutic proteins and bio-imaging fluoroproteins in biomedical field.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Virus de la Hepatitis B/metabolismo , Hepatocitos/metabolismo , Péptidos/química , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/farmacocinética , Línea Celular , Células Cultivadas , Humanos , Péptidos/administración & dosificaciónRESUMEN
To maximize the beneficial effects and minimize the side effect of drugs, DDS (drug delivery system) has been attracted many researchers in the recent drug development. Especially, the in vivo pinpoint delivery system for drugs is very important and key technology for developing the next generations of anti-cancer drugs and gene therapies. Bio-nanocapsule (BNC) is recombinant yeast-derived hepatitis B virus surface antigen particle, which has been used as a recombinant hepatitis B vaccine for the last 20 years in the world. BNC can incorporate various materials (chemical compounds, proteins, genes, siRNA, etc) by the fusion with liposome, and deliver them to the organs and tissues in vivo specifically by the action of bio-recognition molecules on the BNC's surface. The transfection efficiency is significantly higher than that of liposome, because BNC harbors the complete set of hepatitis B virus infection machinery. Recently, we succeeded in the in vivo retargeting of BNC by displaying either antibody or homing peptide, less than 10 amino acid residues for in vivo targeting. BNC is a hybrid of liposome and virus, and very flexible system for in vivo retargeting. BNC might be very promising carriers in the next generation of DDS.
Asunto(s)
Sistemas de Liberación de Medicamentos , Nanocápsulas , Animales , Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Terapia Genética , Vectores Genéticos , Antígenos de Superficie de la Hepatitis B , Vacunas contra Hepatitis B , Humanos , Liposomas , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Vacunas Sintéticas , Virus/genéticaRESUMEN
The investigation of common structural motifs provides additional information on why proteins conserve similar topologies yet may have non-conserved amino acid sequences. Proteins containing the ubiquitin superfold have similar topologies, although the sequence conservation is rather poor. Here, we present novel similarities and differences between the proteins ubiquitin and NEDD8. They have 57% identical sequence, almost identical backbone topology and similar functional strategy, although their physiological functions are mutually different. Using variable pressure NMR spectroscopy, we found that the two proteins have similar conformational fluctuation in the evolutionary conserved enzyme-binding region and contain a structurally similar locally disordered conformer (I) in equilibrium with the basic folded conformer (N). A notable difference between the two proteins is that the equilibrium population of I is far greater for NEDD8 (DeltaG(0)(NI)<5 kJ/mol) than for ubiquitin (DeltaG(0)(NI)=15.2(+/-1.0) kJ/mol), and that the tendency for overall unfolding (U) is also far higher for NEDD8 (DeltaG(0)(NU)=11.0(+/-1.5) kJ/mol) than for ubiquitin (DeltaG(0)(NU)=31.3(+/-4.7) kJ/mol). These results suggest that the marked differences in thermodynamic stabilities of the locally disordered conformer (I) and the overall unfolding species (U) are a key to determine the functional differences of the two structurally similar proteins in physiology.