Investigation of pharmacokinetic properties of a PEGylated bilirubin nanoparticle in male Sprague-Dawley rats using liquid chromatography-quadrupole time-of-flight mass spectrometry.

Polyethylene glycol (PEG) was introduced into synthetic bilirubin 3α and a PEGylated bilirubin 3α nanoparticle (BX-001N, Brixelle®) was developed for the first time.An in vitro microsomal stability study, in vivo PK studies with intravenous bolus (IV) and subcutaneous injection (SC), and a semi-mass balance study of BX-001N were investigated to evaluate its pharmacokinetic (PK) properties in male Sprague-Dawley (SD) rats using developed liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qTOF/MS).Following IV administration at 10 or 30 mg/kg, BX-001N showed very low clearance (0.33-0.67 mL/min/kg) with predominant distribution in the vascular system (Vd = 51.73-83.02 mL/kg). BX-001N was also very stable in vitro liver microsomal stability study.Following SC administration at 10 or 30 mg/kg, the bioavailability of BX-001N in plasma at 10 mg/kg was around 43% and showed the less dose-proportionality at 30 mg/kg dose.BX-001N was mainly excreted via the urinary pathway (86.59-92.99% of total amount of parent drug in excreta; urine and feces) not via the biliary one.

Potentiating the Cross-Reactive IFN-γ T Cell and Polyfunctional T Cell Responses by Heterologous GX-19N DNA Booster in Mice Primed with Either a COVID-19 mRNA Vaccine or Inactivated Vaccine.

Waning vaccine-induced immunity, coupled with the emergence of SARS-CoV-2 variants, has inspired the widespread implementation of COVID-19 booster vaccinations. Here, we evaluated the potential of the GX-19N DNA vaccine as a heterologous booster to enhance the protective immune response to SARS-CoV-2 in mice primed with either an inactivated virus particle (VP) or an mRNA vaccine. We found that in the VP-primed condition, GX-19N enhanced the response of both vaccine-specific antibodies and cross-reactive T Cells to the SARS-CoV-2 variant of concern (VOC), compared to the homologous VP vaccine prime-boost. Under the mRNA-primed condition, GX-19N induced higher vaccine-induced T Cell responses but lower antibody responses than the homologous mRNA vaccine prime-boost. Furthermore, the heterologous GX-19N boost induced higher S-specific polyfunctional CD4+ and CD8+ T cell responses than the homologous VP or mRNA prime-boost vaccinations. Our results provide new insights into booster vaccination strategies for the management of novel COVID-19 variants.

Immunological characteristics of MAV/06 strain of varicella-zoster virus vaccine in an animal model.

BACKGROUND: Varicella-zoster virus (VZV) is a pathogen that causes chickenpox and shingles in humans. Different types of the varicella vaccines derived from the Oka and MAV/06 strains are commercially available worldwide. Although the MAV/06 vaccine was introduced in 1990s, little was known about immunological characteristics. RESULTS: Here, we evaluated B and T cell immune response in animals inoculated with the Oka and MAV/06 vaccines as well as a new formulation of the MAV/06 vaccine. A variety of test methods were applied to evaluate T and B cell immune response. Plaque reduction neutralization test (PRNT) and fluorescent antibody to membrane antigen (FAMA) assay were conducted to measure the MAV/06 vaccine-induced antibody activity against various VZVs. Glycoprotein enzyme-linked immunosorbent assay (gpELISA) was used to compare the degree of the antibody responses induced by the two available commercial VZV vaccines and the MAV/06 vaccine. Interferon-gamma enzyme-linked immunosorbent spot (IFN-γ ELISpot) assays and cytokine bead array (CBA) assays were conducted to investigate T cell immune responses. Antibodies induced by MAV/06 vaccination showed immunogenicity against a variety of varicella-zoster virus and cross-reactivity among the virus clades. CONCLUSIONS: It is indicating the similarity of the antibody responses induced by commercial varicella vaccines and the MAV/06 vaccine. Moreover, VZV-specific T cell immune response from MAV/06 vaccination was increased via Th1 cell response. MAV/06 varicella vaccine induced both humoral and cellular immune response via Th1 cell mediated response.

Optimization of linear double-stranded RNA for the production of multiple siRNAs targeting hepatitis C virus.

RNA interference (RNAi)-based gene silencing possesses great therapeutic potential for inhibiting replication of human viruses such as hepatitis C virus (HCV). However, one of the putative limitations for its use as a therapy is the rapid emergence of escape variants. These contain deletions or mutations within the viral genome sequences complementary to the small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) being used for treatment. As a potential solution to this problem, we constructed an expression system for duplex RNAs harboring two siRNA units using convergent H1 and U6 Pol III promoters. Here, the length and orientation of the transcript, tandem siRNA (tsiRNA), were optimized to be processed by the intracellular ribonuclease Dicer into functional siRNAs targeting different sequences. Assessment in transfected cells indicates that the length of the tsiRNA duplex (40-42 base pairs) is more critical for both siRNA-producing capacity and gene silencing activity than the orientation of each siRNA unit. In Huh7 cells replicating full-length HCV RNA, expression of length-optimized tsiRNA inhibited viral protein levels as efficiently as a single 21-nucleotide siRNA-expression construct, without affecting miRNA maturation or induction of an interferon response. We verified that the anti-viral activity of tsiRNA was achieved by precise cleavage of two target sites. A distinct advantage of this strategy is that each side of the optimized linear duplex RNA could enter into the Dicer-mediated processing machinery, thus likely providing more equal and efficient production of multiple siRNAs required for reducing the chance of viral escape.

Membrane-based hybridization capture of intracellular peptide nucleic acid.

Peptide nucleic acid (PNA) is a chimeric oligonucleotide with nucleotide-derived bases and a peptide backbone. Compared with natural nucleotides, PNA has several advantages, including improved stability and high sequence discrimination during duplex formation. Despite its potential for therapeutic application, analysis technologies have not been generalized, mainly due to ambiguous physiochemical properties resembling those of nucleic acids as well as protein. Here we present a PNA detection method: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrotransfer to a Western blotting membrane and then hybridization with a radiolabeled oligonucleotide probe. This method is useful for evaluating the quality of synthetic PNA and determining its intracellular localization.

Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy.

As recent advancements in the chimeric antigen receptor-T cells have revolutionized the way blood cancers are handled, potential benefits from producing off-the-shelf, standardized immune cells entail the need for development of allogeneic immune cell therapy. However, host rejection driven by HLA disparity in adoptively transferred allogeneic T cells remains a key obstacle to the universal donor T cell therapy. To evade donor HLA-mediated immune rejection, we attempted to eliminate T cell's HLA through the CRISPR/Cas9 gene editing system. First, we screened 60 gRNAs targeting B2M and multiple sets of gRNA each targeting α chains of HLA-II (DPA, DQA and DRA, respectively) using web-based design tools, and identified specific gRNA sequences highly efficient for target deletion without carrying off-target effects. Multiplex genome editing of primary human T cells achieved by the newly discovered gRNAs yielded HLA-I- or HLA-I/II-deficient T cells that were phenotypically unaltered and functionally intact. The overnight mixed lymphocyte reactions demonstrated the HLA-I-negative cells induced decreased production of IFN-γ and TNF-α in alloreactive T cells, and deficiency of HLA-I/II in T cells further dampened the inflammatory responses. Taken together, our approach will provide an efficacious pathway toward the universal donor cell generation by manipulating HLA expression in therapeutic T cells.

Targeted delivery of siRNA against hepatitis C virus by apolipoprotein A-I-bound cationic liposomes.

BACKGROUND/AIMS: Hepatitis C virus (HCV) is one of the major human hepatic RNA viruses. Recently, we developed a liver-specific siRNA delivery technology using DTC-Apo composed of cationic liposomes (DTC) and apolipoprotein A-I (apo A-I). Here, we investigated whether DTC-Apo nanoparticles can systemically deliver siRNA into mouse hepatocytes expressing HCV proteins and inhibit their expression efficiently. METHODS: A transient HCV model was constructed by hydrodynamic injection of plasmid DNA expressing viral structural proteins under hepatic control region and alpha1-antitrypsin promoter elements. Using this model, DTC-Apo containing HCV-core-specific siRNA was intravenously injected to assess antiviral activity as well as the duration of silencing. RESULTS: Post-administration of DTC-Apo/HCV-specific siRNA at a dose of 2mg siRNA/kg inhibited viral gene expression by 65-75% in the liver on day 2. Improved activity (95% knockdown on day 2) without immunotoxicity was obtained by 2'-OMe-modification at two U sequences on its sense strand. Notably, the gene silencing effect of the modified siRNA was still maintained at day 6, while the unmodified one lost RNAi activity after day 4. CONCLUSIONS: Our results suggest that DTC-Apo liposome is a highly potential delivery vehicle to transfer therapeutic siRNA especially targeting HCV to the liver.

Hepatic siRNA delivery using recombinant human apolipoprotein A-I in mice.

Apolipoprotein A-I (apo A-I), the major protein component of high density lipoprotein (HDL), plays a key role in reverse cholesterol transport from peripheral tissues to liver or steroidogenic organs. Class B, type 1 scavenger receptor (SR-BI) is abundantly expressed in these target tissues and recognizes apo A-I of HDL for selective cholesteryl ester uptake. Recently, we reported the liver-targeting potential of plasma-derived apo A-I and the efficient delivery of therapeutic small interfering RNAs (siRNA) assembled with cationic liposome and apo A-I. In this study, we expressed and purified recombinant human apo A-I (rhapo A-I), low endotoxin grade, from an Escherichia coli expression system. The liver-targeting property of rhapo A-I was compared to that of plasma-derived apo A-I. Using a hepatitis C virus mouse model, intravenous administration of virus-specific siRNA with liposome and rhapo A-I significantly inhibited viral protein expression, demonstrating great promise for its use in clinical applications.

Immunostimulatory properties and antiviral activity of modified HBV-specific siRNAs.

Sequence-specific gene silencing by small interfering RNA (siRNA) is an intense area of focus in the development of novel therapeutic agents. Currently, there are two major hurdles to achieving clinically effective siRNA-based therapeutics: establishment of an efficient delivery system that transfers the siRNA to the correct tissue(s); and the reduction of unintended immunotoxicity associated with unmodified siRNA. We have developed a novel liver-specific delivery system of apolipoprotein A-I-decorated cationic lipids (DTC-Apo). Here, we show that intravenous injection of an unmodified hepatitis B virus (HBV)-specific siRNA encapsulated in DTC-Apo activates the innate immune response in mice. However, 2'-O-methyl (2'-OMe) modification of siRNA sense-strand uridine or uridine/adenosine residues efficiently abrogated the immunostimulatory properties of the siRNA and also silenced viral replication. In contrast, pyrimidine modification by 2'-OMe or 2'-fluoro (2'-F) substitution failed to circumvent liposome-induced immune recognition. Our findings provide useful information for the design of chemically-modified siRNAs for in vivo applications.

Inhibition of hepatitis C virus gene expression by small interfering RNAs using a tri-cistronic full-length viral replicon and a transient mouse model.

HCV is an ideal target for siRNA as its genome, a single-stranded RNA, is translated into a single viral polyprotein and replicated into negative-stranded RNA. In the present study, we monitored the effects of 36 different small interfering RNAs (siRNAs) transcribed from a plasmid-derived expression system on the luciferase activities expressed from a full-length HCV replicon, to identify potent siRNA target sites. Delivery of nine selected siRNA expression vectors into human hepatoma cells (Huh7) carrying a genomic HCV replicon resulted in a significant reduction in viral protein and RNA levels. Moreover, synthetic siRNAs directed to target sites (core, NS3, NS4A and NS4B coding regions) in the HCV genome efficiently suppressed viral replication in a dose-dependent manner. A transient mouse model system expressing viral structural proteins in the liver was constructed using the hydrodynamic transfection method to confirm in vivo anti-HCV activity of the selected siRNAs. A 21-nucleotide siRNA, which can hybridize to the HCV core coding region with a single G-U base pair, suppressed weakly transgene expression in mice. However, this anti-viral effect was enhanced upon substitution with a 27-mer duplex RNA. Our results will provide useful information about designing potent siRNAs against variable target sites.

Efficient inhibition of hepatitis B virus replication by small interfering RNAs targeted to the viral X gene in mice.

The hepatitis B virus (HBV), as a major cause of acute and chronic hepatitis in humans, contains a partial double-stranded circular DNA genome of 3.2kb that is transcribed into the 3.5-, 2.4-, 2.1-, and 0.7-kb viral transcripts by the host RNA polymerase II. The HBV X (HBx) gene is consistently expressed in all four HBV viral mRNAs and thus an ideal target for developing viral inhibitors via a gene therapeutic approach. In this study, we show that two HBx-specific small interfering RNAs (siRNA), HBx1 and HBx3, significantly decrease both viral RNA and protein levels, and completely block replication in cultured cells co-transfected with a siRNA expression plasmid and an HBV replication-competent vector. To further confirm these antiviral activities of selected siRNAs in small animals, we established acute and chronic HBV mouse models by hydrodynamic injection of this plasmid containing the full-length HBV genome. Selected HBx-specific siRNAs also induced a significant anti-viral effect in living animals. Our findings should facilitate the development of an alternative therapeutic agent against HBV infection, particularly HBV-derived hepatocellular carcinoma (HCC) in which HBx has been known as one of the major pathological factors.

Comparison of immunogenicity of cell-and egg-passaged viruses for manufacturing MDCK cell culture-based influenza vaccines.

While cell culture-based technology has been recently used for manufacturing influenza vaccines, currently available seed viruses are mostly egg-derived reassortants that are egg-adapted to achieve high virus growth in eggs. For use as viruses for cell culture-based influenza vaccine manufacturing, egg-adapted viral seeds may undergo several passages in manufacturing cell lines. However, the suitability of such cell-passaged viruses for vaccine production remains largely unelucidated. In this study, influenza viruses produced in suspension Madin-Darby canine kidney (MDCK) cell cultures were compared to those produced in embryonated hen's eggs for manufacturing MDCK cell culture-based influenza vaccines through comparability studies of virus productivity and vaccine immunogenicity. The results indicate no change in the amino acid sequence of the main antigens, including hemagglutinin (HA) and neuraminidase (NA), of cell-passaged viruses after three passages in suspension MDCK cells. In lab-scale (3-L) single-use bioreactors, suspension MDCK culture supernatants inoculated with cell-passaged viruses were found to show higher virus productivity, suspension MDCK culture supernatants inoculated with egg-passaged viruses, in respect to the HA titers and HA contents determined by single radial immunodiffusion. Finally, comparable hemagglutination inhibition and influenza-specific IgG titers were determined in the mice immunized with cell culture-based vaccines produced with cell- or egg-passaged viruses. These results indicate that MDCK cell-passaged viruses from egg-adapted viruses, as well as egg-derived seed virus, are suitable for MDCK cell culture-based influenza vaccine production.

Branched, tripartite-interfering RNAs silence multiple target genes with long guide strands.

Structural modifications could provide classical small interfering RNA (siRNA) structure with several advantages, including reduced off-target effects and increased silencing activity. Thus, RNA interference (RNAi)-triggering molecules with diverse structural modifications have been investigated by introducing variations on duplex length and overhang structure. However, most of siRNA structural variants are based on the linear duplex structure. In this study, we introduce a branched, non-linear tripartite-interfering RNA (tiRNA) structure that could induce silencing of multiple target genes. Surprisingly, the gene silencing by tiRNA structure does not require Dicer-mediated processing into smaller RNA units, and the 38-nt-long guide strands can trigger specific gene silencing through the RNAi machinery in mammalian cells. tiRNA also shows improved gene silencing potency over the classical siRNA structure when complexed with cationic delivery vehicles due to the enhanced intracellular delivery. These results demonstrate that tiRNA is a novel RNA nanostructure for executing multi-target gene silencing with increased potency, which could be utilized as a structural platform to develop efficient anticancer or antiviral RNAi therapeutics.

Systemic and specific delivery of small interfering RNAs to the liver mediated by apolipoprotein A-I.

Tissue-targeted delivery of small interfering RNA (siRNA) must be achieved before RNA interference (RNAi) technology can be used in practical therapeutic approaches. In this study, the potential of apolipoprotein A-I (apo A-I) for the systemic delivery of nucleic acids to the liver is demonstrated using real-time in vivo imaging. As a proof of concept, synthetic siRNAs against hepatitis B virus (HBV) were formulated into complexes of apo A-I and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (DTC-Apo) and injected intravenously (i.v.) into a mouse model carrying replicating HBV. We show that administration of these nanoparticles can significantly reduce viral protein expression by receptor-mediated endocytosis. The advantages of the apo A-I-mediated siRNA delivery method are its liver specificity, its effectiveness at low doses (< or = 2 mg/kg) in only a single treatment, and its persistent antiviral effect up to 8 days. The liver-targeted gene silencing was also shown by in vivo images, in which bioluminescent signals emitted from the liver were efficiently reduced after i.v. administration of luciferase-specific siRNA and DTC-Apo lipoplex. Thus, our unique approach to siRNA delivery creates a foundation for the development of a new class of promising therapeutics against hepatitis viruses or hepatocyte genes related to tumor growth.

Identification of proteins highly expressed in the hyphae of Candida albicans by two-dimensional electrophoresis.

The increase in Candida albicans infections is caused by the increase in therapies resulting in immunocompromised patients. One factor required for C. albicans pathogenicity is the morphological transition from yeast to hypha. The protein profiles of whole extracts from yeasts and hyphae were examined using two-dimensional electrophoresis to identify the proteins related to the morphological transition. Over 900 protein spots were visualized by silver staining and 11 spots were increased more than three-fold reproducibly during hyphal differentiation. Six of the 11 spots were identified by peptide mass fingerprints, of which three represented PRA1, two PHR1 and the last TSA1. Vertical streak patterns of Pra1p and Phr1p indicated that post-translational modifications seem to be caused by variable glycosylation. Comparative proteome analysis between the wild-type and the deletion mutants, CAMB43 (deltapra1) and CAS10 (deltaphr1), further confirmed the identity of PRA1 and PHR1. Interestingly, Pra1p was downregulated in phr1-deleted mutants. Only PHR1 transcription was increased, indicating that PRA1 and TSA1 are controlled at the post-translational level.