Yoko Fujita

Bio: Yoko Fujita is an academic researcher from Kyoto University. The author has contributed to research in topics: Virus & Influenza A virus. The author has an hindex of 5, co-authored 10 publications receiving 132 citations.

Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping.

Abstract: Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.

A Monoclonal Antibody to the 15,000 Dalton Protein Associated with Porcine Pulmonary Surfactant

Abstract: From a surface active fraction of porcine lung lavage fluid, separated by discontinuous sucrose density gradient ultracentrifugation, a protein with a nominal molecular weight (MW) of 15,000 daltons was isolated by sequential extraction with several buffers, including one containing deoxycholate. A monoclonal antibody was prepared from a hybrid cell (8B5E) obtained by fusing a myeloma cell, X63.Ag8.653, with spleen cells of BALB/c mice immunized with the protein. With immunoblotting technique, the antibody was found to be specific to the 15,000 dalton protein and did not react with another surfactant-associated protein with a nominal MW of 38,000 daltons. The antibody's IgG subclass was IgG1 and the light chain was kappa. In immunohistochemical studies using biotinylated antibody, peroxidase reaction products were localized selectively at inclusions of alveolar wall cells which were located chiefly at the alveolar corners. These results strongly suggest that this 15,000 dalton protein was localized in inclusions of alveolar wall cells and did not originate from other larger surfactant-associated proteins degraded after secretion into alveolar space.

Pulmonary damage induced in mice by a monoclonal antibody to proteins associated with pig pulmonary surfactant.

Abstract: Severe pulmonary damage was induced in mice inoculated with hybridomas (8B5E) secreting a monoclonal antibody to the 15,000-dalton protein associated with pig pulmonary surfactant. These mice exhibited severe respiratory distress starting 8-9 days after inoculation and died. Microscopically, lungs were airless and congested with hyaline membrane formation in patent terminal airways. Purified antibody from this hybridoma also induced similar damage in mouse lungs. However, neither hybridomas (1B6A) nor purified antibody, which is specific to pig 35,000-dalton protein but not to the mouse counterpart, induced these changes. Electron microscopically, many unexpanded lamellar bodies were seen floating in the edema fluid in these damaged lungs, and fragmentary lipid membranes were found in the electron dense material around these lamellar bodies, suggesting disintegration of these structures. Alveolar epithelial cells were desquamated, leaving the basement membranes bare, and mouse C3 was demonstrated in the d...

Function and regulation of expression of pulmonary surfactant-associated proteins.

Abstract: RESEARCH PAPERS Proteins Ostrich crystallins. Structural characterization of 8-crystallin with enzymic activity Characterization of antibodies to the glycosyl-phosphatidylinositol membrane anchors of mammalian proteins Identification of valine/leucine/isoleucine and threonine/alanine/ glycine proton-spin systems of Escherichia coli adenylate kinase by selective deuteration and selective protonation

The proteins of the surfactant system

Abstract: The structural and functional integrity of pulmonary surfactant depends on several specific proteins. Two of these, SP-A and SP-D, are large and water-soluble, while SP-B and SP-C are small and very hydrophobic. SP-A is an 18-mer of 26 kDa polypeptide chains and contains N-linked oligosaccharides. Structurally, it can be characterized as a collagen/lectin hybrid. Together with SP-B, SP-A is required for conversion of secreted endogenous surfactant to tubular myelin in the alveolar lining. It also regulates surfactant secretion and reuptake of surfactant lipids by type II cells; these functions are probably receptor mediated. SP-D, a 12-mer of 39 kDa polypeptide chains, is a collagenous glycoprotein with structural similarities to C-type lectins. Both SP-A and SP-D stimulate alveolar macrophages. SP-B is a 79-residue polypeptide that contains three intrachain disulphide bridges. It exists mainly as a homodimer, which is strongly positively charged and may selectively remove anionic and unsaturated lipid species from the alveolar surface film, thereby increasing surface pressure. SP-C is a mainly alpha-helical, extraordinarily hydrophobic polypeptide containing 35 amino acid residues and covalently linked palmitoyl groups. Its alpha-helical portion is inserted into surfactant lipid bilayers. SP-C accelerates the adsorption of lipid bilayers to an interfacial monolayer. In babies with respiratory distress syndrome, the clinical response to treatment with surfactant containing SP-B and SP-C is much faster than in babies treated with protein-free synthetic surfactant. We speculate that, in the near future, surfactant preparations based on recombinant hydrophobic proteins will be available for clinical use.

The efficacy and safety of KL4-surfactant in preterm infants with respiratory distress syndrome.

Abstract: The present study was undertaken to determine if a synthetic peptide, KLLLLKLLLLKLLLLKLLLLK (KL4), in which K = lysine and L = leucine, in an aqueous dispersion of phospholipids (DPPC and POPG), would expand pulmonary alveoli and improve gas exchange in premature human infants with respiratory distress syndrome (RDS). The KL4 peptide was synthesized to resemble the amino acid pattern of surfactant protein B (SP-B). Forty-seven infants with RDS were treated within 4 h of birth with the KL4-peptide/phospholipid mixture, called KL4-Surfactant. The average arterial-to-alveolar oxygen tension ratios (a/A O2) of 39 patients included in efficacy analyses rose from pretreatment values of 0.14 +/- 0.02 (mean +/- SEM) to 0.40 +/- 0.04 (normal value > or = 0.40) by 12 h of age. Mean airway pressures and oxygenation index values fell concomitantly, and expansion of the lungs was observed on radiographs. The median duration of mechanical ventilation was 5.0 d. Of the 39 included infants, 29 required only a single dose. Radiographic data indicate that those patients requiring a second instillation of KL4-Surfactant but not showing a sustained rise in a/A O2 ratios did, in fact, exhibit expansion of alveoli in the lung. There were no RDS-related deaths; the incidence of complications was no higher than found in other comparable published studies. The data demonstrate that the synthetic peptide, KL4, which mimics the hydrophobic and hydrophilic pattern of SP-B, when formulated in an aqueous dispersion with the phospholipids DPPC and POPG, creates a strong and durable surfactant activity as judged by expansion of pulmonary alveoli and improvement of gas exchange in infants with RDS.