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.

Relationships of Lower Lung Fibrosis, Pleural Disease, and Lung Mass with Occupational, Household, Neighborhood, and Slate Roof-Dense Area Residential Asbestos Exposure.

This study aimed to evaluate the relationship between various asbestos exposure routes and asbestos-related disorders (ARDs). The study population comprised 11,186 residents of a metropolitan city who lived near asbestos factories, shipyards, or in slate roof-dense areas. ARDs were determined from chest X-rays indicating lower lung fibrosis (LFF), pleural disease (PD), and lung masses (LMs). Of the subjects, 11.2%, 10.4%, 67.2% and 8.3% were exposed to asbestos via occupational, household, neighborhood, and slate roof routes, respectively. The odds ratio (OR) of PD from household exposure (i.e., living with asbestos-producing workers) was 1.9 (95% confidence interval: 0.9⁻4.2), and those of LLF and PD from neighborhood exposure, or residing near asbestos factories) for <19 or >20 years, or near a mine, were 4.1 (2.8⁻5.8) and 4.8 (3.4⁻6.7), 8.3 (5.5⁻12.3) and 8.0 (5.5⁻11.6), and 4.8 (2.7⁻8.5) and 9.0 (5.6⁻14.4), respectively. The ORs of LLF, PD, and LM among those residing in slate-dense areas were 5.5 (3.3⁻9.0), 8.8 (5.6⁻13.8), and 20.5 (10.4⁻40.4), respectively. Substantial proportions of citizens residing in industrialized cities have potentially been exposed to asbestos, and various exposure routes are associated with the development of ARDs. Given the limitations of this study, including potential confounders such as socioeconomic status, further research is needed.

Sequencing and characterization of Varicella-zoster virus vaccine strain SuduVax.

BACKGROUND: Varicella-zoster virus (VZV) causes chickenpox in children and shingles in older people. Currently, live attenuated vaccines based on the Oka strain are available worldwide. In Korea, an attenuated VZV vaccine has been developed from a Korean isolate and has been commercially available since 1994. Despite this long history of use, the mechanism for the attenuation of the vaccine strain is still elusive. We attempted to understand the molecular basis of attenuation mechanism by full genome sequencing and comparative genomic analyses of the Korean vaccine strain SuduVax. RESULTS: SuduVax was found to contain a genome that was 124,759 bp and possessed 74 open reading frames (ORFs). SuduVax was genetically most close to Oka strains and these Korean-Japanese strains formed a strong clade in phylogenetic trees. SuduVax, similar to the Oka vaccine strains, underwent T- > C substitution at the stop codon of ORF0, resulting in a read-through mutation to code for an extended form of ORF0 protein. SuduVax also shared certain deletion and insertion mutations in ORFs 17, 29, 56 and 60 with Oka vaccine strains and some clinical strains. CONCLUSIONS: The Korean VZV vaccine strain SuduVax is genetically similar to the Oka vaccine strains. Further comparative genomic and bioinformatics analyses will help to elucidate the molecular basis of the attenuation of the VZV vaccine strains.

AtHMA1 contributes to the detoxification of excess Zn(II) in Arabidopsis.

AtHMA1 is a member of the heavy metal-transporting ATPase family. It exhibits amino acid sequence similarity to two other Zn(II) transporters, AtHMA2 and AtHMA4, and contains poly-His motifs that are commonly found in Zn(II)-binding proteins, but lacks some amino acids that are typical for this class of transporters. AtHMA1 localizes to the chloroplast envelope. In comparison with wild-type plants, we observed a more pronounced sensitivity in the presence of high Zn(II) concentrations, and increased accumulation of Zn in the chloroplast of T-DNA insertional mutants in AtHMA1. The Zn(II)-sensitive phenotype of AtHMA1 knock-out plants was complemented by the expression of AtHMA1 under the control of its own promoter. The Zn(II)-transporting activity of AtHMA1 was confirmed in a heterologous expression system, Saccharomyces cerevisiae. The sensitivity of yeast to high concentrations of Zn(II) was altered by the expression of AtHMA1 lacking its N-terminal chloroplast-targeting signal. Taken together, these results suggest that under conditions of excess Zn(II), AtHMA1 contributes to Zn(II) detoxification by reducing the Zn content of Arabidopsis thaliana plastids.

Expression of the novel wheat gene TM20 confers enhanced cadmium tolerance to bakers' yeast.

Cadmium causes the generation of reactive oxygen species, which in turn causes cell damage. We isolated a novel gene from a wheat root cDNA library, which conferred Cd(II)-specific tolerance when expressed in yeast (Saccharomyces cerevisiae). The gene, which we called TaTM20, for Triticum aestivum transmembrane 20, encodes a putative hydrophobic polypeptide of 889 amino acids, containing 20 transmembrane domains arranged as a 5-fold internal repeating unit of 4 transmembrane domains each. Expression of TaTM20 in yeast cells stimulated Cd(II) efflux resulting in a decrease in the content of yeast intracellular cadmium. TaTM20-induced Cd(II) tolerance was maintained in yeast even under conditions of reduced GSH. These results demonstrate that TaTM20 enhances Cd(II) tolerance in yeast through the stimulation of Cd(II) efflux from the cell, partially independent of GSH. Treatment of wheat seedlings with Cd(II) induced their expression of TaTM20, decreasing subsequent root Cd(II) accumulation and suggesting a possible role for TaTM20 in Cd(II) tolerance in wheat.

Rice P1B-type heavy-metal ATPase, OsHMA9, is a metal efflux protein.

P(1B)-type heavy-metal ATPases (HMAs) are transmembrane metal-transporting proteins that play a key role in metal homeostasis. Despite their importance, very little is known about their functions in monocot species. We report the characterization of rice (Oryza sativa) OsHMA9, a member of the P(1B)-type ATPase family. Semiquantitative reverse transcription-polymerase chain reaction analyses of seedlings showed that OsHMA9 expression was induced by a high concentration of copper (Cu), zinc (Zn), and cadmium. We also determined, through promoterbeta-glucuronidase analysis, that the main expression was in the vascular bundles and anthers. The OsHMA9:green fluorescence protein fusion was localized to the plasma membrane. Heterologous expression of OsHMA9 partially rescued the Cu sensitivity of the Escherichia coli copA mutant, which is defective in Cu-transporting ATPases. It did not rescue the Zn sensitivity of the zntA mutant, which is defective in Zn-transporting ATPase. To further elucidate the functional roles of OsHMA9, we isolated two independent null alleles, oshma9-1 and oshma9-2, from the T-DNA insertion population. Mutant plants exhibited the phenotype of increased sensitivity to elevated levels of Cu, Zn, and lead. These results support a role for OsHMA9 in Cu, Zn, and lead efflux from the cells. This article is the first report on the functional characterization of a P(1B)-type metal efflux transporter in monocots.