Dendritic-cell-targeting virus-like particles as potent mRNA vaccine carriers.

Messenger RNA vaccines lack specificity for dendritic cells (DCs)-the most effective cells at antigen presentation. Here we report the design and performance of a DC-targeting virus-like particle pseudotyped with an engineered Sindbis-virus glycoprotein that recognizes a surface protein on DCs, and packaging mRNA encoding for the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or for the glycoproteins B and D of herpes simplex virus 1. Injection of the DC-targeting SARS-CoV-2 mRNA vaccine in the footpad of mice led to substantially higher and durable antigen-specific immunoglobulin-G titres and cellular immune responses than untargeted virus-like particles and lipid-nanoparticle formulations. The vaccines also protected the mice from infection with SARS-CoV-2 or with herpes simplex virus 1. Virus-like particles with preferential uptake by DCs may facilitate the development of potent prophylactic and therapeutic vaccines.

Modified lentiviral globin gene therapy for pediatric ß0/ß0 transfusion-dependent ß-thalassemia: A single-center, single-arm pilot trial.

ß0/ß0 thalassemia is the most severe type of transfusion-dependent ß-thalassemia (TDT) and is still a challenge facing lentiviral gene therapy. Here, we report the interim analysis of a single-center, single-arm pilot trial (NCT05015920) evaluating the safety and efficacy of a ß-globin expression-optimized and insulator-engineered lentivirus-modified cell product (BD211) in ß0/ß0 TDT. Two female children were enrolled, infused with BD211, and followed up for an average of 25.5 months. Engraftment of genetically modified hematopoietic stem and progenitor cells was successful and sustained in both patients. No unexpected safety issues occurred during conditioning or after infusion. Both patients achieved transfusion independence for over 22 months. The treatment extended the lifespan of red blood cells by over 42 days. Single-cell DNA/RNA-sequencing analysis of the dynamic changes of gene-modified cells, transgene expression, and oncogene activation showed no notable adverse effects. Optimized lentiviral gene therapy may safely and effectively treat all ß-thalassemia.

In vivo CRISPR gene editing in patients with herpetic stromal keratitis.

In vivo CRISPR gene therapy holds large clinical potential, but the safety and efficacy remain largely unknown. Here, we injected a single dose of herpes simplex virus 1 (HSV-1)-targeting CRISPR formulation in the cornea of three patients with severe refractory herpetic stromal keratitis (HSK) during corneal transplantation. Our study is an investigator-initiated, open-label, single-arm, non-randomized interventional trial at a single center (NCT04560790). We found neither detectable CRISPR-induced off-target cleavages by GUIDE-seq nor systemic adverse events for 18 months on average in all three patients. The HSV-1 remained undetectable during the study. Our preliminary clinical results suggest that in vivo gene editing targeting the HSV-1 genome holds acceptable safety as a potential therapy for HSK.

Targeting herpes simplex virus with CRISPR-Cas9 cures herpetic stromal keratitis in mice.

Herpes simplex virus type 1 (HSV-1) is a leading cause of infectious blindness. Current treatments for HSV-1 do not eliminate the virus from the site of infection or latent reservoirs in the trigeminal ganglia. Here, we target HSV-1 genomes directly using mRNA-carrying lentiviral particles that simultaneously deliver SpCas9 mRNA and viral-gene-targeting guide RNAs (designated HSV-1-erasing lentiviral particles, termed HELP). We show that HELP efficiently blocks HSV-1 replication and the occurrence of herpetic stromal keratitis (HSK) in three different infection models. HELP was capable of eliminating the viral reservoir via retrograde transport from corneas to trigeminal ganglia. Additionally, HELP inhibited viral replication in human-derived corneas without causing off-target effects, as determined by whole-genome sequencing. These results support the potential clinical utility of HELP for treating refractory HSK.

Lentiviral delivery of co-packaged Cas9 mRNA and a Vegfa-targeting guide RNA prevents wet age-related macular degeneration in mice.

Therapeutic genome editing requires effective and targeted delivery methods. The delivery of Cas9 mRNA using adeno-associated viruses has led to potent in vivo therapeutic efficacy, but can cause sustained Cas9 expression, anti-Cas9 immune responses and off-target edits. Lentiviral vectors have been engineered to deliver nucleases that are expressed transiently, but in vivo evidence of their biomedical efficacy is lacking. Here, we show that the lentiviral codelivery of Streptococcus pyogenes Cas9 mRNA and expression cassettes that encode a guide RNA that targets vascular endothelial growth factor A (Vegfa) is efficacious in a mouse model of wet age-related macular degeneration induced by Vegfa. A single subretinal injection of engineered lentiviruses knocked out 44% of Vegfa in retinal pigment epithelium and reduced the area of choroidal neovascularization by 63% without inducing off-target edits or anti-Cas9 immune responses. Engineered lentiviruses for the transient expression of nucleases may form the basis of new treatments for retinal neovascular diseases.

Salinibius halmophilus gen. nov., sp. nov., isolated from a marine solar saltern.

A novel Gram-stain-negative, facultatively anaerobic, flagellated and spiral-shaped bacterium, designated WDS2A16AT was isolated from a marine solar saltern in Weihai, PR China. Growth was observed at 20-40 °C (optimal 33-37 °C), 1-15 % (w/v) NaCl (optimal 3-4 %) and pH 6.0-9.0 (optimal pH 7.5). Major cellular fatty acids (>10 %) were C18 : 1ω7c and C16 : 0. Phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid were detected as the predominant polar lipids. The sole respiratory quinone was Q-8. The DNA G+C content of strain WDS2A16AT was 48.5 mol%. The 16S rRNA gene sequence similarities of WDS2A16AT with other species were less than 91 %. The average nucleotide identity, in silico DNA-DNA hybridization and amino acid identity of strain WDS2A16AT with the most related strain Gynuella sunshinyii YC6258 T were 66.1, 19.3 and 48.1 %, respectively. Comparative analysis of 16S rRNA gene sequences and phenotypic characterization indicated that strain WDS2A16AT represents a novel species in a new genus, for which the name Salinibius halmophilus gen. nov., sp. nov. is proposed. The type strain is WDS2A16AT (=KCTC 52225T=MCCC 1H00139T).

Bacillus marinisedimentorum sp. nov., isolated from marine sediment.

A novel Gram-stain-positive, motile and facultatively anaerobic strain, designated NC2-31T, was isolated from sediment from the coast of Weihai, PR China. Optimal growth occurred at 37 °C, pH 7.5 and with 2.0-3.0 % (w/v) NaCl. MK-7 was the major respiratory quinone. Meso-diaminopimelic acid was a diagnostic diamino acid in the peptidoglycan. The major polar lipids of NC2-31T were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). The genomic DNA G+C content of the strain was 46.3 mol%. The predominant cellular fatty acids (>10.0 %) of NC2-31T were iso-C15 : 0 (18.9 %), anteiso-C15 : 0 (15.8 %), summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) (15.3 %) and iso-C16 : 0 (10.3 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that NC2-31T should be classified as representing a member of the genus Bacillus. Based on data from the current polyphasic study, NC2-31T represents a novel species within the genus Bacillus, for which the name Bacillusmarinisedimentorum sp. nov. is proposed with type strain NC2-31T (=KCTC 33721T=MCCC 1K01239T).

Rhodosalinus sediminis gen. nov., sp. nov., isolated from marine saltern.

A novel Gram-stain-negative, moderately halophilic, motile, facultatively anaerobic and rod-shaped strain, designated WDN1C137T, was isolated from a marine saltern at Wendeng, PR China. Optimal growth occurred at 40 °C, pH 7.5 and with 7.0 % (w/v) NaCl. Q-10 was the sole respiratory quinone. The major cellular fatty acids (>10.0 %) in WDN1C137T were C18 : 1ω7c (46.2 %), cyclo C19 : 0ω8c (18.7 %) and C16 : 0 (12.3 %). The major polar lipids were phosphatidylglycerol, phosphoglycolipid, phosphatidylcholine, one unidentified glycolipid, one unidentified lipid, one unidentified aminolipid and two unidentified phospholipids. The genomic DNA G+C content was 70.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that WDN1C137T shared the highest similarity (94.5 %) to Roseivivax jejudonensis KCTC 42110T, followed by Roseivivax halodurans JCM 10272T (94.2 %) and Roseivivax roseus DSM 23042T (94.1 %). WDN1C137T formed a separate branch from the closely related genera Roseivivax, Loktanella, Paracoccus and Cribrihabitans within the family Rhodobacteraceae, which indicated that it represented a novel genus in the phylogenetic tree. On the basis of the data from the current polyphasic study, the isolate is proposed to represent a novel species of a novel genus within the family Rhodobacteraceae, with the name Rhodosalinus sediminis gen. nov., sp. nov. The type strain of the type species is WDN1C137T (=KCTC 52478T=MCCC 1H00170T).

Marinobacter salexigens sp. nov., isolated from marine sediment.

A novel bacterium, designated as strain HJR7T, was isolated from a marine sediment sample collected from the coastal area of Weihai, China (121° 57' E, 37° 29' N). Cells were Gram-stain-negative, facultative anaerobic, non-motile and rod-shaped. The temperature, pH and NaCl ranges for growth were determined as 4-40 °C, pH 6.5-9.5 and 0.5-15.0 % (w/v), respectively. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain HJR7T belongs to the genus Marinobacter in the family Alteromonadaceae. The most closely related species were Marinobacter aromaticivorans (97.6 % 16S rRNA gene sequence similarity) and Marinobacter maritimus (97.3 % similarity). Ubiquinone 9 (Q-9) was the only respiratory quinone detected in strain HJR7T. The major fatty acids of strain HJR7T were C12 : 0, C16 : 0, C16 : 0 N alcohol, C18 : 1ω9c and C18 : 3ω6, 9, 12c. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, and an unidentified phospholipid. The DNA G+C content of strain HJR7T was 53.7 mol%. On the basis of phylogenetic, genotypic, phenotypic, and chemotaxonomic analyses, strain HJR7T represents a novel species within the genus Marinobacter, for which the name Marinobacter salexigens sp. nov. is proposed. The type strain is HJR7T (=KCTC 52545T=MCCC 1H00176T).

Agarilytica rhodophyticola gen. nov., sp. nov., isolated from Gracilaria blodgettii.

A novel Gram-stain-negative, rod-shaped, non-spore-forming, aerobic, agarolytic bacterium, designated 017T, was isolated from Gracilaria blodgettii collected at the coast of Lingshui county, Hainan province, China. Optimal growth occurred at 28-33 °C (range 15-40 °C), with 3 % (w/v) NaCl (range 2-4 %) and at pH 8.0 (range pH 6.5-8.5). Cells of strain 017T were motile and formed yellow colonies on marine agar 2216. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 017T shared the highest similarity with Teredinibacter turnerae T7902T (94.4 %). The predominant polar lipids of the novel isolate consisted of phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and some other unknown lipids. Major cellular fatty acids (>10 %) were C16 : 0, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c/iso-C15 : 0 2-OH), and the sole respiratory lipoquinone was Q-8. The DNA G+C content of strain 017T was 40.2 mol%. Comparative analysis of 16S rRNA gene sequences and phenotypic characterization indicated that strain 017T represents a novel species in a new genus of the family Cellvibrionaceae, order Cellvibrionales, for which the name Agarilytica rhodophyticola gen. nov., sp. nov. is proposed. The type strain of Agarilytica rhodophyticola is 017T (=KCTC 42584T=MCCC 1H00123T).

Motilimonas eburnea gen. nov., sp. nov., isolated from coastal sediment.

A novel Gram-stain-negative, non-spore-forming, facultatively anaerobic bacterium, designated YH6T, was isolated from marine sediment in Weihai, China. Cells of starin YH6T were motile, straight rods that formed ivory-white colonies on 2216E agar. Optimal growth occurred at 28-33 °C (range 15-37 °C), in the presence of 2-4 % (w/v) NaCl (range 1-8 %) and at pH 7.5-8.5 (range pH 6.5-9.0). The sole respiratory lipoquinone was Q-8, and the major fatty acids (>10 %) were C16 : 0 and summed feature 3 (C16 : 1ω7c/iso-C15 : 0 2-OH). The polar lipids profile of the novel strain consisted of phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and several other unknown lipids (phospholipids, lipid and phosphoaminolipid). The G+C content of the genomic DNA was 46.5 mol%. The closest type strain phylogenetically to strain YH6T was Vibrio variabilis (92.99 % 16S rRNA gene sequence similarity) followed by Paramoritella alkaliphila (92.55 %), Pseudoalteromonas aurantia (92.20 %) and Pseudoalteromonas citrea (92.20 %). Phylogenetic analysis of the 16S rRNA gene sequence placed the novel strain in the order Alteromonadales, class Gammaproteobacteria. On the basis of the 16S rRNA gene sequence data as well as physiological and biochemical characteristics, we concluded that strain YH6T represents a novel species of a new genus. We propose the name of Motilimonas eburnea gen. nov., sp. nov. for this novel species. The type strain of the novel species is YH6T (=KCTC 42594T=MCCC 1H00122T).

Marinicrinis sediminis gen. nov., sp. nov., isolated from marine sediment.

Novel Gram-stain-variable, bent rods or long filaments that were endospore-forming, facultatively anaerobic, oxidase- and catalase-negative, and designated strain NC2-42T, were isolated from sediment on the coast of Weihai, China. Optimal growth occurred at 37 °C, pH 7.5 and with 2-3 % (w/v) NaCl. MK-7 was the sole respiratory quinone and meso-diaminopimelic acid was a diagnostic diamino acid in the peptidoglycan. The polar lipid profile of this novel isolate consisted of phosphatidylglycerol, phosphatidylethanolamine, an unknown phospholipid, an unknown phosphoaminolipid, two unknown glycolipids and an unknown lipid. The major cellular fatty acids in strain NC2-42T were anteiso-C15 : 0, iso-C16 : 0 and C16 : 0. The G+C content of the genomic DNA of strain NC2-42T was 58.11 mol% (HPLC). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NC2-42T showed the highest similarity (92.32 %) to Paenibacillus profundus within the family Paenibacillaceae. Based on data from this taxonomic study using a polyphasic approach, the isolate is proposed to represent a novel species of a new genus within the family Paenibacillaceae, with the name Marinicrinis sediminis gen. nov., sp. nov. The type strain of the type species is NC2-42T (=KCTC 33676T=MCCC 1K01238T).

Aliifodinibius halophilus sp. nov., a moderately halophilic member of the genus Aliifodinibius, and proposal of Balneolaceae fam. nov.

A novel Gram-stain-negative, rod-shaped, facultatively anaerobic, oxidase-negative and catalase-positive bacterium, designated 2W32T, was isolated from a marine solar saltern on the coast of Weihai, Shandong Province, China. Strain 2W32T was tolerant to moderate salt conditions. Optimal growth occurred at 33-37 °C (range 20-45 °C) and pH 7.5-8.0 (range pH 7.0-8.5) with 6-10 % (w/v) NaCl (range 2-18 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 2W32T shared highest similarity with Aliifodinibius sediminis YIM J21T (94.6 %), Aliifodinibius roseus YIM D15T (94.4 %), Fodinibius salinus YIM C003T (93.6 %), Gracilimonas tropica CL-CB462T (88.6 %) and Balneola vulgaris 13IX/A01/164T (86.4 %) and less than 83.0 % similarity with other species of the phylum Bacteroidetes. The isolate and closely related species formed a novel family-level clade in the phylum Bacteroidetes. The polar lipid profile of the novel isolate consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified aminolipid, an unidentified glycolipid and an unidentified lipid. The dominant cellular fatty acids (>10 %) were iso-C15 : 0, iso-C17 : 1ω9c and summed feature 3 (C16:1ω7c and/or iso-C15 : 0 2-OH) and the sole respiratory quinone was menaquinone 7 (MK-7). The DNA G+C content of strain 2W32T was 47.5 mol %. Comparative analysis of 16S rRNA gene sequences and characterization indicated that strain 2W32T represents a novel species within the genus Aliifodinibius, for which the name Aliifodinibius halophilus sp. nov. is proposed. The type strain is 2W32T (=KCTC 42497T=CICC 23869T). In addition, a novel family, Balneolaceae fam. nov., is proposed to accommodate the genera Fodinibius, Aliifodinibius, Gracilimonas and Balneola.

Investigation of two Flacourtiaceae plants: Bennettiodendron leprosipes and Flacourtia ramontchi.

Investigations of two Flacourtiaceae plants, Bennettiodendron leprosipes and Flacourtia ramontchi, resulted in the isolation and structural elucidation of six new constituents including two phenolic glycosides ( 1 and 2), one lignan ( 3), two lignan glycosides ( 4 and 5), and a monoterpene glycoside ( 6), together with 22 known compounds ( 7- 28). The structures of the new compounds were elucidated by spectroscopic analysis and chemical methods. The selected isolates 1, 2, 8- 10, 22- 26, and some phenolic glycosides 29- 42 previously isolated from another Flacourtiaceae plant, Itoa orientalis, were tested against snake venom phosphodiesterase I (PDE I) activity. The result indicated that 22, 30, 32, 34, and 40 exhibited moderate inhibitory activities against PDE I with the values ranging from 13.15 to 20.86 %, and 1, 8, 10, 25, 31, 33, 35, 38, 39, and 41 showed weak inhibitory activity.