Journal: Science Advances

Article Title: An ASXL3–thyroid hormone axis in parvalbumin interneurons controls autism-like behaviors

doi: 10.1126/sciadv.aeh2640

Figure Lengend Snippet: ( A ) Illustration of the molecular strategy for the expression of the intact ASXL3 protein facilitated by the Cfa GEP split intein. ( B ) Diagrammatic depiction of the retro-orbital injection technique applied to neonatal mice at postnatal day 3. Created in BioRender. Weng, W. (2026) https://BioRender.com/znoeri1 . ( C and D ) Immunodetection and quantitative analysis of total ASXL3 protein expression levels. AAV-GFP, injection of AAV-PHP.eB encoding GFP. AAV-Asxl3, injection of AAV-PHP.eB encoding Flag-ASXL3 N -Intein N and Intein C -ASXL3 C -HA. ( E to H ) Representative images and quantification of ASXL3 and PV expression in the RSC following AAV delivery. ( I to K ) Examination and quantification of ASXL3 and PV expression in the hippocampus after AAV administration. ( L and M ) Immunofluorescence staining of neurons in the second and third cortical layers, accompanied by quantitative analysis of neuronal counts. ( N and O ) Immunofluorescence staining of the fifth layer of cortical neurons and its number statistics. Data are presented as mean ± SEM, n = 3 mice per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test, with significance denoted by * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The AAV-PHP.eB-hSyn-Flag-Asxl3 N -Intein N , AAV-PHP.eB-hSyn-Intein C -Asxl3 C - 3HA, and AAV-PHP.eB-hSyn-EGFP were procured from PackGene Biotech for retro-orbital injection.

Techniques: Expressing, Injection, Immunodetection, Analysis, Immunofluorescence, Staining

Journal: Cell Discovery

Article Title: The accessory protein ORF3a hijacks the CLCC1 chloride channel to disrupt ER homeostasis in betacoronavirus pathogenesis

doi: 10.1038/s41421-026-00918-0

Figure Lengend Snippet: a AAV-PHP.eB-based transgene delivery strategy involving retro-orbital injection in mice. EGFP-tagged SARS-CoV-2 ORF3a and EGFP were driven by a CAG promoter. b – d SARS-CoV-2 ORF3a expression leads to weight loss and mortality in mice. The wild-type mice were injected with AAV-PHP.eB at postnatal day 22 (P22) and weighed at P40 ( b ). The mice were sacrificed when substantial weight loss ( c ) and movement impairment ( d ) were observed. The dose of AAV-PHP. eB, 1 × 10 11 vg/20 g mice. e, f SARS-CoV-2 ORF3a interacts with endogenous CLCC1 and induces LC3 lipidation, ER-phagy, nucleophagy, and apoptosis in mouse brains. Four mice were sacrificed 28 days post AAV-PHP.eB injection. Brain lysates were prepared for co-IP experiments ( e ) and immunoblot analysis ( f ). In e , GFP-trap beads captured the EGFP-tagged SARS-CoV-2 ORF3a and EGFP. Red arrows indicate EGFP-tagged SARS-CoV-2 ORF3a as input. The relative expression level of endogenous CLCC1 was calculated and normalized ( e ). In f , antibodies against LC3, FAM134B, LMNB1, and caspase-3 were used to detect LC3-I/LC3-II conversion, ER-phagy, nucleophagy, and caspase-3-dependent apoptosis, respectively. g TEM images of hippocampal regions expressing SARS-CoV-2 ORF3a-EGFP or EGFP. Red arrow, the bleb amid NE; red asterisk, the autolysosome; blue arrow, ER whorl-like membrane stacks. N nucleus, M mitochondria, NE nuclear envelope, G Golgi apparatus; E endoplasmic reticulum, L lysosome. h , i Immunofluorescence analysis of mouse brains expressing SARS-CoV-2 ORF3a-EGFP and EGFP. The mice were sacrificed 20 days post AAV-PHP.eB administration. ER stress, misfolded protein accumulation, and protein degradation were assessed using staining for Bip, ubiquitin, and K48-linked ubiquitin, respectively, in the dentate gyrus and olfactory bulbs. j TUNEL assay of the hippocampal dentate gyrus in mice expressing SARS-CoV-2 ORF3a-EGFP vs EGFP control. k Neuroinflammatory profiling in thalamic regions of ORF3a-EGFP-expressing mice. Microglial activation (Iba1) and astrogliosis (GFAP) were assessed by immunostaining. Scale bar, 200 nm in g ; 5 μm in h and i ; 20 μm in j ; 50 μm in k . In b – d , n = 5. The values are presented as the mean ± SD. ** P < 0.01, by Student’s t -test ( c ) and the Mantel‒Cox test ( d ).

Article Snippet: AAV-PHP.eB virus harboring a GFP and SARS-CoV-2 ORF3a cassette (1 × 10 13 vg/mL) was generated by Packgene Biotech and diluted to a final concentration of 50 × 10 10 vg/600 μL in sterile PBS.

Techniques: Injection, Expressing, Co-Immunoprecipitation Assay, Western Blot, Membrane, Immunofluorescence, Staining, Ubiquitin Proteomics, Olfactory, TUNEL Assay, Control, Activation Assay, Immunostaining

Journal: Cell Reports Medicine

Article Title: Self-sufficient killing of malignant tumors by an engineered cancer-selective gene circuit

doi: 10.1016/j.xcrm.2026.102936

Figure Lengend Snippet: Safety and efficacy of P ETS∗ -driven cell lysis (A) Dose-dependent activation of P ETS∗ by different ETS-related transcription factors (ETS TFs) and fusion gene products. HEK293 cells were co-transfected with a synthetic promoter containing 21 tandem GGAA repeats driving SEAP expression (pQL71; P ETS∗ -SEAP-pA) and different amounts of constitutive expression vectors for ETS TFs (ETV1, pQL89; ETV4, pQL88; ETS1, pYSJ24; ELF4, pYSJ37; ELF3, pQL126; ELF5, pYSJ7; EHF, pYSJ6; ETV5, pYSJ1; ETS2, pYSJ4; ERG, pQL91; ETV2, pYSJ17; ELK1, pYSJ15; ELK3, pYSJ5; FLI1, pQL49; SPI1, pYSJ21; SPIB, pYSJ26; and SPIC, pYSJ27), fusion gene products containing ETS TFs (EWS-FEV, pYSJ29; EWS-FLI1, pQL43; EWS-ERG, pQL97; EWS-ETV1, pQL105; and EWS-ETV4, pQL104), or ETS-unrelated fusion genes (PAX3-FOXO1, pQD321; PAX7-FOXO1, pQD301; BCR-ABL, pSL1014; and AML1-ETO, pQZ197). For phosphorylation-dependent ETS family members (e.g., pELK1), cells were co-transfected with a constitutive expression vector for human insulin receptor activating MAPK signaling (pIR) and stimulated with recombinant human insulin (10 μg/mL). SEAP levels in culture supernatants were scored at 24 h post transfection. Data are presented as means ± SD; n = 3 individual experiments. Heatmap shows average log 2 -fold changes (log 2 FC) of SEAP expression for each condition divided by basal SEAP expression levels in non-transfected HEK293 cells. (B) ETS∗ specificity of the therapeutic gene circuit. Human lung adenocarcinoma (A549, NCI-H1299), adriamycin-resistant breast cancer (MCF7/ADR), colorectal cancer (LoVo), renal clear cell carcinoma (786-0), rhabdomyosarcoma (RD), breast adenocarcinoma (MCF-7), cervical cancer (HeLa), and native HEK293 cells were transduced with lentiviral particles created for P hCMV -, P ETS∗ -specific, or P hCMVmin -driven mCherry expression. Flow cytometry analysis (5 × 10 3 cells per group; gating threshold: 10 3 fluorescence units) was performed at 48 h after transduction. Expression of mCherry (data shown in B) and abnormal ETS TFs (data shown in A) was confirmed by quantitative reverse-transcription PCR (RT-qPCR) analysis. (C) Inactivity of P ETS∗ in healthy tissues in vivo . Constitutive (P hCMV -driven), abnormal ETS TF-selective (P ETS∗ -driven), and transcriptionally inactive (P hCMVmin -driven) expression units for firefly luciferase (FLuc) were transferred into AAV particles of the serotypes 2/9 and PHP.eB. Two weeks after intravenous injection of 1 × 10 11 vg AAV particles into healthy C57BL/6 mice, FLuc levels in different tissues (H, heart; Li, liver; Sp, spleen; Lu, lung; Ki, kidney; Br, brain) were recorded by bioluminescence imaging. AAV particles containing P MusAFP - or P hGfaABC1D -driven constructs with known tumor specificity were used as references. Data are shown as the mean ± SEM of n = 4 mice per group. Raw data showing whole-body distribution and normalized luminescence signals in all mice are shown in E. (D) Comparative analysis of different P ETS∗ -inducible cell-killing strategies. 48 h after transduction of A549 cells with lentivirus particles created for P ETS∗ -specific expression of the adenovirus replication gene (E1A), the ganciclovir-inducible suicide gene (HSV-TK), or the pro-apoptotic gene hBax, cells were supplemented with replication-deficient adenoviruses (AD ΔE1A , 5 MOI) or ganciclovir (GCV, 10 μM). Viability of cells was estimated using the CCK8 assay and by crystal violet staining after another 48 h. Data are presented as means ± SD; n = 3 individual experiments. ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001. (E) P ETS∗ remains inactive in normal proliferating tissues. 5 days after establishing an excisional wound healing model in mice, 5 μg of plasmid DNA containing P hSurv -, P hE2F -, or P ETS∗ -driven mCherry expression units were intradermally injected into scar tissues. A P hCMV -driven expression vector was used as positive control for constitutive expression. A P hCMVmin -driven vector was used as negative control indicative for basal expression. Microscopic analysis of DAPI-stained skin tissues (scale bars, 200 μm) was performed at 48 h post transfection. Representative images for n = 3 mice are shown.

Article Snippet: AAV2/9-P ETS∗ -FLuc, AAV2/9-P MusAFP -Fluc, AAV2/9-P hCMV -FLuc, AAV2/9-P hCMVmin -FLuc, AAV-PHP.eB-P ETS∗ -FLuc, AAV-PHP.eB-P hCMV -Fluc, AAV-PHP.eB-P hCMVmin -Fluc and AAV-PHP.eB-P hGfaABC1D -Fluc were produced by PackGene Biotech (Guangzhou, China) using the transfer plasmids pQL120, pQL90, pYW475, pQL121 and pYW470 , respectively.

Techniques: Lysis, Activation Assay, Transfection, Expressing, Phospho-proteomics, Plasmid Preparation, Recombinant, Transduction, Flow Cytometry, Fluorescence, Reverse Transcription, Quantitative RT-PCR, In Vivo, Luciferase, Injection, Imaging, Construct, CCK-8 Assay, Staining, Positive Control, Negative Control

Journal: Nature Communications

Article Title: Incorporating AI-optimized zinc finger proteins enhances the efficiencies and targeting ranges of miniature base editors

doi: 10.1038/s41467-026-75912-x

Figure Lengend Snippet: a SMN2 locus DNA sequence with annotated PAM, protospacer, ZFP binding site, and the targeting base. Targeted nucleotide (A4) editing efficiencies at SMN2 exon 7 by dCas12f-ABE and zmBEs in 293XT cells were measured via HTS ( b ); bystander editing is shown in ( c ). Error bars indicate SD from five independent experiments. Statistical tests used Dunnett T3 tests after Welch and Brown-Forsythe ANOVA. d 293XT cells were transfected with dCas12f-ABE, with or without the indicated ZFP and matched sgRNA. SMN2 exon 7 inclusion was assessed by RT-PCR. Unedited cells served as a mock; GAPDH as an endogenous control. e Quantified SMN2 exon 7 inclusion from ( d ). Error bars represent SD from three independent experiments. Statistics used Tukey tests after ordinary one-way ANOVA. f The top 25 PLM-predicted ZFP variants were cloned into dCas12f-ABE. Resultant zmBEs were transfected into 293XT cells with the SMN2 sgRNA. T:A(6)>C:G substitutions were analyzed by HTS. Data represent three independent experiments. Color coding marks ZFP mutation positions; dashed line represents activity of zmBE with original ZFP. Bottom : Schematic of SMN2 -targeting ZFP. g sgRNAs with different scaffolds were co-transfected with zmBE 1 into 293XT cells. T:A(6)>C:G editing efficiencies were determined by HTS. Error bars indicate SD from three independent experiments. Statistics used Tukey tests after ordinary one-way ANOVA. h Schematic of AAV vector containing zmABE and SMN2 -targeting sgRNA. i Schematic illustrating AAV delivery into SMA mice via CSF and IV injection. j Righting reflex time of SMA mice on P12. AAV-zmABE: n = 15; mock (untreated SMA mice): n = 15. Lines denote medians; each dot represents one mouse. Statistics by Mann-Whitney test, two-sided. k SMA mouse survival curves (AAV-zmABE: n = 15; mock: n = 15). Medians are indicated. Statistical analysis by Mantel-Cox test, two-sided. l T:A(6)>C:G editing was quantified by HTS in GFP-positive spinal cord nuclei from AAV-zmABE-treated SMA mice ( n = 5). Mock: untreated SMA mice ( n = 10). Each dot denotes one animal. Dashed and dotted lines indicate medians and quartiles, respectively. Statistical analysis by Mann-Whitney test, two-sided. Source data are provided as a Source Data file.

Article Snippet: ZmABE and SMN2 sgRNA contained PHP.eB AAV particles (1 × 1013 vg/mL; sequence was listed in Supplementary Data ) were produced by PackGene Biotech (China).

Techniques: Sequencing, Binding Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Control, Clone Assay, Mutagenesis, Activity Assay, Plasmid Preparation, IV Injection, MANN-WHITNEY