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Comprehensive genome editing and signaling pathway engineering evolve potent and safe adenoviral producer cell

ABSTRACT: Dramatic attention has been drawn to adenovirus as a vehicle for vaccine and cancer therapy, due to the capabilities of annihilating tumor cells and stimulating host immune response. However, few producer cells are perfect for adenoviral manufacture. Herein, a novel genome editing strategy by CRISPR-Cas9 technology and sgRNA library not only modulated the cellular gene expression profile systemically that enhanced subgroup B and C adenovirus yields by 3~7 folds, but also substituted naïve adenovirus sequence in HEK293 cells with a recombinant E1 transgene, drastically suppressing the production of clinically pathogenic replication-competent adenovirus. Moreover, directed evolution towards a serum-free gradient in bioreactor generated an adaptive clone MC09 with both safety and potency for scalable adenoviral manufacture in large-scale clinical applications. Molecularly, combined inhibition of carbon metabolism, apoptosis and lysosome pathways curtailed adenoviral yield in MC09 cells, as demonstrated by triple knockdown of H6PD, Lamin A/C and caspase-3. Concomitantly, overexpression of these molecules further boosted biosynthesis of adenoviral vectors, highlighting a cooperative effect among multiple pathways to amplify adenovirus production.

ORGANISM(S): Homo Sapiens

SUBMITTER: Hu Zhou

PROVIDER: PXD069719 | iProX | Tue Oct 21 00:00:00 GMT+01:00 2025

REPOSITORIES: iProX

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Asymmetrically-substituted quadruplex-binding napththalene diimides showing potent activity in models for pancreatic cancer

Project description:Targeting quadruplexes in cancer genes and pathways by a novel class of asymmetrically-substituted napththalene diimides (SOP1812) with potent cellular and antitumour activity in models for pancreatic cancer.

2020-06-04 | GSE151741 | GEO

Assembly of a nuclear lamin coil 2 fragment

Project description:Nuclear structure and function are governed by lamins, which are intermediate filaments mostly consisting of α-helices. Different lamin assembly models have been proposed based on low resolution or fragmented structures. However, their assembly mechanisms at the molecular level are poorly understood. The structure shows the anti-parallel arrangement of two coiled-coil dimers, which is important for the assembly process. We further discovered a new interaction of a coil 2 by using chemical cross-linking and mass analysis, of which the results were deposited in the PRIDE identifier PXD013144. Here we showed that a cysteine-substituted coil 2 R388C segment (286-400 amino acid residues of lamin) was dimerized by the chemical crosslinking. Our findings also provide a molecular basis for the assembly mechanisms of other intermediate filaments.

2025-12-15 | PXD014029 | Pride

Loss of Lamin A/C in myeloid cells promotes lung metastasis through Gfi-1 and C/EBPe-mediated granulocytic differentiation

Project description:The immune suppressive tumor microenvironment contributes to metastatic spread. In particular, tumor-associated myeloid cells, which differ from normal myeloid cells, suppress cytotoxic T lymphocyte-mediated anti-tumor immunity. However, the underlying molecular mechanisms for tumor-associated myeloid lineage differentiation and functional properties are not well understood. Here we report a lack of Lamin A/C, a nuclear lamina protein associated with chromatin remodeling, in tumor-associated granulocytic myeloid cells. Using mouse models of myeloid specific Lamin A/C knockout, and mouse models of triple negative breast cancer (TNBC), we discovered that the loss of Lamin A/C drives CD11b+Ly6G+ granulocytic lineage differentiation. Mechanistically, loss of Lamin A/C increased H3K4me3 and led to the up-regulation of transcription factors C/EBPe and Gfi-1, which are critical for granulocytic lineage differentiation. In addition, loss of Lamin A/C changes the production of inflammatory chemokines and decreases anti-tumor immunity, leading to increased lung metastasis. Our data provide mechanistic understanding of myeloid lineage differentiation and function in the immune suppressive microenvironment in TNBC metastasis.

2019-12-28 | GSE141124 | GEO

Deleterious assembly of the lamin A/C mutant p.S143P causes ER stress in familial dilated cardiomyopathy

Project description:Mutation of the LMNA gene, encoding nuclear lamin A and lamin C (hereafter lamin A/C), is a common cause of familial dilated cardiomyopathy (DCM). Among Finnish DCM patients, the founder mutation c.427T>C (p.S143P) is the most frequently reported genetic variant. Here, we show that p.S143P lamin A/C is more nucleoplasmic and soluble than wild-type lamin A/C and accumulates into large intranuclear aggregates in a fraction of cultured patient fibroblasts as well as in cells ectopically expressing either FLAG- or GFP-tagged p.S143P lamin A. In fluorescence loss in photobleaching (FLIP) experiments, non-aggregated EGFP-tagged p.S143P lamin A is significantly more dynamic. In in vitro association studies, p.S143P lamin A failed to form appropriate filament structures but instead assembled into disorganized aggregates similar to those observed in patient cell nuclei. A whole genome expression analysis revealed an elevated unfolded protein response (UPR) in cells expressing p.S143P lamin A/C. Additional endoplasmic reticulum (ER) stress induced by tunicamycin reduced the viability of mutant lamin expressing cells further. In summary, p.S143P lamin A/C affects normal lamina structure and influences the cellular stress response, homeostasis and viability. Total RNA obtained from 4 control and 7 patient cell lines

2016-06-10 | E-GEOD-82290 | biostudies-arrayexpress

Potent and uniform fetal hemoglobin induction via base editing.

Project description: Not available

| S-EPMC10722557 | biostudies-literature

Secretory MPP3 reinforce myeloid differentiation trajectory and amplify myeloid cell production

Project description:Secretory MPP3 reinforce myeloid differentiation trajectory and amplify myeloid cell production

| PRJNA753832 | ENA

A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2alpha [ChIP-Seq]

Project description:Lamins are components of the peripheral nuclear lamina and interact with heterochromatic genomic regions, termed lamina-associated domains (LADs). In contrast to Lamin B11, lamin A/C also localizes throughout the nucleus, where it associates with the chromatin-binding protein lamina-associated polypeptide (LAP) 2alpha. Here we show lamin A/C also interacts with euchromatin, as determined by chromatin immunoprecipitation analyses of eu- and heterochromatin-enriched samples. By way of contrast, Lamin B11 was only found associated with heterochromatin. Euchromatic regions occupied by lamin A/C overlap with those bound by LAP2alpha, the depletion of which shifts binding of lamin A/C towards more heterochromatic regions. These alterations in lamin A/C chromatin interaction affect epigenetic histone marks in euchromatin without significantly affecting gene expression, while loss of lamin A/C in heterochromatic regions increased gene expression. Our data show a novel role of nucleoplasmic lamin A/C and LAP2alpha in regulating euchromatin. Examination of Lamin A/C, Lamin B1 and Lap2a DNA binding in Lap2alpha +/+ and Lap2a -/- cells and according changes in Histone modifications and gene expression

2016-01-19 | E-GEOD-70147 | biostudies-arrayexpress

Precise Gene Editing of Pathogenic Lamin A Mutations Corrects Cardiac Disease

Project description:Mutations in the lamin A (LMNA) gene, which encodes the lamin A and C proteins, cause severe human diseases collectively known as laminopathies. These conditions are often devastating and lack effective therapies. In this study, we developed precise base editing (BE) strategies targeting the human LMNA gene variants L35P and R249Q, which cause Congenital Muscular Dystrophy (CMD) and Dilated Cardiomyopathy with Conduction Defects (DCM-CD), respectively. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carrying the R249Q mutation displayed nuclear aberrations, DNA damage, and abnormal Ca2+ transients. Similarly, L35P iPSC-CMs exhibited abnormal contraction, DNA damage, and reduced lamin A/C protein expression. We also generated novel “humanized” mouse models carrying these pathogenic human mutations. R249Q homozygous mice exhibited cardiac conduction abnormalities, cardiac arrhythmias, and premature death. Mice with the homozygous L35P mutation displayed severe muscle-wasting and reduced lifespan, while heterozygous L35P mice displayed dilated cardiomyopathy (DCM). We developed an adenine base editing (ABE) approach for correcting the R249Q mutation and a cytosine base editing (CBE) strategy for the L35P variant. Precise correction of these mutations in iPSC-CMs successfully rescued all of the in vitro abnormalities. Furthermore, delivery of the BE components using adeno-associated virus (AAV) prevented the pathological phenotypes and extended longevity of mice carrying the LMNA L35P and the R249Q mutations. These results demonstrate the efficacy of ABE and CBE in correcting pathogenic LMNA mutations that cause cardiac disease, highlighting BE as a promising therapeutic approach for human laminopathies.

2025-09-23 | GSE308777 | GEO

Potent and uniform fetal hemoglobin induction via base editing [Cut & Run]

Project description:Genetic manipulations to increase fetal hemoglobin (HbF, α2γ2) in postnatal red blood cells (RBCs) can alleviate β-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease, which creates uncontrolled indel mixtures, or adenine base editors (ABE), which generate more precise nucleotide changes. The most potent modification was ABE generation of γ-globin (HBG1/HBG2) −175 A>G, which creates a promoter binding motif for the transcriptional activator TAL1. In erythroid colonies with >87.5% on-target edits, those with −175 A>G expressed 81 ± 7% HbF, versus 17 ± 11% in unedited controls. In comparison, HbF levels were lower and more variable in erythroid cells modified with either of two Cas9 nuclease strategies with similar editing efficiencies, being 32 ± 19% when a BCL11A repressor binding motif in the γ-globin promoter was targeted and 52 ± 13% when the +58 BCL11A erythroid enhancer was targeted. Contrary to currently accepted models of γ-globin regulation, HbF levels varied significantly with different Cas9 indels that disrupted the γ-globin promoter BCL11A binding motif. The −175 A>G base edit also induced HbF more potently than did the Cas9 nuclease approaches in RBCs generated after transplantation of modified normal or SCD patient CD34+ cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 nuclease can cause unexpected variations in biological outcomes that can be circumvented by base editing, with important implications for therapeutic gene editing efforts.

2023-04-28 | GSE228819 | GEO

Project description:Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease that is frequently caused by a de novo point mutation at position 1824 in LMNA. This mutation activates a cryptic splice donor site in exon 11, and leads to an in-frame deletion within the prelamin A mRNA and the production of a dominant negative lamin A protein, known as progerin. Here we show that HGPS cells experience genome-wide alterations in patterns of H3K27me3 deposition, changes in the associations of genomic loci with nuclear lamin A/C, and, at late passages, genome-wide loss of spatial compartmentalization of active and inactive chromatin domains that characterizes chromosome folding in normal cells. We further demonstrate that the H3K27me3 changes associate with gene expression alterations in HGPS cells. Our results support a model that the accumulation of progerin in the nuclear lamina leads to altered H3K27me3 marks in heterochromatin, possibly through the down-regulation of EZH2, and disrupts heterochromatin-lamina interactions. These changes may then lead to the genomic disorganization and changes in transcriptional regulation we observe in HGPS fibroblasts. ChIP-Seq was performed for H3K27me3 and lamin A/C in the human genome in three primary fibroblast cell lines: an HGPS patient (HGPS), normal cells from the father of the HGPS patient (Father), and age-matched normal cells (Age Control). Two biological replicates were performed.

2012-10-30 | E-GEOD-41757 | biostudies-arrayexpress

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