Biology subjects

Sherman, L.

Publications and source records attributed to Sherman, L..

4 recordsLinked to original sources

bioRxiv2025

Bottlebrush Polymer Conjugates for Enhanced Antisense Oligonucleotide Therapy in Myotonic Dystrophy Type 1

Oligonucleotides are a promising genetic medicine for myotonic dystrophy type 1 (DM1), the most common adult-onset muscular dystrophy. However, poor muscle distribution of nucleic acid drugs after systemic administration has hindered drug development, and no curative treatment exists. Additionally, DM1 pathology requires drug localization to the nucleus, where pathogenic mutant RNA is trapped, posing challenges after endocytosis and endosomal escape. Here, we show that a locked nucleic acid oligonucleotide targeting mutant CUGexp RNA tracts, conjugated to a bottlebrush polymer, exhibited improved muscle distribution and potent correction of DM1-associated splicing at low nanomolar doses in a DM1 mouse model. Significant improvements in myotonia, body weight, and grip strength were observed. The conjugates were well tolerated after 12 weeks of weekly intravenous dosing. These results suggest that bottlebrush polymer bioconjugates may overcome key limitations of traditional antisense drugs for muscular dystrophies, with the potential as potent, durable, and cost-efficient DM1 therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/668589v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@e3961corg.highwire.dtl.DTLVardef@194225dorg.highwire.dtl.DTLVardef@5c470dorg.highwire.dtl.DTLVardef@3cc46a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Large-scale manipulation of radial positioning does not affect most aspects of genome organization

Although the spatial organization of the genome is closely linked to biological function, genome structure is highly stochastic. Within this heterogeneity, specific function-associated structural elements must be maintained, even when the nucleus is deformed due to physiological physical constraints. The radial positioning of genomic loci - their distance from the nuclear periphery - has long been considered an important feature of genome organization which is correlated with both structural elements and genomic activity. In the current study, we developed an experimental system for manipulating the radial positioning of the genome, by expressing the sperm-specific protein Prm1 in somatic cells. By microscopy, we observe that initial Prm1 nuclear foci develop within 72 hours into a large Prm1 focus occupying most of the nuclear interior, while the entire genome is driven towards the nuclear periphery, resulting in a 3-5 fold reduction in the volume that the genome occupies. Noting that this system enables isolation of a pure population of cells with reorganized nuclei, we then used Hi-C to study the effects of this perturbation. Remarkably, we find that interaction patterns are largely robust to this major nuclear reorganization, with minor changes which mostly reflect a strengthening of heterochromatin self-interactions. Our experimental system provides means for manipulating nuclear organization in a reproducible manner, potentially allowing to examine radial positioning decoupled from other features of genome organization. Highlighting the complementary nature of microscopic and genomic methods, our work further suggests a remarkable resilience of genome structure such that large-scale nuclear changes, including chromosome compression and changes in radial positioning, can occur without extensive alteration of functional genome organization.

genomics↗

bioRxiv2022

Bone Marrow- and Umbilical Cord-Derived Mesenchymal Stem Cell Secretome Alters Gene Expression and Upregulates Motility of Human Endometrial Stromal Cells

IntroductionCyclic regeneration of the endometrium, and its repair after parturition or injury, are crucial for successful reproduction. Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSC) and umbilical cord (UC-MSC) facilitate tissue repair via their secretome, which contains growth factors and cytokines that promote wound healing. Despite the implication of MSCs in endometrial regeneration and repair, the mechanisms remain unclear. This study tested the hypothesis that the secretome of MSCs from human BM and UC upregulates human endometrial stromal cell (HESC) proliferation, migration and invasion, and activates pathways to increase HESC motility. MethodsMSCs were purchased from ATCC (BM-MSC-1) and cultured from the BM aspirate of three healthy female donors (BM-MSC-2-4), and from umbilical cords of two healthy male term infants (UC-MSC-1-2). Indirect co-culture of MSCs and hTERT-immortalized HESCs via a transwell system studied the effect of the BM-MSC and UC-MSC secretome on HESC proliferation, migration, and invasion. To study the effect of the MSC secretome on HESC gene expression, HESCs were exposed to the BM-MSC secretome via indirect co-culture for 24 h. Total RNA was extracted from HESCs for RNA sequencing (RNA-Seq). Differentially expressed genes (DEG) and significantly altered pathways were identified. MSigDB was used to identify the top 15 enriched biological pathways (padj < 0.05). RT-qPCR was performed to validate changes in mRNA expression of DEG common to both BM-MSC exposures. Given robust upregulation of CCL2 mRNA expression in HESCs exposed to the BM- and UC-MSC secretomes, transwell migration and invasion assays were performed to determine the effect of recombinant CCL2 on HESC motility. Statistical significance was defined as p<0.05. ResultsIndirect co-culture of HESCs with BM- or UC-MSCs resulted in significant increase in HESC migration and invasion regardless of the source of MSCs. However, effects on cellular proliferation varied among the MSC donors. Exposure of HESCs to the secretome of BM-MSCs changed the expression of 10,139 genes with FDR < 0.05. There was overlap among 4350 genes between HESCs exposed to BM-MSC-1 and BM-MSC-2. Within four biological pathways enriched in HESCs, 4 genes (CCL2, HGF, PLAU, and BDKRB2) were differentially expressed in HESCs that had been cocultured with BM-MSC-1 and BM-MSC-2. qRT-PCR showed significantly increased mRNA expression of CCL2 in HESCs exposed to BM-MSC-1 (5-fold) and BM-MSC-2 (7.7-fold). In contrast, the increase in HGF expression was significant after exposure to BM-MSC-2 (1.8-fold) but not BM-MSC-1. Exposure to the UC-MSC secretome had similar effects on HESC-derived CCL2 and HGF levels. CCL2 expression was significantly increased (6.5-fold) by UC-MSC-2 but not by UC-MSC-1; HGF expression was significantly increased (1.6-fold) by UC-MSC-2 but not by UC-MSC-1. Validation studies indicated that exposure to recombinant CCL2 for 48 hours significantly increased HESC migration (1.2-fold) and invasion (1.4-fold). These data suggest that CCL2 is a key factor in mediating MSC-induced HESC motility. ConclusionIncreased HESC motility by the secretome of BM- and UC-MSC appears to be mediated by paracrine and autocrine mechanisms, in part by upregulated CCL2 expression in HESC. Together, our data support the potential for leveraging the MSC secretome as a novel cell-free therapy in the treatment of disorders of endometrial regeneration.

cell biology↗

bioRxiv2021

Generation and diversification of recombinant monoclonal antibodies for studying mitosis

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitotic cell division; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

cell biology↗