Biology subjects

Lippmann, E.

Publications and source records attributed to Lippmann, E..

15 recordsLinked to original sources

bioRxiv2025

Evaluating the effects of aging on biodistribution and gene silencing activity of lipid-siRNA conjugates delivered into cerebrospinal fluid

Aging is the primary risk factor for chronic neurodegenerative diseases and is associated with alterations to cerebrospinal fluid (CSF) flow and clearance. CSF delivery is currently the most clinically advanced route of administration for oligonucleotide therapeutics, but it remains poorly understood how aging, which is rarely incorporated into clinical trials, impacts biodistribution, gene silencing activity, and potential toxicity of these compounds. Here, we evaluated a lipid-siRNA conjugate (L2-siRNA) for potential age-related changes to CSF-mediated delivery, mRNA silencing, and safety. We found that L2-siRNA exhibited comparable biodistribution and on-target silencing of Huntingtin (Htt) between young and aged mice in all tested regions of the central nervous system (CNS) and across extended time points. Examining transport along CSF efflux routes revealed uptake in deep cervical lymph nodes and dura. Further, L2-siRNA did not generate detectable toxicity in the CNS or periphery of aged mice. A subset of studies benchmarked L2-siRNA against a C16 lipid-siRNA conjugate that recently entered clinical trials. Collectively, these results provide valuable insight into siRNA conjugate biodistribution and activity in the CNS in the context of aging and further establish the performance of L2-siRNA under conditions relevant to the treatment of neurodegenerative diseases.

bioengineering↗

bioRxiv2025

Precision targeting of C3+ reactive astrocyte subpopulations with endogenous ADAR in an iPSC-derived model

Astrocytes play pivotal roles in maintaining neural architecture and function. However, their pronounced heterogeneity, especially in reactive states where distinct subtypes can adopt potentially opposing functions (e.g., neuroprotective vs. neuroinflammatory), complicates our understanding of their net contributions to neurological disorders. A critical challenge arises because these functionally distinct subpopulations often coexist, and the lack of precise tools to separately monitor or manipulate them has significantly hindered efforts to dissect their specific roles in disease progression. Here, we address this gap by developing and optimizing fluorescent RNA sensors mediated by endogenous adenosine deaminase acting on RNA (ADAR) for application in induced pluripotent stem cell (iPSC)-derived astrocytes. We employed a streamlined screening methodology to enhance sensor specificity and functionality for complement component 3 (C3), a key marker predominantly associated with neuroinflammatory astrocytes, thus enabling subtype-specific tracking and providing a crucial tool for distinguishing these cells within heterogeneous populations. By integrating the biological complexity of astrocytes with the technological precision of ADAR-mediated sensing, this study establishes a robust framework for investigating astrocyte dynamics.

neuroscience↗

bioRxiv2025

Substrate stiffness and shear stress collectively regulate the inflammatory phenotype in cultured human brain microvascular endothelial cells

Brain endothelial cells experience mechanical forces in the form of blood flow-mediated shear stress and underlying matrix stiffness, but intersectional contributions of these factors towards blood-brain barrier (BBB) impairment and neurovascular dysfunction have not been extensively studied. Here, we developed in vitro models to examine the sensitivity of primary human brain microvascular endothelial cells (BMECs) to substrate stiffness, with or without exposure to fluid shear stress. Using a combination of molecular profiling techniques, we show that BMECs exhibit an inflammatory signature at both the mRNA and protein level when cultured on gelatin substrates of intermediate stiffness ([~]30 kPa) versus soft substrates ([~]6 kPa). Exposure to modest fluid shear stress (1.7 dyne/cm2) partially attenuated this signature, including reductions in levels of soluble chemoattractants and surface ICAM-1. Overall, our results indicate that increased substrate stiffness promotes an inflammatory phenotype in BMECs that is dampened in the presence of fluid shear stress.

neuroscience↗

bioRxiv2025

Intravenous lipid-siRNA conjugate mediates gene silencing at the blood-brain barrier and blood-CSF barrier

Barriers of the central nervous system (CNS), such as the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB), regulate the two-way exchange of material between the blood and CNS. These barriers pose a considerable challenge for efficacious delivery of intravenously administered therapies into the CNS, motivating exploration of their function and ways to modulate their properties. While the BBB and BCSFB can become dysfunctional in patients with chronic CNS diseases, few studies have focused on strategies for targeting these interfaces. Here, we showed that an intravenously administered albumin-binding lipid-siRNA conjugate was delivered to and silences genes within brain endothelial cells and choroid plexus epithelial cells, which comprise the BBB and BCSFB, respectively. A single intravenous dose of lipid-siRNA conjugate was delivered to [~]100% of brain endothelial cells and major choroid plexus cell types, without any substantial delivery into brain parenchymal tissue. Sustained gene silencing was achieved in both brain endothelial cells (over two weeks) and bulk choroid plexus tissues (up to one month). Moreover, single cell RNA sequencing demonstrated gene knockdown in capillaries, venous endothelial cells, and choroid plexus epithelial cells without silencing genes in parenchymal cell populations. Collectively, this work establishes an effective nonviral framework to mediate gene inhibition in the brain barriers. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/642142v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@b00a52org.highwire.dtl.DTLVardef@175a363org.highwire.dtl.DTLVardef@39be75org.highwire.dtl.DTLVardef@1072093_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Anatomically and Physiologically Accurate Engineered Neurovascular Unit and Blood-Brain Barrier Model Using Microvessels Isolated from Postmortem Human Brain Tissue

Brain vasculature is a complex and heterogeneous physiological structure that serves specialized roles in maintaining brain health and homeostasis. There is substantial interest in developing representative human models of the brain vasculature for drug screening and disease modeling applications. Many contemporary strategies have focused on culturing neurovascular cell types in hydrogels and microdevices, but it remains challenging to achieve anatomically relevant vascular structures that have physiologically similar function to their in vivo counterparts. Here, we present a strategy for isolating microvessels from cryopreserved human cortical tissue and culturing these vessels in a biomimetic gelatin-based hydrogel contained in a microfluidic device. We provide histological evidence of arteriole and capillary architectures within hydrogels, as well as anastomosis to the hydrogel edges allowing lumen perfusion. In capillaries, we demonstrate restricted diffusion of a 10 kDa dextran, indicating intact passive blood-brain barrier function. We anticipate this bona fide human brain vasculature-on-a-chip will be useful for various biotechnology applications.

bioengineering↗

bioRxiv2024

Lipid-siRNA conjugate accesses a perivascular transport mechanism and achieves widespread and durable knockdown in the central nervous system

Short-interfering RNA (siRNA) has gained significant interest for treatment of neurological diseases by providing the capacity to achieve sustained inhibition of nearly any gene target. Yet, efficacious drug delivery throughout deep brain structures of the CNS remains a considerable hurdle for intrathecally administered therapeutics. We herein describe an albumin-binding lipid-siRNA conjugate that transports along meningeal and perivascular CSF pathways, leading to broad dispersion throughout the CNS parenchyma. We provide a detailed examination of the temporal kinetics of gene silencing, highlighting potent knockdown for up to five months from a single injection without detectable toxicity. Single-cell RNA sequencing further demonstrates gene silencing activity across diverse cell populations in the parenchyma and at brain borders, which may provide new avenues for neurological disease-modifying therapies.

bioengineering↗

bioRxiv2024

Arteriolar degeneration and stiffness in cerebral amyloid angiopathy are linked to β-amyloid deposition and lysyl oxidase.

Cerebral amyloid angiopathy (CAA) is a vasculopathy characterized by vascular {beta}-amyloid (A{beta}) deposition on cerebral blood vessels. CAA is closely linked to Alzheimers disease (AD) and intracerebral hemorrhage. CAA is associated with the loss of autoregulation in the brain, vascular rupture, and cognitive decline. To assess morphological and molecular changes associated with the degeneration of penetrating arterioles in CAA, we analyzed post-mortem human brain tissue from 26 patients with mild, moderate, and severe CAA end neurological controls. The tissue was optically cleared for three-dimensional light sheet microscopy, and morphological features were quantified using surface volume rendering. We stained A{beta}, vascular smooth muscle (VSM), lysyl oxidase (LOX), and vascular markers to visualize the relationship between degenerative morphological features, including vascular dilation, dolichoectasia (variability in lumenal diameter) and tortuosity, and the volumes of VSM, A{beta}, and LOX in arterioles. Atomic force microscopy (AFM) was used to assess arteriolar wall stiffness, and we identified a pattern of morphological features associated with degenerating arterioles in the cortex. The volume of VSM associated with the arteriole was reduced by around 80% in arterioles with severe CAA and around 60% in cases with mild/moderate CAA. This loss of VSM correlated with increased arteriolar diameter and variability of diameter, suggesting VSM loss contributes to arteriolar laxity. These vascular morphological features correlated strongly with A{beta} deposits. At sites of microhemorrhage, A{beta} was consistently present, although the morphology of the deposits changed from the typical organized ring shape to sharply contoured shards with marked dilation of the vessel. AFM showed that arteriolar walls with CAA were more than 400% stiffer than those without CAA. Finally, we characterized the association of vascular degeneration with LOX, finding strong associations with VSM loss and vascular degeneration. These results show an association between vascular A{beta} deposition, microvascular degeneration, and increased vascular stiffness, likely due to the combined effects of replacement of VSM by {beta}-amyloid, cross-linking of extracellular matrices (ECM) by LOX, and possibly fibrosis. This advanced microscopic imaging study clarifies the association between A{beta} deposition and vascular fragility. Restoration of physiologic ECM properties in penetrating arteries may yield a novel therapeutic strategy for CAA.

neuroscience↗

bioRxiv2023

IQGAP2 regulates blood-brain barrier immune dynamics

Brain endothelial cells (BECs) play an important role in maintaining central nervous system (CNS) homeostasis through blood-brain barrier (BBB) functions. BECs express low baseline levels of adhesion receptors, which limits entry of leukocytes. However, the molecular mediators governing this phenotype remain mostly unclear. Here, we explored how infiltration of immune cells across the BBB is influenced by the scaffold protein IQ motif containing GTPase activating protein 2 (IQGAP2). In mice and zebrafish, we demonstrate that loss of Iqgap2 increases infiltration of peripheral leukocytes into the CNS under homeostatic and inflammatory conditions. Using single-cell RNA sequencing and immunohistology, we further show that BECs from mice lacking Iqgap2 exhibit a profound inflammatory signature, including extensive upregulation of adhesion receptors and antigen-processing machinery. Human tissue analyses also reveal that Alzheimers disease is associated with reduced hippocampal IQGAP2. Overall, our results implicate IQGAP2 as an essential regulator of BBB immune privilege and immune cell entry into the CNS.

neuroscience↗

bioRxiv2022

Generalized strategy for engineering mammalian cell-compatible RNA-based biosensors from random sequence libraries

Fluorescent RNA-based biosensors are useful tools for real-time detection of molecules in living cells. These biosensors typically consist of a chromophore-binding aptamer and a target-binding aptamer, whereby the chromophore-binding aptamer is destabilized until a target is captured, which causes a conformational change to permit chromophore binding and an increase in fluorescence. The target-binding region is typically fabricated using known riboswitch motifs, which are already known to have target specificity and undergo structural changes upon binding. However, known riboswitches only exist for a limited number of molecules, significantly constraining biosensor design. To overcome this challenge, we designed a framework for producing mammalian cell-compatible biosensors using aptamers selected from a large random library by capture-SELEX. As a proof-of-concept, we generated and characterized a fluorescent RNA biosensor against L-dopa, the precursor of several neurotransmitters. Overall, we suggest that this approach will have utility for generating RNA biosensors that can reliably detect custom targets in mammalian cells.

molecular biology↗

bioRxiv2022

Reactive astrocytes transduce blood-brain barrier dysfunction through a TNFα-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin

Astrocytes are critical components of the neurovascular unit that support blood-brain barrier (BBB) function in brain microvascular endothelial cells (BMECs). Transformation of astrocytes to a reactive state in response to injury and disease can be protective or harmful to BBB function, but the underlying mechanisms for these effects remain mostly unclear. Using a human induced pluripotent stem cell (iPSC)-derived coculture model of BMEC-like cells and astrocytes, we found that tumor necrosis factor alpha (TNF) transitions astrocytes to an inflammatory reactive state through activated STAT3 signaling, whereby the resultant astrocytes disrupt passive BBB function and induce vascular cell adhesion molecule 1 (VCAM-1) expression in the BMEC-like cells. These associations between inflammatory reactive astrocytes, STAT3 activation, and vascular VCAM-1 expression were corroborated in human postmortem tissue. Bioinformatic analyses coupled with CRISPR interference techniques in the iPSC model revealed that inflammatory reactive astrocytes transduce BBB disruption in part through SERPINA3, which encodes alpha 1-antichymotrypsin (1ACT), a secreted serine protease inhibitor associated with aging, neuroinflammation, and Alzheimers disease. In murine ex vivo cortical explant cultures, shRNA-mediated silencing of Serpina3n in astrocytes reduced vascular VCAM-1 expression after TNF challenge. Further, direct treatment with recombinant Serpina3n in both ex vivo explant cultures and the brain in vivo (via intracerebroventricular injection into wild-type mice) was sufficient to induce vascular VCAM-1 expression and reduce tight junction integrity. Overall, our results define the TNF-STAT3 signaling axis as a driver of an inflammatory reactive astrocyte subtype responsible for BBB dysfunction. Our results also identify 1ACT as an explicit mediator of BBB damage and suggest that inhibition of 1ACT expression or activity could represent a therapeutic avenue for reversing BBB deficits in aging and neurodegenerative disease.

neuroscience↗

bioRxiv2021

Rapid Prototyping of Cell Culture Microdevices Using Parylene-Coated 3D Prints

Fabrication of microfluidic devices by photolithography generally requires specialized training and access to a cleanroom. As an alternative, 3D printing enables cost-effective fabrication of microdevices with complex features that would be suitable for many biomedical applications. However, commonly used resins are cytotoxic and unsuitable for devices involving cells. Furthermore, 3D prints are generally refractory to elastomer polymerization such that they cannot be used as master molds for fabricating devices from polymers (e.g. polydimethylsiloxane, or PDMS). Different post-print treatment strategies, such as heat curing, ultraviolet light exposure, and coating with silanes, have been explored to overcome these obstacles, but none have proven universally effective. Here, we show that deposition of a thin layer of parylene, a polymer commonly used for medical device applications, renders 3D prints biocompatible and allows them to be used as master molds for elastomeric device fabrication. When placed in culture dishes containing human neurons, regardless of resin type, uncoated 3D prints leached toxic material to yield complete cell death within 48 hours, whereas cells exhibited uniform viability and healthy morphology out to 21 days if the prints were coated with parylene. Diverse PDMS devices of different shapes and sizes were easily casted from parylene-coated 3D printed molds without any visible defects. As a proof-of-concept, we rapid prototyped and tested different types of PDMS devices, including triple chamber perfusion chips, droplet generators, and microwells. Overall, we suggest that the simplicity and reproducibility of this technique will make it attractive for fabricating traditional microdevices and rapid prototyping new designs. In particular, by minimizing user intervention on the fabrication and post-print treatment steps, our strategy could help make microfluidics more accessible to the biomedical research community.

bioengineering↗

bioRxiv2021

Albumin-binding Aptamer Chimeras for Improved siRNA Bioavailability

Short interfering RNAs (siRNAs) are potent nucleic acid-based drugs designed to target disease driving genes that may otherwise be undruggable with small molecules. However, the potential of administering therapeutic siRNA in vivo is limited by poor pharmacokinetic properties, including rapid renal clearance and nuclease degradation. Nanocarriers have traditionally been explored as means to overcome these challenges, but they have intrinsic downsides such as dose-limiting toxicity and synthetic complexity. Backpacking on natural carriers such as albumin, which is present at high concentration and has a long half-life in serum, is an effective way to modify pharmacokinetics of biologic drugs that otherwise have poor bioavailability. In this work, we sought to develop albumin-binding aptamer-siRNA chimeras to improve the bioavailability of siRNA. We used a Systematic Evolution of Ligands through Exponential Enrichment (SELEX) approach to obtain RNA aptamers with modified bases that bind albumin with high affinity. We then fused the aptamers directly to an siRNA to generate the chimera structure. These aptamer-siRNA chimeras are stable in serum, exhibit potent gene knockdown capabilities in vitro, and display extended circulation time in vivo. We suggest that this albumin-binding aptamersiRNA chimera approach is a promising strategy for drug delivery applications.

bioengineering↗

bioRxiv2021

Influence of substrate stiffness on barrier function in an iPSC-derived in vitro blood-brain barrier model

IntroductionVascular endothelial cells respond to a variety of biophysical cues such as shear stress and substrate stiffness. In peripheral vasculature, extracellular matrix (ECM) stiffening alters barrier function, leading to increased vascular permeability in atherosclerosis and pulmonary edema. The effect of ECM stiffness on blood-brain barrier (BBB) endothelial cells, however, has not been explored. To investigate this topic, we incorporated hydrogel substrates into an in vitro model of the human BBB. MethodsInduced pluripotent stem cells were differentiated to brain microvascular endothelial-like (BMEC-like) cells and cultured on hydrogel substrates of varying stiffness. Cellular changes were measured by imaging, functional assays such as transendothelial electrical resistance (TEER) and p-glycoprotein efflux activity, and bulk transcriptome readouts. ResultsThe magnitude and longevity of TEER in iPSC-derived BMEC-like cells is enhanced on compliant substrates. Quantitative imaging shows that BMEC-like cells form fewer intracellular actin stress fibers on substrates of intermediate stiffness (20 kPa relative to 1 kPa and 150 kPa). Chemical induction of actin polymerization leads to a rapid decline in TEER, agreeing with imaging readouts. P-glycoprotein activity is unaffected by substrate stiffness. Modest differences in RNA expression corresponding to specific signaling pathways were observed as a function of substrate stiffness. ConclusionsiPSC-derived BMEC-like cells exhibit differences in passive but not active barrier function in response to substrate stiffness. These findings may provide insight into BBB dysfunction during neurodegeneration, as well as aid in the optimization of more complex threedimensional neurovascular models utilizing compliant hydrogels.

bioengineering↗

bioRxiv2021

Influence of basal media composition on barrier fidelity within human pluripotent stem cell-derived blood-brain barrier models

It is increasingly recognized that brain microvascular endothelial cells (BMECs), the principle component of the blood-brain barrier (BBB), are highly sensitive to soluble cues from both the bloodstream and the brain. This concept extends in vitro, where the extracellular milieu can also influence BBB properties in cultured cells. However, the extent to which baseline culture conditions can affect BBB properties in vitro remains unclear, which has implications for model variability and reproducibility, as well as downstream assessments of molecular transport and disease phenotypes. Here, we explore this concept by examining BBB properties within human induced pluripotent stem cell (iPSC)-derived BMEC-like cells cultured under serum-free conditions in different basal media with fully defined compositions. We demonstrate notable differences in both passive and active BBB properties as a function of basal media composition. Further, RNA sequencing and phosphoproteome analyses revealed alterations to various signaling pathways in response to basal media differences. Overall, our results demonstrate that baseline culture conditions can have a profound influence on the performance of in vitro BBB models, and these effects should be considered when designing experiments that utilize such models for basic research and preclinical assays.

neuroscience↗

bioRxiv2020

CRISPR-mediated isogenic cell-SELEX approach for generating highly specific aptamers against native membrane proteins

IntroductionThe generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated via CRISPR techniques. MethodsUsing a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells. Results10 rounds of cell-SELEX were conducted via simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor. ConclusionsOur data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach should be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface.

bioengineering↗