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 Alzheimer's 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.

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, achieving efficacious drug delivery throughout deep brain structures of the CNS remains a considerable hurdle. We herein describe a lipid-siRNA conjugate that, following delivery into the cerebrospinal fluid (CSF), is transported effectively through perivascular spaces, enabling broad dispersion within CSF compartments and through 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.

Albumin-binding RNAi Conjugate for Carrier Free Treatment of Arthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are joint diseases that are associated with pain and lost quality of life. No disease modifying OA drugs are currently available. RA treatments are better established but are not always effective and can cause immune suppression. Here, an MMP13-selective siRNA conjugate was developed that, when delivered intravenously, docks onto endogenous albumin and promotes preferential accumulation in articular cartilage and synovia of OA and RA joints. MMP13 expression was diminished upon intravenous delivery of MMP13 siRNA conjugates, consequently decreasing multiple histological and molecular markers of disease severity, while also reducing clinical manifestations such as swelling (RA) and joint pressure sensitivity (RA and OA). Importantly, MMP13 silencing provided more comprehensive OA treatment efficacy than standard of care (steroids) or experimental MMP inhibitors. These data demonstrate the utility of albumin 'hitchhiking' for drug delivery to arthritic joints, and establish the therapeutic utility of systemically delivered anti-MMP13 siRNA conjugates in OA and RA. Editorial summary: Lipophilic siRNA conjugates optimized for albumin binding and "hitchhiking" can be leveraged to achieve preferential delivery to and gene silencing activity within arthritic joints. Chemical stabilization of the lipophilic siRNA enables intravenous siRNA delivery without lipid or polymer encapsulation. Using siRNA sequences targeting MMP13, a key driver of arthritis-related inflammation, albumin hitchhiking siRNA diminished MMP13, inflammation, and manifestations of osteoarthritis and rheumatoid arthritis at molecular, histological, and clinical levels, consistently outperforming clinical standards of care and small molecule MMP antagonists.

Albumin-Binding Aptamer Chimeras for Improved siRNA Bioavailability.

Introduction: 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, therapeutic potential of siRNA in vivo is limited by poor pharmacokinetic properties, including rapid renal clearance and nuclease degradation. 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. Methods: A Systematic Evolution of Ligands through Exponential Enrichment (SELEX) approach was used to obtain modified RNA-binding aptamers, which were then fused directly to siRNA via in vitro transcription. Molecular and pharmacokinetic properties of the aptamer-siRNA chimeras were subsequently measured in vitro and in vivo. Results: In vitro assays show that albumin-binding aptamers are stable in serum while maintaining potent gene knockdown capabilities in the chimera format. In vivo, the absolute circulation half-life of the best-performing aptamer-siRNA chimera (Clone 1) was 1.6-fold higher than a scrambled aptamer chimera control. Conclusions: Aptamer-siRNA chimeras exhibit improved bioavailability without compromising biological activity. Hence, this albumin-binding aptamer-siRNA chimera approach may be a promising strategy for drug delivery applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00718-y.

Reactive astrocytes transduce inflammation in a blood-brain barrier model through a TNF-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin.

Astrocytes are critical components of the neurovascular unit that support blood-brain barrier (BBB) function. Pathological transformation of astrocytes to reactive states can be protective or harmful to BBB function. Here, using a human induced pluripotent stem cell (iPSC)-derived BBB co-culture model, we show that tumor necrosis factor (TNF) transitions astrocytes to an inflammatory reactive state that causes BBB dysfunction through activation of STAT3 and increased expression of SERPINA3, which encodes alpha 1-antichymotrypsin (α1ACT). To contextualize these findings, we correlated astrocytic STAT3 activation to vascular inflammation in postmortem human tissue. Further, in murine brain organotypic cultures, astrocyte-specific silencing of Serpina3n reduced vascular inflammation after TNF challenge. Last, treatment with recombinant Serpina3n in both ex vivo explant cultures and in vivo was sufficient to induce BBB dysfunction-related molecular changes. Overall, our results define the TNF-STAT3-α1ACT signaling axis as a driver of an inflammatory reactive astrocyte signature that contributes to BBB dysfunction.

Caveolae-Mediated Transport at the Injured Blood-Brain Barrier as an Underexplored Pathway for Central Nervous System Drug Delivery.

Drug delivery to the central nervous system (CNS) is generally hindered by the selectivity of the blood-brain barrier (BBB). However, there is strong evidence that the integrity of the BBB is compromised under certain pathological conditions, potentially providing a window to deliver drugs to injured brain regions. Recent studies suggest that caveolae-mediated transcytosis, a transport pathway suppressed in the healthy BBB, becomes elevated as an immediate response to ischemic stroke and at early stages of aging, where it may precede irreversible neurological damage. This article reviews early-stage caveolar transcytosis as a novel and promising drug delivery opportunity. We propose that albumin-binding and nanoparticle approaches have the potential to leverage this window of transcellular BBB disruption for trafficking therapeutic agents into the CNS.