Exofection as a Therapeutic Modality: Restoring P-gp Activity via Trophoblast-Derived EV in Neuroinflammatory Disorders

Kammala, A. K.; Tatiparthy, M.; Sreenivasa Murthy, S. G. S.; Garza, K.; Budhwani, S.; Richardson, L. S.; Menon, R.; Krishnan, B.

doi:10.64898/2026.04.02.716001

bioRxiv · 10.64898/2026.04.02.716001

Exofection as a Therapeutic Modality: Restoring P-gp Activity via Trophoblast-Derived EV in Neuroinflammatory Disorders

Kammala, A. K.·Tatiparthy, M.·Sreenivasa Murthy, S. G. S.·Garza, K.·Budhwani, S.·Richardson, L. S.·Menon, R.·Krishnan, B.

View original source ↗Export metadata ↓

Abstract

BackgroundP-glycoprotein (P-gp/ABCB1) is a key efflux transporter that maintains barrier integrity by clearing xenobiotics and toxic metabolites. At the feto-maternal interface, trophoblast-derived extracellular vesicles (CTC-EVs) naturally and transiently transfer functional P-gp to maternal decidual cells, restoring lost and or reduced P-gp function (exofection) to sustain pregnancy homeostasis. A similar loss of P-gp at the blood brain barrier (BBB) contributes to impaired amyloid-{beta} (A{beta}) clearance and neuroinflammation in Alzheimers disease. We investigated whether CTC-EV-mediated exofection could restore P-gp function in human brain endothelial cells (hBECs) and enhance A{beta} clearance under inflammatory and neurodegenerative conditions. MethodsCTC-EVs were isolated and characterized by nanoparticle tracking analysis and western blotting for P-gp and EV markers. Transcriptomic profiling of CTC-EVs identified enrichment of transporter-related genes, including solute carriers and ABC transporters, along with inflammatory mediators. Network analysis revealed coordinated modules linking EV cargo to transporter regulation, endocytosis/trafficking pathways, and inflammatory remodeling processes converging on BBB efflux activity. hBECs were exposed to LPS (500 ng/mL, 48 h) with or without CTC-EVs. P-gp expression was assessed by immunofluorescence (mean fluorescence intensity, MFI) and western blotting, while functional efflux was measured using Calcein-AM assays. A{beta} oligomer transport was evaluated using a transwell hBEC model. In vivo, 3xTg-AD mice received intravenous CTC-EVs (1x10L/day for 5 days), followed by assessment of P-gp expression, A{beta} burden, and neuroinflammatory markers. Pharmacokinetic studies in P-gp knockout mice were conducted to confirm functional transporter recovery. ResultsLPS exposure significantly reduced P-gp expression in hBECs (41.3% decrease in MFI, p=0.0084), which was restored by CTC-EVs (46.7% increase vs. LPS, p=0.0121). Exofection increased P-gp by a 2.1-fold following EV treatment as determined by western blot. Functional assays demonstrated enhanced efflux, with a 38.5% reduction in intracellular Calcein fluorescence (p<0.001). Network-informed mechanisms supported coordinated regulation of transporter and trafficking pathways. CTC-EVs improved A{beta} transport across inflamed hBEC monolayers. In vivo, EV-treated 3xTg-AD mice exhibited increased P-gp expression in the frontal cortex (38.6%) and hippocampus (42.1%), reduced A{beta} plaque burden (27.9%), and decreased inflammatory markers (IL-1{beta} and TNF-, p<0.05). In P-gp knockout mice, EVs reduced brain drug accumulation by 22.4% (p=0.032), confirming restoration of transporter function. ConclusionCTC derived EVs are natural carriers of functional transporter proteins and restore efflux capacity in compromised endothelial barriers. Integration of transcriptomic and network analyses highlights coordinated regulation of transporter, trafficking, and inflammatory pathways underlying exofection. This reproductive biology inspired strategy offers a promising therapeutic approach for enhancing A{beta} clearance and mitigating neuroinflammation in Alzheimers disease.

Explore related subjects

pharmacology and toxicology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Kammala, A. K., Tatiparthy, M., Sreenivasa Murthy, S. G. S., Garza, K., Budhwani, S., Richardson, L. S., Menon, R., Krishnan, B.. 2026-04-06. Exofection as a Therapeutic Modality: Restoring P-gp Activity via Trophoblast-Derived EV in Neuroinflammatory Disorders. https://doi.org/10.64898/2026.04.02.716001

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Denaturation-sensitive BRET probes enable intracellular thermal shift measurements across diverse protein targets and biomolecular complexes

Thermal shift assays have revolutionized drug discovery, but can be limited in sensitivity and scalability. Combining the unique attributes of differential scanning fluorimetry (DSF) and cellular thermal shift (CETSA), we report the methodological framework for the bioluminescence resonance energy transfer shift assay (BRETSA). BRETSA utilizes denaturation sensitive BRET probes, in combination with luciferase-tagged target proteins, to track and longitudinally record thermal shift in living cells. Denaturation profiles were successfully measured for over 500 globular proteins, spanning 24 target families in diverse cellular compartments. Among these, target engagement was confirmed for over 200 proteins; including validated drug targets as well as understudied members of the kinase subfamily. Leveraging orthosteric, allosteric, and covalent ligands in potency studies, this method offers improved sensitivity and scalability compared to other thermal shift methods. We extend the utility of BRETSA to interrogate target engagement at biomolecular complexes and query cooperative binding for molecular glues and emerging tricomplex drug modalities.

pharmacology and toxicology↗

bioRxiv2026

Peptide Sequence Determines the Efficacy of Single-Dose AAV-Vectorized GLP-1R Agonists in Diet-Induced Obese Mice

Background: Glucagon-like peptide-1 receptor (GLP-1R) agonists such as semaglutide produce substantial weight loss and glycemic improvement, but their long-term efficacy depends on sustained adherence to chronic dosing. Adeno-associated virus (AAV) gene therapy could, in principle, provide durable GLP-1R agonist (GLP-1RA) exposure from a single administration, but the degree of peptide engineering required for a vectorized GLP-1RA transgene to be efficacious in vivo is unknown. Methods: We generated four self-complementary AAV (scAAV) constructs encoding: native human GLP-1(7-36) (GLP-1), a DPP-IV-resistant Gly8-GLP-1(7-36) variant (Gly8), Exendin-4 (Ex4), or an Exendin-4 human IgG4 Fc fusion protein (Ex4-Fc). All four constructs were validated to secrete functional GLP-1R agonists in vitro using a conditioned-media potency assay. Subsequently, these constructs were packaged into PHP.eB capsids and administered as a single intravenous dose (5x1012 vg/kg) to diet-induced obese (DIO) mice alongside a vehicle control. Body weight, body composition, food intake, fasting plasma insulin, and oral glucose tolerance were assessed over a 3-week period. At the end of the study, tissues were harvested for assessments of plasma/liver lipids, tissue transgene transcript and peptide levels. Results: HEK293 cells transfected with the four AAV constructs all secreted active peptides/proteins as assessed by a cell-based GLP-1R activation assay, although the effective concentration of the GLP-1 constructs in the media spanned nearly four orders of magnitude 72 hours after transfection. In diet-induced obese (DIO) mice, Ex4 and Ex4-Fc vectors, but not GLP-1 or Gly8 vectors, produced significant reductions in body weight of up to 22.6% of starting body weight out to 21 days after a single dose. In addition, reductions in food intake, fat mass, and fasting insulin relative to vehicle were also observed for Ex4 and Ex4-Fc. Furthermore, Ex4-Fc improved oral glucose tolerance while reducing plasma cholesterol and liver triglycerides relative to vehicle. Although transcript levels did not differ appreciably among the four constructs in either brain or liver, measurable peptide product in plasma and brain lysate tracked the rank order of the in vitro supernatant screen, with GLP-1 and Gly8 groups indistinguishable from endogenous background. Conclusions: PHP.eB scAAV Ex4 and Ex4-Fc showed robust efficacy on multiple metabolic endpoints including weight loss and glucose tolerance. In contrast, vectorized GLP-1 and Gly8 showed no in vivo efficacy despite comparable levels of viral transcript in the brain and the liver. Vectorized GLP-1 and Gly8 also performed poorly compared to exendin-4-based constructs in vitro, suggesting that conditioned-media potency screening is a valid early filter for construct selection in vectorized-peptide AAV development. Engineering peptides to improve translation and secretion efficiency, in addition to circulating plasma stability, could further optimize vectorized GLP-1RA therapeutics.

pharmacology and toxicology↗

bioRxiv2026

Preclinical Evaluation of a Novel BOC-Modified Triazoloacridinone (TRA-01AR) in Combination with 5-Fluorouracil in Colorectal Cancer

5-Fluorouracil (5-FU) remains a cornerstone of colorectal cancer (CRC) therapy, yet its clinical efficacy is limited by resistance, systemic toxicity, and unfavorable pharmacokinetics. Strategies that enhance tumor sensitivity while maintaining tolerability are therefore of considerable interest. Here, we evaluated 5-FU in combination with two triazoloacridinones, C-1305 and a novel BOC-modified derivative, TRA-01AR, using integrated cellular, transcriptomic, bioenergetic, and in vivo approaches. Both compounds induced cytotoxic effects across cancer cell models and produced overlapping but distinct transcriptional responses, primarily affecting DNA replication and cell cycle pathways. Bioenergetic profiling revealed a shift toward oxidative phosphorylation accompanied by reduced glycolysis, indicating modulation of cellular metabolism relevant to chemotherapy response. Interestingly, TRA-01AR exhibited delayed cytotoxicity relative to C-1305, although the underlying mechanism remains to be defined. In vivo, the combination of 5-FU with C-1305 significantly reduced tumor weight in HT-29 xenografts, whereas 5-FU combined with TRA-01AR did not result in a statistically significant reduction compared to 5-FU alone. TRA-01AR was associated with a more favorable systemic profile and increased intratumoral blood flow compared with C-1305. Collectively, these findings indicate that triazoloacridinones modulate transcriptional and metabolic programs relevant to the 5-FU response. While C-1305 demonstrated greater antitumor activity under the tested conditions, TRA-01AR exhibited distinct biological effects, including improved tolerability and vascular modulation. Further studies are required to clarify TRA-01AR's mechanism of action and assess its potential relevance in CRC-specific therapies.

pharmacology and toxicology↗