Biology subjects

Sahay, G.

Publications and source records attributed to Sahay, G..

6 recordsLinked to original sources

bioRxiv2026

Lipid nanoparticle induced model endosomal membrane disruption

Endosomal escape remains the central bottleneck limiting lipid nanoparticle (LNP)-mediated mRNA delivery. In this study, we use giant unilamellar vesicles (GUVs) mimicking the endosomal membrane to directly visualize the interaction and membrane disruptions caused by two model LNPs constituted by two different ionizable lipids: LP01 and BiP-20 (a Branched ionizable Phospholipid) that are both benchmark ionizable lipids for in vivo gene editing in liver. Despite identical TNS-measured apparent pKa (6.6) for both lipids in our conditions, LP01 LNP showed maximal membrane engagement at pH 5.8, while BiP20 LNP required pH 5.1, demonstrating that bulk pKa alone cannot predict the pH at which functional membrane interaction occurs. Using confocal microscopy, we show that the LNP engagement with the endosomal membrane proceeds through first electrostatic attraction, clustering, membrane disruption by forming highly curved morphologies, and finally leading to the rupture of the whole endosomal mimicking membrane. Notably, the mRNA cargo is released only upon complete lysis of the membrane. This indicates that the membrane destabilization and cargo release are mechanistically distinct steps. These findings establish a cell-free platform for dissecting individual sub-steps of endosomal escape.

biophysics↗

bioRxiv2026

An mRNA-Lipid Nanoparticle Platform Encoding the Conserved Outer Membrane Protein BamA Elicits Broadly Cross-Reactive Systemic and Mucosal Antibodies Against Antimicrobial-Resistant Neisseria gonorrhoeae

Neisseria gonorrhoeae (Ng) is the causative agent of gonorrhea, and the global spread of antimicrobial-resistant strains makes vaccine development a public health priority. Although messenger RNA (mRNA) vaccines have transformed protection against viral diseases, the platform remains in its infancy against pathogenic bacteria. Here, we evaluated the immunogenicity and protective efficacy of an mRNA-lipid nanoparticle (LNP) vaccine encoding the highly conserved outer membrane antigen BamA in the female mouse model of lower genital tract infection. We delivered BamA mRNA-LNPs via intramuscular (IM) or intranasal (IN) routes, with or without CpG ODN 2395, and measured antigen-specific antibody responses in serum and vaginal lavage samples. Both routes elicited robust BamA-specific antibodies that recognized diverse Ng isolates, including ceftriaxone-resistant strains. However, neither route accelerated bacterial clearance nor reduced bioburden, nor did either generate serum bactericidal activity. These findings show that BamA mRNA-LNPs are immunogenic but, as formulated, are not protective, and they pave the way for modifications to the construct, adjuvant, and route. To our knowledge, this is the first evaluation of an mRNA vaccine against Ng, establishing the platform as an amenable approach for gonococcal antigen testing. ImportanceNeisseria gonorrhoeae is a high-priority public health threat, and no licensed gonococcal vaccine exists. mRNA-lipid nanoparticle vaccines have transformed antiviral immunization but remain largely untested against bacterial pathogens. This study is the first to evaluate an mRNA vaccine against N. gonorrhoeae, using the conserved, essential outer membrane antigen BamA. We show that BamA mRNA-LNPs delivered by intramuscular or intranasal routes elicit robust, broadly cross-reactive antibodies that recognize diverse isolates, including ceftriaxone-resistant strains, even though the tested formulations did not confer protection in mice. By establishing mRNA-LNPs as a viable platform for gonococcal antigen testing, this work lays the foundation for optimizing constructs, adjuvants, and routes for developing mRNA-based vaccines against gonorrhea and other antimicrobial-resistant bacteria.

microbiology↗

bioRxiv2024

Synthesis of ionizable lipopolymers using split-Ugi reaction for pulmonary delivery of various size RNAs and gene editing

We present an efficient approach for synthesizing cationic poly(ethylene imine) derivatives using the multicomponent split-Ugi reaction to rapidly create a library of complex functional ionizable lipopolymers. We synthesized a diverse library of 155 polymers, formulated them into polyplexes to establish structure-activity relationships crucial for endosomal escape and efficient transfection. After discovering a lead structure, lipopolymer-lipid hybrid nanoparticles are introduced to preferentially deliver to and elicit effective mRNA transfection in lung endothelium and immune cells, including T cells with low in vivo toxicity. The lipopolymer-lipid hybrid nanoparticles showed 300-fold improvement in systemic mRNA delivery to the lung compared to in vivo-JetPEI(R). Lipopolymer-lipid hybrid nanoparticles demonstrated efficient delivery of mRNA-based therapeutics for treatment of two different disease models. Lewis Lung cancer progression was significantly delayed after treatment with loaded IL-12 mRNA in U155@lipids after repeated i.v. administration. Systemic delivery of human CFTR (hCFTR) mRNA resulted in production of functional form of CFTR protein in the lungs. The functionality of hCFTR protein was confirmed by restoration of CFTR- mediated chloride secretion in conductive airway epithelia in CFTR knockout mice after nasal instillation of hCFTR mRNA loaded U155@lipids. We further showed that, U155@lipids nanoparticles can deliver complex CRISPR-Cas9 based RNA cargo to the lung, achieving 5.6 {+/-} 2.4 % gene editing in lung tissue. Moreover, we demonstrated successful PD-1 gene knockout of T cells in vivo. Our results highlight a versatile delivery platform for systemic delivering of mRNA of various sizes for gene therapy for a variety of therapeutics.

bioengineering↗

bioRxiv2024

Microfluidic platform enables shear-less aerosolization of lipid nanoparticles for messenger RNA inhalation

Leveraging the extensive surface area of the lungs for gene therapy, inhalation route offers distinct advantages for delivery. Clinical nebulizers that employ vibrating mesh technology are the standard choice for converting liquid medicines into aerosols. However, they have limitations when it comes to delivering mRNA through inhalation, including severe damage to nanoparticles due to shearing forces. Here, we introduce a novel microfluidic aerosolization platform (MAP) that preserves the structural and physicochemical integrity of lipid nanoparticles, enabling safe and efficient mRNA delivery to the respiratory system. Our results demonstrated the superiority of the novel MAP over the conventional vibrating mesh nebulizer, as it avoided problems such as particle aggregation, loss of mRNA encapsulation, and deformation of nanoparticle morphology. Notably, aerosolized nanoparticles generated by the microfluidic device led to enhanced transfection efficiency across various cell lines. In vivo experiments with mice that inhaled these aerosolized nanoparticles revealed successful, lung-specific mRNA transfection without observable signs of toxicity. This pioneering MAP represents a significant advancement for the pulmonary gene therapy, enabling precise and effective delivery of aerosolized nanoparticles.

bioengineering↗

bioRxiv2020

Illuminating endosomal escape of polymorphic lipid nanoparticles that boost mRNA delivery

Lipid-based nanoparticles (LNPs) for the delivery of mRNA have jumped to the forefront of non-viral gene delivery. Despite this exciting development, poor endosomal escape after LNP cell entry remains an unsolved, rate-limiting bottleneck. Here we report the use of galectin 8-GFP (Gal8-GFP) cell reporter system to visualize the endosomal escaping capabilities of LNPs encapsulating mRNA. LNPs substituted with phytosterols in place of cholesterol exhibited various levels of Gal8 recruitment in the Gal8-GFP reporter system. In live-cell imaging, the results of the LNPs containing {beta}-sitosterol (LNP-Sito) showed a 10-fold increase in detectable endosomal perturbation events when compared to the standard cholesterol LNPs (LNP-Chol), suggesting the superior capability of LNP-Sito to escape from endosomal entrapment. Trafficking studies of these LNPs showed strong localization with late endosomes. This highly sensitive and robust Gal8-GFP reporter system can be a valuable tool to elucidate intricacies of LNP trafficking and ephemeral endosomal escape events, enabling the advancement in gene delivery.

bioengineering↗

bioRxiv2020

Rapid generation of circulating and mucosal decoy ACE2 using mRNA nanotherapeutics for the potential treatment of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters through the airways and infects the lungs, causing lethal pulmonary damage in vulnerable patients. This virus contains spike proteins on its envelope that binds to human angiotensin-converting enzyme 2 (hACE2) expressed on the surface of airway cells, enabling entry of the virus for causing infection1,2. In severe cases, the virus enters the circulatory system, contributing to multiorgan failure. Soluble form of hACE2 binds to SARS-CoV-2 spike protein and prevents viral entry into target cells3. Moreover, soluble recombinant ACE2 ameliorates lung injury4 but its short half-life limits its therapeutic utility5. Here, we engineered synthetic mRNA to encode a soluble form of hACE2 (hsACE2) to prevent viral infection. Novel lipid nanoparticles (LNPs) were used to package mRNA and transfect mammalian cells for enhanced production of secreted proteins. Intravenously administered LNP led to hepatic delivery of the mRNA. This elicited secretion of hsACE2 into the blood circulation within 2 h, and levels of circulating hsACE2 peaked at 6 h and gradually decreased over several days. Since the primary site of entry and pathogenesis for SARS-CoV-2 is the lungs, we instilled LNPs into the lungs and were able to detect hsACE2 in the bronchoalveolar lavage fluid within 24 h and lasted for 48 h. Through co-immunoprecipitation, we found that mRNA-generated hsACE2 was able to bind with the receptor binding domain of the SARS-CoV-2 spike protein. Furthermore, hsACE2 was able to strongly inhibit (over 90%) SARS-CoV-2 pseudovirus infection. Our proof of principle study shows that mRNA-based nanotherapeutics can be potentially deployed for pulmonary and extrapulmonary neutralization of SARS-CoV-2 and open new treatment opportunities for COVID-19.

bioengineering↗