Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

Patel, Siddharth; Ashwanikumar, N.; Robinson, Ema; Xia, Yan; Mihai, Cosmin; Griffith, Joseph P.; Hou, Shangguo; Esposito, Adam A.; Ketova, Tatiana; Welsher, Kevin; Joyal, John L.; Almarsson, Ӧrn; Sahay, Gaurav

doi:10.1038/s41467-020-14527-2

Cited by 289 publications

(310 citation statements)

References 72 publications

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“…Recently, we discovered a simple substitution of cholesterol to β-sitosterol within LNP formulations boosts intracellular delivery of mRNA 22,23 . We compared the enhanced LNP formulation (eLNP: containing β-sitosterol) with the conventional LNP (containing cholesterol) as the delivery vector for hsACE2 mRNA.…”

Section: Mainmentioning

confidence: 99%

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

Kim

Mukherjee

Nelson

et al. 2020

Preprint

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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.

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Section: Mainmentioning

confidence: 99%

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

Kim

Mukherjee

Nelson

et al. 2020

Preprint

Self Cite

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“…The addition of cholesterol into the formulation has been shown to the enhance transfection efficiency of lipid-base nanoparticles, potentially by promoting membrane fusion and endosomal escape and, as expected, the percentage of cholesterol has a considerable influence on intracellular gene delivery (Pozzi et al, 2012). Patel et al reported that the inclusion of a naturally occurring cholesterol analogous (C-24 alkyl phytosterols) into lipid-based nanoformulations enhances mRNA delivery (Patel et al, 2020). In this regard, the length of the alkyl tail, the flexibility of sterol ring and the polarity associated with the hydroxyl group were found essential to maintain a high transfection efficiency.…”

Section: Helper Lipids and Stealth Lipidsmentioning

confidence: 85%

Advances in Lipid Nanoparticles for mRNA-Based Cancer Immunotherapy

2020

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Over the past decade, messenger RNA (mRNA) has emerged as potent and flexible platform for the development of novel effective cancer immunotherapies. Advances in non-viral gene delivery technologies, especially the tremendous progress in lipid nanoparticles' manufacturing, have made possible the implementation of mRNA-based antitumor treatments. Several mRNA-based immunotherapies have demonstrated antitumor effect in preclinical and clinical studies, and marked successes have been achieved most notably by its implementation in therapeutic vaccines, cytokines therapies, checkpoint blockade and chimeric antigen receptor (CAR) cell therapy. In this review, we summarize recent advances in the development of lipid nanoparticles for mRNA-based immunotherapies and their applications in cancer treatment. Finally, we also highlight the variety of immunotherapeutic approaches through mRNA delivery and discuss the main factors affecting transfection efficiency and tropism of mRNA-loaded lipid nanoparticles in vivo.

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“…With a near atomic resolution (57), EM is an irreplaceable tool in studying the physio-chemical properties of NP and quantifying their voyage through the endo-lysosomal pathway (30,37,(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74). EM can even detect a low number (few hundreds) of single nanoparticles escaping endosomal structures, and since it is a label-free method, it will localise and quantify NP generally untraceable by standard light microscopy methods.…”

Section: Electron Microscopy and Cryo-electron Microscopymentioning

confidence: 99%

“…Various papers study the endosomal escape of NP using several independent microscopic techniques (30,58,68,140,141). However, a correlative approach is more desirable, as it Up to date, the only CLEM approach used to quantitatively study endo-lysosomal tracking of NP has been developed by Han et al (74) (Fig.…”

Section: Correlative Imagingmentioning

confidence: 99%