ArticlePublisher preview available

Modulating Lipid Nanoparticles with Histidinamide‐Conjugated Cholesterol for Improved Intracellular Delivery of mRNA

Wiley
Advanced Healthcare Materials
Authors:
Onesun Jung
Onesun Jung
Onesun Jung
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Hye‐youn Jung
Hye‐youn Jung
Hye‐youn Jung
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Le Thi Thuy at Chungnam National University
Le Thi Thuy
Minyoung Choi
Minyoung Choi
Minyoung Choi
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Show all 12 authorsHide
Read publisher preview
Download citation
To read the full-text of this research, you can request a copy directly from the authors.

Abstract and Figures

Recently, mRNA‐based therapeutics, including vaccines, have gained significant attention in the field of gene therapy for treating various diseases. Among the various mRNA delivery vehicles, lipid nanoparticles (LNPs) have emerged as promising vehicles for packaging and delivering mRNA with low immunogenicity. However, while mRNA delivery has several advantages, the delivery efficiency and stability of LNPs remain challenging for mRNA therapy. In this study, an ionizable helper cholesterol analog, 3β[L‐histidinamide‐carbamoyl] cholesterol (Hchol) lipid is developed and incorporated into LNPs instead of cholesterol to enhance the LNP potency. The pKa values of the Hchol‐LNPs are ≈6.03 and 6.61 in MC3‐ and SM102‐based lipid formulations. Notably, the Hchol‐LNPs significantly improve the delivery efficiency by enhancing the endosomal escape of mRNA. Additionally, the Hchol‐LNPs are more effective in a red blood cell hemolysis at pH 5.5, indicating a synergistic effect of the protonated imidazole groups of Hchol and cholesterol on endosomal membrane destabilization. Furthermore, mRNA delivery is substantially enhanced in mice treated with Hchol‐LNPs. Importantly, LNP‐encapsulated SARS‐CoV‐2 spike mRNA vaccinations induce potent antigen‐specific antibodies against SARS‐CoV‐2. Overall, incorporating Hchol into LNP formulations enables efficient endosomal escape and stability, leading to an mRNA delivery vehicle with a higher delivery efficiency.
Screening of LNPs with various ratios of cholesterol to Hchol. A) Schematic illustration of FLuc mRNA‐LNPs formulated with cholesterol and Hchol. B) In vitro transfection efficiency of Hchol‐LNPs in HepG2. The luciferase expression of MC3‐Hchol100 and SM102‐Hchol100 was normalized to MC3‐chol and SM102‐chol, respectively (mean ± standard deviation, n = 3). C) In vivo activity of MC3‐chol and MC3‐Hchol100. LNPs were intramuscularly administered. The total flux of each LNP at each time point was normalized to the total flux obtained from MC3‐chol.
Screening of LNPs with various ratios of cholesterol to Hchol. A) Schematic illustration of FLuc mRNA‐LNPs formulated with cholesterol and Hchol. B) In vitro transfection efficiency of Hchol‐LNPs in HepG2. The luciferase expression of MC3‐Hchol100 and SM102‐Hchol100 was normalized to MC3‐chol and SM102‐chol, respectively (mean ± standard deviation, n = 3). C) In vivo activity of MC3‐chol and MC3‐Hchol100. LNPs were intramuscularly administered. The total flux of each LNP at each time point was normalized to the total flux obtained from MC3‐chol.
… 
Physicochemical properties of LNPs formulated with cholesterol and Hchol. A) Size and PDI (mean ± standard deviation, n = 3). B) Zeta potential (mean ± standard deviation, n = 3). C) Encapsulation efficiency (mean ± standard deviation, n = 3). D) pKa value determined by TNS assay (mean ± standard deviation, n = 3). E) cryo‐TEM results. The scale bar is 50 nm. F) Cell viability over NIH3T3 and HeLa cells (mean ± standard deviation, n = 3).
Physicochemical properties of LNPs formulated with cholesterol and Hchol. A) Size and PDI (mean ± standard deviation, n = 3). B) Zeta potential (mean ± standard deviation, n = 3). C) Encapsulation efficiency (mean ± standard deviation, n = 3). D) pKa value determined by TNS assay (mean ± standard deviation, n = 3). E) cryo‐TEM results. The scale bar is 50 nm. F) Cell viability over NIH3T3 and HeLa cells (mean ± standard deviation, n = 3).
… 
Intracellular delivery and endosomal escape of Hchol‐LNPs. A) Confocal microscope image showing subcellular localization of Cy5‐GFP mRNA‐LNPs in HeLa cells. B) Fluorescence‐activated cell sorting (FACS) results show uptake of LNPs and protein expression. C) Hemolysis assay of LNPs in acidic and neutral pH conditions. Data presented as mean ± SD of three separate experiments. **p < 0.005. ***p <0.001, student's t‐test.
Intracellular delivery and endosomal escape of Hchol‐LNPs. A) Confocal microscope image showing subcellular localization of Cy5‐GFP mRNA‐LNPs in HeLa cells. B) Fluorescence‐activated cell sorting (FACS) results show uptake of LNPs and protein expression. C) Hemolysis assay of LNPs in acidic and neutral pH conditions. Data presented as mean ± SD of three separate experiments. **p < 0.005. ***p <0.001, student's t‐test.
… 
In vivo activity evaluation of SM102‐chol and SM102‐Hchol100. A) Whole‐body imaging of the mice at indicated time points after intravenous (i.v.) and intramuscular (i.m.) FLuc mRNA‐LNPs injection. Quantitative analysis of the fluorescence signal measured in C57BL/6 mice injected with 0.1 mg kg⁻¹ mRNA‐LNPs by B) intravenous and C) intramuscular routes. D) Cumulative amount of protein by calculating the area‐under‐the‐curve (AUC) in mice injected with 0.1 mg kg⁻¹ mRNA‐LNPs after intravenous and intramuscular injections. Significance was calculated by student's t‐test and data are presented as mean ± SD (n = 3–6, ns: no significant difference, *p < 0.05, **p < 0.005, ***p <0.001, student's t‐test).
In vivo activity evaluation of SM102‐chol and SM102‐Hchol100. A) Whole‐body imaging of the mice at indicated time points after intravenous (i.v.) and intramuscular (i.m.) FLuc mRNA‐LNPs injection. Quantitative analysis of the fluorescence signal measured in C57BL/6 mice injected with 0.1 mg kg⁻¹ mRNA‐LNPs by B) intravenous and C) intramuscular routes. D) Cumulative amount of protein by calculating the area‐under‐the‐curve (AUC) in mice injected with 0.1 mg kg⁻¹ mRNA‐LNPs after intravenous and intramuscular injections. Significance was calculated by student's t‐test and data are presented as mean ± SD (n = 3–6, ns: no significant difference, *p < 0.05, **p < 0.005, ***p <0.001, student's t‐test).
… 
Analysis of inflammatory response and immune response of SM102‐chol and SM102‐Hchol100. A) The relative mRNA expression levels of inflammation‐related genes were compared in cells treated with SM102‐chol and SM102‐Hchol100 by quantitative PCR (qPCR) analysis. Significance was calculated by student's t‐test and data are presented as mean ± SD (n = 3–6, ns: no significant difference, **p < 0.01. ***p < 0.001, student's t‐test). B) Serum immune responses against SARS‐CoV‐2 after intramuscular vaccination with SM102‐chol and SM102‐Hchol100 encapsulating SARS‐CoV‐2 spike mRNA. ELISA measured the SARS‐CoV‐2 S1‐ and S2‐specific IgG titers. The antibody titers were quantified as AUC based on titration curves in Figure S7 in the Supporting Information. The normalized AUC was calculated by normalizing the value with the AUC determined in the PBS group. Significance was calculated by one‐way analysis of variance (ANOVA) and error bars represent the SEM (n = 5, *p < 0.05, ****p < 0.0001).
Analysis of inflammatory response and immune response of SM102‐chol and SM102‐Hchol100. A) The relative mRNA expression levels of inflammation‐related genes were compared in cells treated with SM102‐chol and SM102‐Hchol100 by quantitative PCR (qPCR) analysis. Significance was calculated by student's t‐test and data are presented as mean ± SD (n = 3–6, ns: no significant difference, **p < 0.01. ***p < 0.001, student's t‐test). B) Serum immune responses against SARS‐CoV‐2 after intramuscular vaccination with SM102‐chol and SM102‐Hchol100 encapsulating SARS‐CoV‐2 spike mRNA. ELISA measured the SARS‐CoV‐2 S1‐ and S2‐specific IgG titers. The antibody titers were quantified as AUC based on titration curves in Figure S7 in the Supporting Information. The normalized AUC was calculated by normalizing the value with the AUC determined in the PBS group. Significance was calculated by one‐way analysis of variance (ANOVA) and error bars represent the SEM (n = 5, *p < 0.05, ****p < 0.0001).
… 
Figures - available from: Advanced Healthcare Materials
This content is subject to copyright. Terms and conditions apply.
A preview of this full-text is provided by Wiley.
Learn more
Content available from Advanced Healthcare Materials 
This content is subject to copyright. Terms and conditions apply.
RESEARCH ARTICLE
www.advhealthmat.de
Modulating Lipid Nanoparticles with
Histidinamide-Conjugated Cholesterol for Improved
Intracellular Delivery of mRNA
Onesun Jung, Hye-youn Jung, Le Thi Thuy, Minyoung Choi, Seongyeon Kim,
Hae-Geun Jeon, Jihyun Yang, Seok-Min Kim, Tae-Don Kim, Eunjung Lee,*
Yoonkyung Kim,* and Joon Sig Choi*
Recently, mRNA-based therapeutics, including vaccines, have gained
significant attention in the field of gene therapy for treating various diseases.
Among the various mRNA delivery vehicles, lipid nanoparticles (LNPs) have
emerged as promising vehicles for packaging and delivering mRNA with low
immunogenicity. However, while mRNA delivery has several advantages, the
delivery efficiency and stability of LNPs remain challenging for mRNA therapy.
In this study, an ionizable helper cholesterol analog,
3𝜷[L-histidinamide-carbamoyl] cholesterol (Hchol) lipid is developed and
incorporated into LNPs instead of cholesterol to enhance the LNP potency.
The pKavalues of the Hchol-LNPs are ≈6.03 and 6.61 in MC3- and
SM102-based lipid formulations. Notably, the Hchol-LNPs significantly
improve the delivery efficiency by enhancing the endosomal escape of mRNA.
Additionally, the Hchol-LNPs are more effective in a red blood cell hemolysis
at pH 5.5, indicating a synergistic effect of the protonated imidazole groups of
Hchol and cholesterol on endosomal membrane destabilization. Furthermore,
mRNA delivery is substantially enhanced in mice treated with Hchol-LNPs.
Importantly, LNP-encapsulated SARS-CoV-2 spike mRNA vaccinations induce
potent antigen-specific antibodies against SARS-CoV-2. Overall, incorporating
Hchol into LNP formulations enables efficient endosomal escape and stability,
leading to an mRNA delivery vehicle with a higher delivery efficiency.
1. Introduction
The rapid development of RNA therapy has considerably in-
creased the possibilities to cure previously untreatable diseases.[1]
O. Jung, L. T. Thuy, M. Choi, S. Kim, E. Lee, J. S. Choi
Department of Biochemistry
College of Natural Sciences
Chungnam National University
Daejeon , Republic of Korea
E-mail: ejung@cnu.ac.kr;joonsig@cnu.ac.kr
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/./adhm.
DOI: 10.1002/adhm.202303857
One key technology driving this progress is
lipid nanoparticles (LNPs), a drug delivery
system that consists of a lipid core sur-
rounded by a protective lipid layer.[2]This
system protects therapeutic agents from
degradation and clearance by the immune
system and improves their pharmacokinet-
ics and biodistribution.[3]LNPs are biocom-
patible and biodegradable, making them
a promising alternative to traditional drug
delivery agents such as polymeric nanopar-
ticles and liposomes. The formulation of
LNPs involves microfluidic mixing of lipid
components, including ionizable lipids,
helper lipids, cholesterol, and polyethylene
glycol (PEG)-lipids.[4]These components
are self-assembled into core–shell nanopar-
ticles at low pH, with the core containing
nucleic acid being complexed with ioniz-
able lipids, cholesterol, and helper lipids
(1,2-distearoyl-sn-glycero-3-phosphocholine
(DSPC) and 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine (DOPE)) to improve
the transfection efficacy and structural
integrity.
LNPs effectively deliver nucleic acids
such as mRNA for vaccine and gene
therapy applications.[2c,5]Additionally, LNPs can encapsulate a
wide range of therapeutic agents, including small molecules and
genetic materials, and target specific cells or tissues.[2c,6]Hence,
they have considerable potential as versatile platforms for drug
H.-youn Jung, H.-G. Jeon, J. Yang, Y. Kim
Infectious Disease Research Center
Korea Research Institute of Bioscience and Biotechnology
Daejeon , Republic of Korea
E-mail: ykim@kribb.re.kr
S.-M. Kim, T.-D. Kim
Immunotherapy Research Center
Korea Research Institute of Bioscience and Biotechnology
Daejeon , Republic of Korea
T.-D.Kim,Y.Kim
Bioscience Major
KRIBB School
Korea University of Science and Technology (UST)
Daejeon , Republic of Korea
Adv. Healthcare Mater. 2024,13,  ©  Wiley-VCH GmbH
2303857 (1 of 11)
... Xu et al. introduced an innovative mechanism via lipid-based nanoscale molecular machines, which utilized a light-induced nanomechanical action to destabilize endo-lysosomal compartments, enhancing the cytosolic delivery of therapeutic agents [23]. Building on this concept of improving delivery efficacy, Choi et al. further advanced LNP technology by incorporating histidinamideconjugated cholesterol into LNP formulations, thereby improving mRNA delivery by facilitating endosomal escape [37]. This modification led to potent immune responses and increased stability of the mRNA within biological systems. ...
Rational Design of Lipid-Based Vectors for Advanced Therapeutic Vaccines
Article
Full-text available
  • May 2024
Recent advancements in vaccine delivery systems have seen the utilization of various materials, including lipids, polymers, peptides, metals, and inorganic substances, for constructing non-viral vectors. Among these, lipid-based nanoparticles, composed of natural, synthetic, or physiological lipid/phospholipid materials, offer significant advantages such as biocompatibility, biodegradability, and safety, making them ideal for vaccine delivery. These lipid-based vectors can protect encapsulated antigens and/or mRNA from degradation, precisely tune chemical and physical properties to mimic viruses, facilitate targeted delivery to specific immune cells, and enable efficient endosomal escape for robust immune activation. Notably, lipid-based vaccines, exemplified by those developed by BioNTech/Pfizer and Moderna against COVID-19, have gained approval for human use. This review highlights rational design strategies for vaccine delivery, emphasizing lymphoid organ targeting and effective endosomal escape. It also discusses the importance of rational formulation design and structure–activity relationships, along with reviewing components and potential applications of lipid-based vectors. Additionally, it addresses current challenges and future prospects in translating lipid-based vaccine therapies for cancer and infectious diseases into clinical practice.
View
Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery
Article
Full-text available
  • Oct 2022
  • NAT PROTOC
A new methodology termed selective organ targeting (SORT) was recently developed that enables controllable delivery of nucleic acids to target tissues. SORT lipid nanoparticles (LNPs) involve the inclusion of SORT molecules that accurately tune delivery to the liver, lungs and spleen of mice after intravenous administration. Nanoparticles can be engineered to target specific cells and organs in the body by passive, active and endogenous targeting mechanisms that require distinct design criteria. SORT LNPs are modular and can be prepared using scalable, synthetic chemistry and established engineering formulation methods. This protocol provides detailed procedures, including the synthesis of a representative ionizable cationic lipid, preparation of multiple classes of SORT LNPs by pipette, vortex and microfluidic mixing methods, physical characterization, and in vitro/in vivo mRNA delivery evaluation. Depending on the scale of the experiments, the synthesis of the ionizable lipid requires 4-6 d; LNPs can be formulated within several hours; LNP characterization can be completed in 2-4 h; and in vitro/in vivo evaluation studies require 1-14 d, depending on the design and application. Our strategy offers a versatile and practical method for rationally designing nanoparticles that accurately target specific organs. The SORT LNPs generated as described in this protocol can therefore be applied to multiple classes of LNP systems for therapeutic nucleic acid delivery and facilitate the development of protein replacement and genetic medicines in target tissues. This protocol does not require specific expertise, is modular to various lipids within defined physicochemical classes, and should be accomplishable by researchers from various backgrounds.
View
Lipid nanoparticle-mediated lymph node-targeting delivery of mRNA cancer vaccine elicits robust CD8+ T cell response
Article
Full-text available
  • Aug 2022
  • P NATL ACAD SCI USA
The targeted delivery of messenger RNA (mRNA) to desired organs remains a great challenge for in vivo applications of mRNA technology. For mRNA vaccines, the targeted delivery to the lymph node (LN) is predicted to reduce side effects and increase the immune response. In this study, we explored an endogenously LN-targeting lipid nanoparticle (LNP) without the modification of any active targeting ligands for developing an mRNA cancer vaccine. The LNP named 113-O12B showed increased and specific expression in the LN compared with LNP formulated with ALC-0315, a synthetic lipid used in the COVID-19 vaccine Comirnaty. The targeted delivery of mRNA to the LN increased the CD8+ T cell response to the encoded full-length ovalbumin (OVA) model antigen. As a result, the protective and therapeutic effect of the OVA-encoding mRNA vaccine on the OVA-antigen-bearing B16F10 melanoma model was also improved. Moreover, 113-O12B encapsulated with TRP-2 peptide (TRP2180-188)-encoding mRNA also exhibited excellent tumor inhibition, with the complete response of 40% in the regular B16F10 tumor model when combined with anti-programmed death-1 (PD-1) therapy, revealing broad application of 113-O12B from protein to peptide antigens. All the treated mice showed long-term immune memory, hindering the occurrence of tumor metastatic nodules in the lung in the rechallenging experiments that followed. The enhanced antitumor efficacy of the LN-targeting LNP system shows great potential as a universal platform for the next generation of mRNA vaccines.
View
Lipid Nanoparticle Technologies for Nucleic Acid Delivery: A Nanoarchitectonics Perspective
Article
Full-text available
Lipid‐based nanoparticles have emerged as a clinically viable platform technology to deliver nucleic acids for a wide range of healthcare applications. Within this scope, one of the most exciting areas of recent progress and future innovation potential lies in the material science of lipid‐based nanoparticles, both to refine existing nanoparticle strategies and to develop new ones. Herein, the latest efforts to develop next‐generation lipid‐based nanoparticles are covered by taking a nanoarchitectonics perspective and the design, nucleic acid encapsulation methods, scalable production, and application prospects are critically analyzed for three classes of lipid‐based nanoparticles: 1) traditional lipid nanoparticles (LNPs); 2) lipoplexes; and 3) bicelles. Particular focus is placed on rationalizing how molecular self‐assembly principles enable advanced functionalities along with comparing and contrasting the different nanoarchitectures. The current development status of each class of lipid‐based nanoparticle is also evaluated and possible future directions in terms of overcoming clinical translation challenges and realizing new application opportunities are suggested.
View
An imidazole modified lipid confers enhanced mRNA-LNP stability and strong immunization properties in mice and non-Human primates
Article
Full-text available
  • May 2022
  • BIOMATERIALS
The mRNA vaccine technology has promising applications to fight infectious diseases as demonstrated by the licensing of two mRNA-based vaccines, Comirnaty® (Pfizer/BioNtech) and Spikevax® (Moderna), in the context of the Covid-19 crisis. Safe and effective delivery systems are essential to the performance of these vaccines and lipid nanoparticles (LNPs) able to entrap, protect and deliver the mRNA in vivo are considered by many as the current “best in class”. Nevertheless, current mRNA/LNP vaccine technology has still some limitations, one of them being thermostability, as evidenced by the ultracold distribution chain required for the licensed vaccines. We found that the thermostability of mRNA/LNP, could be improved by a novel imidazole modified lipid, DOG-IM4, in combination with standard helper lipids. DOG-IM4 comprises an ionizable head group consisting of imidazole, a dioleoyl lipid tail and a short flexible polyoxyethylene spacer between the head and tail. Here we describe the synthesis of DOG-IM4 and show that DOG-IM4 LNPs confer strong immunization properties to influenza HA mRNA in mice and macaques and a remarkable stability to the encapsulated mRNA when stored liquid in phosphate buffered saline at 4 °C. We speculate the increased stability to result from some specific attributes of the lipid's imidazole head group.
View
Advances in COVID-19 mRNA vaccine development
Article
Full-text available
  • Mar 2022
To date, the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has determined 399,600,607 cases and 5,757,562 deaths worldwide. COVID-19 is a serious threat to human health globally. The World Health Organization (WHO) has declared COVID-19 pandemic a major public health emergency. Vaccination is the most effective and economical intervention for controlling the spread of epidemics, and consequently saving lives and protecting the health of the population. Various techniques have been employed in the development of COVID-19 vaccines. Among these, the COVID-19 messenger RNA (mRNA) vaccine has been drawing increasing attention owing to its great application prospects and advantages, which include short development cycle, easy industrialization, simple production process, flexibility to respond to new variants, and the capacity to induce better immune response. This review summarizes current knowledge on the structural characteristics, antigen design strategies, delivery systems, industrialization potential, quality control, latest clinical trials and real-world data of COVID-19 mRNA vaccines as well as mRNA technology. Current challenges and future directions in the development of preventive mRNA vaccines for major infectious diseases are also discussed.
View
Modification of Lipid-Based Nanoparticles: An Efficient Delivery System for Nucleic Acid-Based Immunotherapy
Article
Full-text available
  • Mar 2022
  • MOLECULES
Lipid-based nanoparticles (LBNPs) are biocompatible and biodegradable vesicles that are considered to be one of the most efficient drug delivery platforms. Due to the prominent advantages, such as long circulation time, slow drug release, reduced toxicity, high transfection efficiency, and endosomal escape capacity, such synthetic nanoparticles have been widely used for carrying genetic therapeutics, particularly nucleic acids that can be applied in the treatment for various diseases, including congenital diseases, cancers, virus infections, and chronic inflammations. Despite great merits and multiple successful applications, many extracellular and intracellular barriers remain and greatly impair delivery efficacy and therapeutic outcomes. As such, the current state of knowledge and pitfalls regarding the gene delivery and construction of LBNPs will be initially summarized. In order to develop a new generation of LBNPs for improved delivery profiles and therapeutic effects, the modification strategies of LBNPs will be reviewed. On the basis of these developed modifications, the performance of LBNPs as therapeutic nanoplatforms have been greatly improved and extensively applied in immunotherapies, including infectious diseases and cancers. However, the therapeutic applications of LBNPs systems are still limited due to the undesirable endosomal escape, potential aggregation, and the inefficient encapsulation of therapeutics. Herein, we will review and discuss recent advances and remaining challenges in the development of LBNPs for nucleic acid-based immunotherapy.
View
The role of lipid components in lipid nanoparticles for vaccines and gene therapy
Article
  • Jul 2022
  • ADV DRUG DELIVER REV
Lipid nanoparticles (LNPs) play a key role in mRNA vaccines against COVID-19. In addition, many preclinical and clinical studies, including the siRNA-LNP product, Onpattro®, highlight that LNPs unlock the potential of nucleic acid-based therapies and vaccines. To understand how this ‘key’ works, we need to learn about the building blocks that constitute LNPs. In this review, we discuss what each lipid component adds to the LNP delivery platform in terms of size, structure, stability, apparent pKa, nucleic acid encapsulation efficiency, cellular uptake, and endosomal escape. To explore this, we present findings from the liposome field and from landmark and recent articles in the LNP literature. We also discuss challenges and strategies related to in vitro/in vivo studies of LNPs based on fluorescence readouts, immunogenicity/reactogenicity, and LNP delivery beyond the liver. How these fundamental challenges are pursued, including what lipid components are added and combined, will likely determine the scope of LNP-based gene therapies and vaccines for treating various diseases.
View
Hydroxycholesterol substitution in Ionizable lipid nanoparticles for mRNA delivery to T cells
Article
  • May 2022
Delivery of nucleic acids, such as mRNA, to immune cells has become a major focus in the past decade with ionizable lipid nanoparticles (LNPs) emerging as a clinically-validated delivery platform. LNPs-typically composed of ionizable lipids, cholesterol, phospholipids, and polyethylene glycol-have been designed and optimized for a variety of applications including cancer therapies, vaccines, and gene editing. However, LNPs have only recently been investigated for delivery to T cells, which has various therapeutic applications including the engineering of T cell immunotherapies. While several LNP formulations have been evaluated for mRNA delivery, recent work has demonstrated that the utilization of cholesterol analogs may enhance mRNA delivery. Other studies have shown that cholesterols modified with hydroxyl groups can alter endocytic recycling mechanisms. Here, we engineered a library of LNPs incorporating hydroxycholesterols to evaluate their impact on mRNA delivery to T cells by leveraging endosomal trafficking mechanisms. Substitution of 25% and 50% 7α-hydroxycholesterol for cholesterol in LNPs enhanced mRNA delivery to primary human T cells ex vivo by 1.8-fold and 2.0-fold, respectively. Investigation of endosomal trafficking revealed that these modifications also increase late endosome production and reduce the presence of recycling endosomes. These results suggest that hydroxyl modification of cholesterol molecules incorporated into LNP formulations provides a mechanism for improving delivery of nucleic acid cargo to T cells for a range of immunotherapy applications.
View
The clinical progress of mRNA vaccines and immunotherapies
Article
  • May 2022
The emergency use authorizations (EUAs) of two mRNA-based severe acute respiratory syndrome coronavirus (SARS-CoV)-2 vaccines approximately 11 months after publication of the viral sequence highlights the transformative potential of this nucleic acid technology. Most clinical applications of mRNA to date have focused on vaccines for infectious disease and cancer for which low doses, low protein expression and local delivery can be effective because of the inherent immunostimulatory properties of some mRNA species and formulations. In addition, work on mRNA-encoded protein or cellular immunotherapies has also begun, for which minimal immune stimulation, high protein expression in target cells and tissues, and the need for repeated administration have led to additional manufacturing and formulation challenges for clinical translation. Building on this momentum, the past year has seen clinical progress with second-generation coronavirus disease 2019 (COVID-19) vaccines, Omicron-specific boosters and vaccines against seasonal influenza, Epstein–Barr virus, human immunodeficiency virus (HIV) and cancer. Here we review the clinical progress of mRNA therapy as well as provide an overview and future outlook of the transformative technology behind these mRNA-based drugs. Anderson and colleagues discuss the progress and challenges of using mRNA for vaccines and immunotherapy.
View
Targeting strategies for mRNA delivery
Article
  • Jun 2022
mRNA gene therapy has recently emerged as a candidate to enable multiple therapeutic applications including protein replacement therapy, vaccine immunology, and regenerative medicine. Despite the extensive therapeutic potential, the successful clinical translation of mRNA gene therapies has been very limited in practice due to the inadequate understanding of how to target various organs or cell type for protein expression. Multiple studies in the past decade have demonstrated carrier material properties and routes of administration as significant parameters influencing the expression profile of mRNA therapeutics. However, the disparate nature of these reports has prevented critical and global understanding of how these factors contribute to organ targeting for mRNA delivery. Elucidation of trends and commonalities in materials achieving tissue specific mRNA delivery may enable the realization of the medical and commercial promise of therapeutic mRNA medicines. The purpose of this review is to provide a thorough and robust meta-analysis of the various materials that have been successfully used to target different organs for mRNA delivery. The article summarizes the distinct properties of the materials used as well as evaluates various routes of administration of mRNA therapeutics and the applications that can be achieved. This review will therefore serve as useful guide for the community in the development of future materials for mRNA delivery to enable the full potential of this nucleic acid modality for gene therapy.
View