May 19, 2024

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COVID-19 vaccine: SARS-CoV-2 mRNA delivery system for clinical trials

COVID-19 vaccine: SARS-CoV-2 mRNA delivery system for clinical trials


COVID-19 vaccine: SARS-CoV-2 mRNA delivery system for clinical trials.


Current development of lipid nanoparticles for clinical trials of SARS-CoV-2


The earliest mRNA transfection reagent is quaternized cation DOTAP combined with ionizable and fused DOPE, using DNA transfection [85], used to transfect mRNA in many cell types [86].

Although effective in vitro, the permanent cationic quaternary ammonium groups allow these large-sized lipid complexes to be rapidly cleared from the circulation and their usual target organs, lungs, and exhibit toxicity.


The pioneer of LNP today is the stable plasmid-lipid particle (SPLP), which is formed by the combination of DOPE ionized by the fusion gene and the quaternized cationic lipid DODAC. DODAC electrostatically binds and encapsulates the plasmid DNA, and then uses hydrophilicity.


PEG coating makes it stable in aqueous media and restricts protein-cell interactions when administered in vivo [87]. DOPE can be protonated in endosomes after cellular uptake. Because of its cone shape, it can form endosome dissolving ion pairs with endosomal phospholipids to promote endosome release, which is a key event for successful delivery [17].


Then SPLP was further developed into stable nucleic acid lipid particles (SNALP) containing siRNA, including four lipids: ionized rather than quaternized cationic lipids, saturated bilayers to form quaternized zwitterionic lipids, DSPC, and cholesterol And PEG lipids [88].


In addition to electrostatic binding with nucleic acids, the ionizable lipids in SNALPs act as fusion lipids, protonate in endosomes, and form membrane-unstable ion pairs with endosomal phospholipids. It is currently known that DSPC helps to form a stable double layer under the surface of PEG [89].


Cholesterol plays a variety of roles, including filling intergranular spaces, limiting LNP-protein interactions, and possibly promoting membrane fusion [90]. Ionizable lipids are neutral at physiological pH to eliminate any cationic charge in the circulation, but they are protonated in the endosome at pH ~ 6.5 to promote the release of the endosome and play a central role.


The development of the first siRNA product clinically approved in 2018 mainly focused on optimizing ionizable lipids, followed by the ratio of the four lipids used in PEG lipids and LNP, as well as LNP assembly and production procedures. According to the molecular shape hypothesis [12,91], it was found that the optimal number of unsaturated bonds in the C18 tail is the dilinoleic acid tail connected to the dimethylamine head group via an ether [88].


However, the introduction of a single linker at the tail of linoleic acid, which has an optimized carbon number from the dimethylamine head group to the linker, results in the pKa of the ionizable lipid DLin-MC3-DMA in LNP close to 6.4 [92,93]. The final step of optimization is to adjust the lipid molar ratio of MC3/DSPC/cholesterol/PEG-lipid to 50/10/38.5/1.5.


The overall optimization process from DLin-DMA to DLin-MC3-DMA requires screening of more than 300 ionizable lipids among thousands of formulations, resulting in a 200-fold increase in potency and a corresponding reduction in effective dose to achieve the target Gene> 80% lasting inhibition and allowable therapeutic window, Onpattro™ was clinically approved in 2018 [94,95].


This MC3 formulation developed for siRNA is the basis for the subsequent development of LNPs, as described below (Figure 1). After being approved for the delivery of SARS-CoV-2 mRNA vaccines, LNPs are now being used urgently.



COVID-19 vaccine: SARS-CoV-2 mRNA delivery system for clinical trials.

Figure 1 The structure of mRNA lipid nanoparticles. Recent studies using cryo-electron microscopy [96], small-angle neutron scattering, and small-angle X-ray scattering [89] have shown that mRNA lipid nanoparticles include low-copy number mRNA (1-10), and that mRNA is related to the central core of LNP. The ionizable lipid binding. Polyethylene glycol (PEG) lipids form the surface of lipid nanoparticles (LNP) and at the same time form a double-layer DSPC. Charged and uncharged cholesterol and ionizable lipids can be distributed throughout the LNP. Schematic diagrams of other delivery systems are provided in a recent review [14].


Moderna has conducted several preclinical [97,98,99] and clinical studies [97,100] using MC3 in the above Onpattro formulation to deliver nucleoside modified mRNA encoding immunogens. In these relatively new studies of ionizable lipids and MC3, MC3 was later identified as ionizable lipids [42,101].

This new category includes Lipid H [42], which is the ionizable lipid SM-102 in Moderna’s SARS-CoV-2 product mRNA-1273 [41] (Table 2).


Using a nucleoside-modified mRNA encoding immunogen for Zika virus, MC3 LNP can protect immunocompromised mice lacking type I and type II interferon (IFN) signals from a 10 µg dose once or a 2 µg dose primary immunization-boost The lethal attack of immune design [99].

Similar results were obtained in mice with normal immune function that were pre-administered with anti-ifnar1 blocking antibody to establish a lethal model.

In a series of influenza studies that delivered nucleoside-modified mRNA encoding hemagglutinin (HA) immunogens, MC3 LNP delivered intradermally was able to completely protect mice from lethal attacks as low as 0.4 µg in a single dose, although Even with a single dose of up to 10 µg, weight loss will occur after challenge [97].

A single dose of 50 µg or 100 µg in ferrets produced higher HAI (hemagglutination inhibition test) titers, and a single dose of 200 or 400 µg in non-human primates also produced higher HAI titers.

In a few (23) human subjects who received a dose of 100 µg, at the beginning of the study, all subjects had HAI titers> 40 (who protection correlation), which was more than 4 times higher than baseline.

In a larger phase 1 trial, using the same MC3 LNPs to deliver two different nucleoside-modified mRNA-encoded HA immunogens, intramuscular injection of 100 µg of H10N8 immunogen resulted in 100% of 23 subjects HAI titer> 40 [100]. Although there were no life-threatening adverse events, 3 of the 23 subjects experienced severe grade 3 adverse events.

After the occurrence of grade 3 adverse events in 2 of the 3 subjects (in accordance with the study suspension rule), the planned 400 µg dosing was discontinued.

At lower doses, the frequency and severity of adverse events were reduced, but at least one adverse event occurred in almost every subject. These studies are promising, but they also emphasize that the therapeutic window for obtaining protective immunity at a dose that does not cause a problematic number of adverse events is relatively narrow.

This is reminiscent of the narrow therapeutic window of the MC3 precursor DLin-DMA, which requires increased efficacy to reduce the dose and still achieve effective gene knockdown.


Table 2 Ionizable lipids used in lipid nanoparticles. A key feature of the ionizable lipids used in lipid nanoparticles is that the pKa of the ionizable lipids in LNP should be in the range of 6-7 as determined by the TNS dye binding test. The pKa of most ionizable groups calculated theoretically is in the range of 8-9.5, as shown below on the nitrogen atom, using commercial software to theoretically estimate these values ​​in an aqueous medium. The decrease of pKa by 2-3 points from the theoretical value to the TNS value is due to the higher solvation energy of the protons in the lipid phase, resulting in the pH value in the lipid phase higher than the water phase by 2-3 points. The pH value is measured during the TNS measurement. [102].

COVID-19 vaccine: SARS-CoV-2 mRNA delivery system for clinical trials.



Since siRNA products require repeated administrations to treat chronic diseases, there is concern that the slow degradation of dilinoleic acid alkyl tails in MC3 will lead to accumulation and potential toxicity of repeated administrations.

The biodegradable version of MC3, lipid 319 (Table 2), is produced by substituting one of the two double bonds in each alkyl chain with a primary ester that is easily degraded by esterases in the body [68].

It was observed that the half-life of lipid 319 in the liver was less than 1 hour, while it maintained a gene silencing efficiency similar to MC3 in the liver.

The degradation products, as well as their secretion and the non-toxic nature of lipid 319 are confirmed in vivo. The study of Lipid 319 was cited in the preclinical and clinical studies of SARS-CoV-2 as the Acuitas LNP category used in BioNTech[49] and CureVac[53,69] products, although it was tested in Imperial College London[60 The self-amplified RNA delivered by Acuitas LNP in] is cited as being included in a recent patent application [59], represented here by Acuitas’ Lipid A9 (Table 2).

Recently, the identity of the Acuitas ionizable lipid in BNT162b2 approved by BioNTech was disclosed as ALC-0315 [40] (Table 2). An important aspect of these LNPs is that they were developed by screening mRNA expression in the liver after IV administration, and may not have been fully optimized for intramuscular administration of mRNA-based vaccines.


Moderna recently developed a new class of ionizable lipids to replace MC3, mainly due to the above-mentioned problems related to the slow degradation of MC3, but it also strives to improve its effectiveness by achieving larger branches than dilinoleic acid MC3 alkyl tails. [42,101].

This type of new lipid has an ethanolamine ionizable head group, which is connected to a single saturated tail containing a primary degradable ester (such as Maier 2013), and is connected to a second saturated tail. The latter uses a less degradable secondary ester in After 7 carbon atoms, it branches into two saturated C8 tails, such as lipid 5 [101] (Table 2).

Optimized for IV administration to the liver, it was found that similar lipid H[42] or SM-102 is the best choice for intramuscular (IM) administration of vaccines. Branch increase is a common feature pursued by Acuitas, because Lipid A9 has a total of 5 branch chains [59] (Table 2), while Moderna LNPs have 3 branch chains. Increased branching is thought to produce ionizable lipids with more cone-shaped structures.

Therefore, when paired with anionic phospholipids in the endosome, greater membrane damage will occur, following the molecular shape assumptions outlined decades ago [12,91]. When administered by IV, lipid 5 was not detected in the liver at 24h, while MC3 was present in the liver at 71% of its initial dose, verifying the degradability of Lipid 5.

After IV administration, Lipid 5 is 3 times more potent than MC3 in expressing luciferase in mice, and hEPO is 5 times more potent than MC3 in non-human primates. These increases in potency are consistent with the increased release of endosomes, which may be caused by increased release of endosomes. For Lipid 5, up to 15% of the mRNA in the cell is released from the endosomes, compared to 2.5% for MC3. The latter is the same as the previous one. The results of MC3 measured with siRNA are similar [106].

However, in these endosomal release experiments, the cellular uptake of MC3 was 4 times that of Lipid 5, so the absolute amounts of mRNA released by these two LNPs in the cytoplasm were similar.

In the intramuscular administration of the vaccine, the same ionizable lipid pool was examined, and it was also found to be degradable and quickly eliminated due to primary esters, and for influenza nucleoside modified mRNA%, compared with MC3, the protein expression Or immunogenicity, the titer is usually increased by 3-6 times. % Encoded immunogen in mice, although the immunogenicity in non-human primates is the same as MC3 at a dose of 5 µg priming-booster [42].

Lipid H or SM-102 (Table 2) was identified as the best drug candidate, which was only structurally different from lipid 5, which was determined to be the best for IV administration by two-carbon replacement of primary esters.

Lipid 5 LNP has a pKa of 6.56, while lipid H LNP has a pKa of 6.68, indicating that a slight increase in pKa may be beneficial for IM compared to IV administration, although the difference is within the variability range of the test.

Histological examination of the intramuscular injection site in rats showed that Lipid HLNPs attracted less inflammatory infiltration of neutrophils and macrophages than MC3, which may reduce the reactogenicity of the injection site in human trials. [42].



MRNA lipid nanoparticles currently in clinical trials of SARS-CoV-2


5.1. BioNTech/Pfizer


Acuitas ALC-0315 (Table 2) combined with DSPC, cholesterol and PEG lipids is the delivery system in the BioNTech SARS-COV-2 trial [40]. CureVac and Imperial College London may also use ALC-0315, or may use A9 (Table 2).


BioNTech started to develop its SARS-CoV-2 vaccine using four mRNA-encoded immunogens, two of which are nucleoside modified, one is unmodified, and one is self-amplified. There are two reports of nucleoside-modified mRNA: BNT162b1 is a short ~1kb sequence encoding the receptor binding domain of the spike protein. It is modified by the foldon trimer domain and is shown to increase immunogenicity through multivalence. The longer 4.3kb BNT162b2 encodes a diproline stabilized full-length membrane-bound spike protein. BNT162b2 has recently received emergency approvals from the European Union and the United States.


In a preclinical study, after a single dose of 0.2, 1, and 5 µg BNT162b2 in mice, binding antibodies and neutralizing titers could be detected. An order of magnitude increase from the lowest dose to the highest dose caused strong antigen-specific Th1 IFNγ and IL-2 responses in CD4 + and CD8 + splenocytes with very low Th2 cytokine levels [49].

The draining lymph nodes also contained a large number of germinal center B cells and increased counts of CD4 + and CD8 + follicular helper T (Tfh) cells, which were previously identified as part of the LNP induced by the mRNA LNP vaccine alone [33].

In non-human primates, the binding antibody and neutralization titers caused by the initial-booster dose of 30 µg or 100 µg are more than 10 times that of the human recovery period test group, and produce a strong Th1 biased T cell response. The response is thought to be important for preventing vaccine-related enhancement of respiratory diseases [107].

In a limited number (6) of challenged rhesus monkeys, no virus titers were detected in two doses of 100 µg in bronchoalveolar lavage and nasal swabs.


A phase 1 clinical trial of the smaller mRNA-encoding immunogen BNT162b1 is planned to give doses of 10, 30 and 100 µg on day 1 and day 21. The antibody binding and neutralizing titers induced by a medium dose of 30 µg were 30 times and 3 times that of the human convalescent group, respectively. Due to severe injection site pain after the first administration, a booster dose of 100 µg was not given.

100% of subjects in the 30 µg booster group reported pain at the injection site, but the severity was mild or moderate. After the second vaccination with a dose of 30 µg, almost all subjects experienced mild or moderate systemic adverse events, fever, chills, or fatigue. The test also proved the strong response of peripheral blood mononuclear cells to Th1 biased T cells [50].


A phase 2 trial compared BNT162b1 and BNT162b2 in groups of young (18-55 years) and older (65-85 years) subjects [51]. The titers of binding and neutralizing antibodies in elderly subjects were slightly lower, but still higher than those in the recovery period. Compared with younger subjects, the severity of adverse reactions in elderly subjects was also reduced.

Compared with BNT162b1, the frequency of systemic adverse events (fever, chills, fatigue) found in BNT162b2 was significantly reduced by about twice. It is the increased tolerance of BNT162b2 that motivated its choice to enter the Phase 3 trial.

It recently announced a 94% effectiveness, because 162 cases of COVID-19 occurred in the placebo group, and after receiving two doses of 30µg BNT162b2 Only 8 cases were found in the vaccination group[3]


5.2. Moderna


In Moderna’s research, the nucleoside-modified mRNA-encoding immunogen is a transmembrane-anchored diproline stabilized pre-fusion spike, with a natural furin cleavage site, and an LNP based on the prototype MC3 LNP Medium delivery, just using Lipid H (SM-102) instead of MC3 [41, 42].

The mRNA LNP (mRNA-1273) induced neutralizing antibodies in several mouse species when injected at a dose of 1 µg on days 1 and 21, but did not induce neutralizing antibodies at a dose of 0.1 µg [44]. The T cell response seems to be a balanced Th1/Th2 response.

In a mouse-adapted virus challenge model, the virus titers in the mouse lung and turbinate decreased to baseline after two doses of 1 μg, but at 0.1 μg. It did not decrease after the medicine. In rhesus monkeys, two doses of 100 µg produced high binding and neutralizing titers and Th1 biased responses in peripheral blood, which also involved a strong Tfh response [45].

The titers and T cell responses of the two 10 µg dose groups were significantly reduced. Similarly, a 100µg dose can reduce the virus titer in bronchoalveolar lavage fluid and nasal swabs to baseline, while 10µg only decreases in the lungs.


In the phase 1 study, 15 patients in each group received 2 doses of 25, 100, or 250 µg, 4 weeks apart, and the binding and neutralization titers were about 10 times higher than the recovery period of the 100 µg dose, which is approximately equivalent to the recovery period of 25 µg [46 ].

All subjects in the 100µg and 250µg dose groups reported soliciting adverse events, and 3 of the 14 subjects in the 250µg group reported severe adverse events and discontinued the drug.

In the subsequent phase 1 study of elderly patients (56-71 years and older), it was found that the binding antibody titers produced by the 25µg and 100µg doses were higher than the recovery plasma, while the neutralization titers of the 100µg dose were equivalent but lower than The recovery period for a dose of 25 µg [43]. Most patients (about 80%) still have adverse events after the second vaccination, even in the elderly group. Peripheral blood analysis showed that CD4 T cell response was Th1 biased.

Compared with the 25μg dose, the 100μg dose has a higher neutralizing titer. Therefore, this dose was selected for the phase 3 trial. The interim results showed that 90 COVID-19 cases in the placebo group and 5 cases in the vaccination group had 94.5% Effectiveness [2].

An independent committee conducted an interim analysis of Moderna’s Phase 3 trial and found that serious adverse events included fatigue in 9.7% of participants, muscle pain in 8.9%, arthralgia in 5.2%, and headache in 4.5%. In Pfizer/BioNTech In the Phase 3 trial, fatigue was 3.8% and headache was 2% [108].


5.3. CureVac


The CureVac mRNA LNP (CVnCoV) is a non-chemically modified sequence engineered mRNA that encodes a diproline-stabilized full-length S protein delivered in Acuitas LNP, possibly using ionizable lipid ALC-0315.

When using a 2μg dose in mice, the number of weeks between two administrations was checked, ranging from 1-4 weeks, and it was found that longer intervals in Balb/c mice produced higher titers and T cell responses And a balanced Th1/Th2 response [53].

A second dose is required to produce neutralizing antibodies, and two doses of 0.25 µg are not sufficient to produce neutralizing antibodies.

In Syrian golden hamsters, two doses of 10 µg (instead of 2 µg) were able to reduce the virus titer in the lungs (not the turbinates) to baseline.


In a phase 1 clinical trial that examined a dose of 2-12 µg, only the highest dose of 12 µg was found to reach a neutralizing titre at the convalescent serum level, leading to the inclusion of higher doses of 16 and 20 µg in the ongoing phase 2 trial [52].

All patients who received the 12 µg dose experienced systemic adverse events after each administration, most of which were moderate and severe, while> 80% of patients had local injection site pain at mild and moderate levels.


5.4. TranslateBio


Translate Bio uses unmodified mRNA to encode a double-mutated form of the diproline stabilized spike protein, and delivers it via an LNP cited as an ionizable lipid C12-200 [109], which may have been recently released from ICE- [110] Or cysteine-based [55] Candidate for synthesis of ionizable lipid family.

In Balb/c mice, two doses in the range of 0.2–10 µg resulted in binding and neutralization titers much higher than the recovery level. In non-human primates, the titers produced by doses of 15, 45 and 135 µg all exceeded the human recovery period [56]. The immune response also has Th1 bias.


5.5. Arcturus


Arcturus uses self-amplified, full-length, unmodified mRNA to encode the pre-fusion SARS-CoV-2 full-length spike protein, which is delivered via LNP, which uses ionizable lipids and thioesters, via two additional ester groups Connect the amine-containing head group to the lipid tail. The two possible ionizable lipids in this family are lipid 10a (Table 4 of [111]) or lipid 2,2(8,8)4 C CH3 (page 33 of [57]) (Table 2) .

The latter has three branches, similar to Moderna Lipid H, but has a degradable thioester attached to the head group. The characteristics of self-amplified mRNA were observed, that is, more than 1 week after IM administration, the expression of luciferase reporter gene was maintained at a fairly constant level, while the expression of conventional mRNA declined rapidly [58].


Vaccination alone produced a surprising weight loss and clinical score increase in C57BL/6 mice. In mice, a single dose of 2μg or 10μg (instead of 0.2μg) is required to achieve a neutralization titer higher than 100 in a Th1 biased response and a high level of antigen-specific T cell response.

In the K18-hACE2 lethal mouse challenge model, a single administration of 2 µg or 10 µg also has a 100% protective effect, 100% survival, no weight loss, and lung and brain virus titers reduced to baseline levels. Arcturus has completed a phase 1 clinical trial with a dose of 1-10 µg, and selected 7.5 µg for a phase 3 trial [112].


5.6. Imperial College


The pre-fusion stable spike protein encoded by self-amplified mRNA delivered by Imperial College London using Acuitas LNP is described in the patent [59] represented by Lipid A9 [60] (Table 2).

After two injections of 0.01 µg to 10 µg in Balb/c mice, higher dose-dependent antibody and neutralizing titers were obtained.

This response has a strong Th1 bias. Compared with the lower 0.1 and 0.01 µg doses, the 10 and 1 µg doses produced an antigen-specific splenocyte response that was three times higher. Phase 1 clinical trials of this vaccine are about to begin.


5.7. Chulalongkorn University, University of Pennsylvania


Chulalongkorn University, in cooperation with the University of Pennsylvania, is using Genevant LNP (perhaps CL1 lipid) to develop a natural spiked immunogen nucleoside modified mRNA LNP [61].

Their goal is to start phase 1 clinical trials in the first quarter of 2021, and to distribute the vaccine to Thailand and seven surrounding low- to middle-income countries in the fourth quarter of 2021.


5.8. Providence Therapeutics


The notification that Providence Therapeutics was granted a license from Health Canada to conduct human clinical trials of the PTX-COVID-19B mRNA LNP vaccine [113].

After C57BL6 mice received a dose of 20 µg according to the primary immunization-boost immunization regimen, three mRNA candidates encoding the receptor binding domain (full-length spikes with or without mutations at the furin cleavage site) were subjected to preclinical studies [ 114].

The results of preclinical data from Genevant’s undisclosed lipids (which may be similar to CL1 in Table 2) showed robust neutralizing titers for full-length and furin mutant payloads, similar to the data observed in [115].

The Phase 1 clinical trial is scheduled to begin in the first quarter of 2021, and the production and distribution of the vaccine — awaiting regulatory approval — will take place in the same year.


5.9. Storage and distribution


Most RNA LNPs made in the laboratory can be stable for several days at 4 °C, but then show an increase in volume and a gradual loss of biological activity, such as luciferase expression [116]. In previous siRNA LNP preparations, it was generally observed that the size of LNP aggregation increased over time [117].

In order to stabilize the mRNA LNP vaccine for storage and distribution, a frozen form has so far been required. Moderna COVID-19 vaccine needs to be stored at -25 ℃~-15 ℃, but it can be stable for up to 30 days between 2 ℃ and 8 ℃, and it can be stable for up to 12 h between 8 ℃ and 25 ℃[118] .

The Pfizer/BioNTech COVID-19 vaccine needs to be stored at -80 ℃ to -60 ℃, then thawed and stored at 2 ℃ to 8 ℃ for up to 5 days, and then diluted with normal saline before injection [119].


Compared with the conventional freezing temperature required for Moderna vaccine, the dry ice temperature required for Pfizer vaccine during distribution and storage is more difficult to reach.

The reason behind these temperature differences is not obvious, because both vaccines contain similar high concentrations of sucrose as a cryoprotectant. Moderna mRNA LNPs are frozen in Tris and acetate buffers [41], while the Pfizer/BioNTech vaccine only uses phosphate buffer [40]. It is known that phosphate buffer is not ideal for freezing because it is easy to precipitate and causes a sudden change in pH at the beginning of ice crystallization [120,121].

Lyophilization has always been challenging for mRNA LNPs [116]. However, Arcturus stated that their COVID-19 mRNA vaccine is stable in the freeze-dried form, which may greatly simplify the distribution, although the temperature stability of this freeze-dried formulation has not been disclosed [122].





Original source: Automated Manufacture of Autologous CD19 CAR-T Cells for Treatment of Non-hodgkin Lymphoma.Front.Immunol.11:1941. DOI:10.3389/fimmu.2020.01941

COVID-19 vaccine: SARS-CoV-2 mRNA delivery system for clinical trials.

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