Nonviral delivery of self-amplifying RNA vaccines
Open accessAbstract
Despite more than two decades of research and development on nucleic acid vaccines, there is still no commercial product for human use. Taking advantage of the recent innovations in systemic delivery of short interfering RNA (siRNA) using lipid nanoparticles (LNPs), we developed a self-amplifying RNA vaccine. Here we show that nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA. This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors. Given the many positive attributes of nucleic acid vaccines, our results suggest that a comprehensive evaluation of nonviral technologies to deliver self-amplifying RNA vaccines is warranted.
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Written from the paper’s full text.
TL;DR
Nonviral delivery of a 9-kb self-amplifying RNA encapsulated in lipid nanoparticles (LNP/RNA) increased reporter gene expression and immunogenicity compared with unformulated RNA. In mice and cotton rats, LNP/RNA elicited potent RSV-F-specific humoral and cellular responses and protection from RSV challenge comparable to viral replicon particle delivery.
Key findings
- LNP encapsulation protected self-amplifying RNA from RNase degradation and improved in vivo expression: 0.1 μg LNP/RNA increased serum SEAP over 1 μg naked RNA and reduced animal-to-animal variability.
- After two intramuscular immunizations, 0.1 μg LNP/RNA encoding RSV-F elicited F-specific IgG GMT 26,170 versus 2,292 for 1 μg naked RNA; 1 μg LNP/RNA was equivalent to 1 × 10⁶ IU VRPs (GMT 10,478), and seroconversion occurred after a single LNP/RNA dose as low as 0.01 μg RNA.
- In cotton rats, LNP/RNA boosted F-specific IgG approximately 8-fold (GMT 4,355 vs. 526) and RSV neutralization titers 10-fold (GMT 1,493 vs. 154) relative to the same dose of naked RNA, equivalent to 5 × 10⁶ IU VRPs (GMTs 5,861 and 1,690); all self-amplifying RNA vaccines reduced lung viral load >1,000-fold versus controls after intranasal RSV challenge.
- LNP/RNA elicited slightly elevated F-specific IgG2a relative to IgG1 and potent antigen-specific IFN-γ-producing CD4⁺ and CD8⁺ T cell responses; functional immune responses against HIV antigens were also observed (Fig. S5).
Methods
- Preclinical vaccine study using a self-amplifying RNA based on an alphavirus genome, produced by T7 RNA polymerase transcription from linear pDNA and encapsulated in lipid nanoparticles via an ethanol dilution process using DLinDMA, DSPC, cholesterol, and PEG-DMG 2000.
- Female BALB/c mice (8–10 wk, ~20 g) and female cotton rats were immunized intramuscularly on days 0 and 21; cotton rats were challenged intranasally with 1 × 10⁵ pfu RSV 4 wk after the final immunization.
- Main analyses included serum SEAP and luciferase bioluminescence, RSV-F-specific IgG/IgG1/IgG2a ELISA, RSV neutralization titers, T cell cytokine flow cytometry, and lung viral load; statistics used one-way ANOVA or Kruskal-Wallis with Dunn’s posttest at 95% confidence.
Limitations
- Stated: immunogenicity at submilligram doses in larger species “remains to be directly tested”; the mechanism of action is not fully elucidated, and studies are underway to identify transfected cells and explain why LNP delivery gives longer expression than VRP delivery.
- Stated: production of larger (~9 kb) self-amplifying RNA adds challenges, though the authors call them “not insurmountable.”
- Evident from the text: all efficacy and challenge data are from mice and cotton rats, with no human or nonhuman primate data reported for this LNP/RNA vaccine.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
18 of 59 cited sources checked · 11 supported · 0 flagged · accuracy not yet measured
- Premisecitation§ Nonviral delivery of self-amplifying RNA vaccines
Naked RNA vaccines suffer from limited potency, in part due to RNA instability in vivo, related to the presence of degradative enzymes in tissues.
“However, naked RNA vaccines suffer from limited potency, in part due to RNA instability in vivo, related to the presence of degradative enzymes in tissues (15).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [15] source
- Premisecitation
For infant RSV vaccine development, safety considerations favor replicating (or self-amplifying) vaccine candidates over subunit approaches.
“However, for infant RSV vaccine development, safety considerations favor replicating (or selfamplifying) vaccine candidates over subunit approaches (33).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [33] source
- Premisecitation§ Nonviral delivery of self-amplifying RNA vaccines
EP-mediated delivery of pDNA has been shown to be broadly effective.
“Similarly, EP-mediated delivery of pDNA has been shown to be broadly effective (26) and thus was used as the benchmark for pDNA delivery.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [26] source
- Premisecitation
VRPs have been shown to be potent in nonhuman primates and humans at a 100-fold higher dose (1 × 10^8 IU).
“VRPs have been shown to be potent in nonhuman primates and humans at a 100-fold higher dose (1 × 10 8 IU) (22,25), and thus we anticipate that this unique RNA vaccine will be immunogenic at submilligram doses in larger species, but this remains to be directly tested.”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [22] source
Materials and Methods reports a VRP dose of 1 × 10^8 IU in rhesus macaques, and the Results and Discussion report immune responses and protection with a VRP-prime/Env-protein-boost regimen. The copy does not establish potency in humans or that this dose was 100-fold higher than a comparison dose; it also reports that VRP alone did not protect the macaques. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [25] source
- Premisecitation
The current costs of clinical manufacturing of pDNA are on the order of $50-100 per milligram (1-10 g scale).
“The current costs of clinical manufacturing of pDNA are on the order of $50-100 per milligram (1-10 g scale) (26).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [26] source
- Findingown data§ Nonviral delivery of self-amplifying RNA vaccines
Nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA.
“Here we show that nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA.”
Quote in the paperSubstantial claim - Findingown data§ Nonviral delivery of self-amplifying RNA vaccines
The immune responses elicited by the LNP/RNA vaccine technology were comparable to a viral delivery technology.
“This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors.”
Quote in the paperSubstantial claim - Findingown data§ Nonviral delivery of self-amplifying RNA vaccines
The LNP/RNA vaccine technology elicited broad, potent, and protective immune responses.
“This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors.”
Quote in the paperSubstantial claim - Findingown data
Naked control RNA was degraded by RNase A.
“In contrast, the naked control RNA was degraded by RNase A (Fig. 1C, lane 3 compared with lane 2).”
Quote in the paperSubstantial claim - Findingnone given
The immunogenicity and protective efficacy generated by 1 μg LNP/RNA was equivalent to the responses elicited by the VRP delivery technology.
“Importantly, the immunogenicity and protective efficacy generated by 1 μg LNP/RNA was equivalent to the responses elicited by the VRP delivery technology.”
Quote in the paperSubstantial claim - Findingown data
85-98% of the RNA was encapsulated in the LNP/RNA formulations.
“A fluorescence-based (RiboGreen) assay to detect free RNA in solution after LNP encapsulation demonstrated that 85-98% of the RNA was encapsulated (Table S1).”
Quote in the paperSubstantial claim - Findingown data
The size distributions of the LNP/RNA formulations were characterized by a single peak with a low polydispersity index, indicating a relatively monodisperse size distribution.
“The size distributions were characterized by a single peak with a low polydispersity index, indicating a relatively monodisperse size distribution.”
Quote in the paperNot a substantial claim - Findingcitation
The unformulated RNA vaccine elicited serum F-specific IgG and RSV neutralizing antibodies after two vaccinations.
“As described for other naked self-amplifying RNA vaccines (12), the unformulated RNA vaccine elicited serum F-specific IgG and RSV neutralizing antibodies after two vaccinations (Fig. 5 A and B).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [12] source
- Findingown data
The LNP/RNA elicited slightly elevated titers of F-specific IgG2a relative to IgG1, consistent with a TH1 helper T cell phenotype.
“The LNP/RNA (Fig. 4 B and C) elicited slightly elevated titers of Fspecific IgG2a relative to IgG1, consistent with a T H 1 helper T cell phenotype.”
Quote in the paperNot a substantial claim - Findingown data
Seroconversion was established after a single vaccination with LNP/RNA at doses as low as 0.01 μg of RNA.
“Seroconversion was established after a single vaccination with LNP/RNA at doses as low as 0.01 μg of RNA (Fig. S3).”
Quote in the paperSubstantial claim - Findingown data
10 μg of RNA elicited higher F-specific IgG titers than 1 × 10^6 IU of VRPs.
“In an additional experiment, in which a broader dose range of the LNP/RNA vaccine candidate was explored (Fig. 4), 10 μg of RNA elicited higher F-specific IgG titers than 1 × 10 6 IU of VRPs.”
Quote in the paperSubstantial claim - Findingown data
When pDNA was formulated in the LNPs at an equivalent dose of 0.1 μg, IgG titers (GMT <25) were substantially lower than those elicited by LNP/RNA.
“When pDNA was formulated in the LNPs at an equivalent dose of 0.1 μg (Fig. 3), IgG titers (GMT <25) were substantially lower than those elicited by LNP/RNA.”
Quote in the paperSubstantial claim - Findingown data
The F-specific IgG response to 1 μg of LNP/RNA was equivalent to that elicited by 1 × 10^6 IU of VRPs (GMTs of 10,478).
“The F-specific IgG response to 1 μg of LNP/RNA was equivalent to that elicited by 1 × 10 6 IU of VRPs (GMTs of 10,478).”
Quote in the paperSubstantial claim - Findingown data
The LNP/RNA was a potent inducer of antigen-specific IFN-γ producing CD4+ and CD8+ T cell responses.
“Consistent with this phenotype, the LNP/RNA was a potent inducer of antigen-specific IFN-γ producing CD4 + and CD8 + T cell responses (Fig. 4D and Fig. S4).”
Quote in the paperSubstantial claim - Findingown data
A single bilateral i.m. injection of a low dose (1 μg) of naked self-amplifying RNA resulted in measurable but highly variable bioluminescence at day 7.
“A single bilateral i.m. injection of a low dose (1 μg) of naked self-amplifying RNA resulted in measurable but highly variable bioluminescence at day 7 (Fig. 2A), relative to the encapsulated LNP/RNA.”
Quote in the paperSubstantial claim - Findingown data
After two immunizations, LNP/RNA (0.1 μg RNA) was significantly more immunogenic than naked RNA (1 μg), as measured by F-specific IgG geometric mean titers (GMT of 26,170 vs. 2,292).
“After two immunizations, LNP/RNA (0.1 μg RNA) was significantly more immunogenic than naked RNA (1 μg), as measured by the F-specific IgG geometric mean titers (GMT of 26,170 vs. 2,292; Fig. 3).”
Quote in the paperSubstantial claim - Findingown data
No IL2+ TNFα+, IL2+ IFNγ+, IL2+, or IL5+ CD8 T cells were detected.
“No IL2 + TNFα + , IL2 + IFNγ + , IL2 + , or IL5 + CD8 T cells were detected.”
Quote in the paperNot a substantial claim - Findingnone given
In other studies, the LNP/RNA vaccine also elicited functional immune responses against antigens from HIV.
“In other studies, the LNP/RNA vaccine also elicited functional immune responses against antigens from HIV (Fig. S5).”
Quote in the paperNot a substantial claim - Premisecitation§ Nonviral delivery of self-amplifying RNA vaccines
Direct injection of mRNA or pDNA into the skeletal muscle of a mouse resulted in expression of the encoded protein.
“In 1990, Wolff et al. (5) demonstrated that direct injection (”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [5] source
- Premisecitation§ Nonviral delivery of self-amplifying RNA vaccines
mRNA vaccines have been formulated with synthetic delivery vehicles such as liposomes and cationic polymers to increase potency.
“Hence, mRNA vaccines have been formulated with synthetic delivery vehicles such as liposomes (16) and cationic polymers (17) to increase potency.”
Quote in the paperNot a substantial claim0 of 2 sources checked - Interpretationreasoning§ Nonviral delivery of self-amplifying RNA vaccines
The LNP/RNA vaccine technology avoids the inherent limitations of viral vectors.
“This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors.”
Quote in the paperSubstantial claim - Interpretationown data
Factors other than antigen load and persistence govern immune responses.
“Given that electroporated pDNA produced the highest and most prolonged levels of gene expression in situ (Fig. 2D), these data suggest that factors other than antigen load and persistence govern immune responses.”
Quote in the paperSubstantial claim - Interpretationown data
The LNP delivery system, extensively explored for systemic delivery of siRNA, can be used for delivery of self-amplifying RNA vaccines.
“This report demonstrates that the LNP delivery system, extensively explored for systemic delivery of siRNA, can be used for delivery of self-amplifying RNA vaccines.”
Quote in the paperSubstantial claim - Interpretationown data
Encapsulation was required for efficient delivery of functional RNA into muscle.
“Encapsulation was also required for efficient delivery of functional RNA into muscle.”
Quote in the paperSubstantial claim - Premisecitation
The ionizable cationic lipid DLinDMA is highly effective at delivering siRNA systemically in rodents and nonhuman primates.
“The ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA) (27), which is highly effective at delivering siRNA systemically in rodents and nonhuman primates, was used as the main component of the lipid nanoparticles.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [27] source
- Interpretationown data
The particle size and RNA encapsulation data indicate that the ethanol dilution process produces consistent LNP/RNA particles.
“These particle size and RNA encapsulation data indicate that the ethanol dilution process produces consistent LNP/RNA particles.”
Quote in the paperSubstantial claim - Interpretationreasoning
The lack of immune response elicited by LNP delivery of pDNA versus the response to i.m. injection of LNP/RNA and EP delivery of pDNA could potentially be attributed to inefficient transport of pDNA across the nuclear membrane.
“The lack of immune response elicited by LNP delivery of pDNA (Fig. 2) versus the response to i.m. injection of LNP/RNA and EP delivery of pDNA could potentially be attributed to inefficient transport of pDNA across the nuclear membrane.”
Quote in the paperSubstantial claim - Premisecitation
The current projection for the cost of mRNA manufacture is comparable.
“The current projection for the cost of mRNA manufacture is comparable (9), which makes a very compelling commercial case for extensive evaluation of nonviral delivery of self-amplifying RNA and testing in larger animal species.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [9] source
- Interpretationreasoning
Delivering self-amplifying RNA with a synthetic delivery system avoids anti-vector immunity that currently limits the general utility of viral vectors.
“(iii) avoidance of anti-vector immunity that currently limits the general utility of viral vectors”
Quote in the paperSubstantial claim - Premisecitation
The nuclear barrier for pDNA delivery can be overcome by using relatively high pDNA doses and EP to facilitate pDNA delivery to the nucleus.
“This barrier can be overcome by using relatively high pDNA doses and EP to facilitate pDNA delivery to the nucleus (34).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [34] source
- Premisecitation
The recent pioneering work of others on the clinical production of smaller (~2 kb) nonamplifying mRNA has established the feasibility of large-scale production of mRNA using an enzymatic transcription reaction.
“The recent pioneering work of others (6) on the clinical production of smaller (∼2 kb) nonamplifying mRNA has established the feasibility of large-scale production of mRNA using an enzymatic transcription reaction.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [6] source
- Interpretationreasoning
This technology has potential as a platform to address multiple disease targets.
“Thus, this technology has potential as a platform to address multiple disease targets.”
Quote in the paperNot a substantial claim- Rests on an assumption: The immunogenicity and protection observed in mice and cotton rats are predictive of efficacy in humans.
- Rests on an assumption: The SAM vaccine platform can be applied to multiple disease targets.
- May combine two claims. 38
- Classified as probably not a substantial claim. 9
May combine two claims.
Premisecitation§ Nonviral delivery of self-amplifying RNA vaccinespDNA vaccines have proven to be a flexible platform and are broadly effective in small animal models, but have generally lacked potency in human clinical trials.
“Although plasmid DNA (pDNA) vaccines have proven to be a flexible platform and are broadly effective in small animal models, they have generally lacked potency in human clinical trials (2).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [2] source
Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).
Premisecitation§ Nonviral delivery of self-amplifying RNA vaccinesNew technologies will be required to address changing vaccine needs.
“However, with increasing life expectancy in high-income countries and newly emerging infections and poverty in low-income countries, new technologies will be required to address changing vaccine needs (1).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [1] source
May combine two claims.
Premisecitation§ Nonviral delivery of self-amplifying RNA vaccinesVRPs are potent vaccines in mice, nonhuman primates, and humans.
“VRPs are potent vaccines in mice (10,11), nonhuman primates (11,22), and humans (25).”
Quote in the paperSubstantial claim2 of 4 sources checked- cites [10] source
- cites [11] source
- cites [22] source
The Discussion (page unavailable) reports complete protection of rhesus macaques given an intramuscular VRP prime plus Env-protein boost, supporting the nonhuman-primate portion attributed to this source. It also says none of the macaques given VRP alone were protected, so the unqualified description of VRPs as potent vaccines overstates this study. The separate mice and humans citations are not being judged here. (LLM, conf=0.85)
Experimental — accuracy not yet measured - cites [25] source
May combine two claims.
Premisecitation§ Nonviral delivery of self-amplifying RNA vaccinesThere are limited published data on the in vivo delivery of self-amplifying RNA using nonviral delivery strategies, and none has taken advantage of the recently developed, clinically suitable delivery systems for siRNA.
“There are limited published data on the in vivo delivery of self-amplifying RNA using nonviral delivery strategies (3,14,18), and none has taken advantage of the recently developed, clinically suitable delivery systems for siRNA (19,20).”
Quote in the paperSubstantial claim2 of 5 sources checkedMay combine two claims.
Premisecitation§ Nonviral delivery of self-amplifying RNA vaccinesInjection of naked mRNA or self-amplifying RNA in vivo induces gene expression and generates immune responses, with self-amplifying RNA being more efficient for gene expression in situ.
“Injection of naked mRNA or self-amplifying RNA in vivo induces gene expression and generates immune responses (4, 11-13), with self-amplifying RNA being more efficient for gene expression in situ (13,14).”
Quote in the paperSubstantial claim2 of 5 sources checked- cites [4]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [11] source
- cites [12] source
- cites [13] source
The in vivo gene-expression results report that injected replicative mRNA produced substantially more luciferase than non-replicative mRNA in muscle. The Discussion reports an immune response from prior CEA mRNA injections, but this study did not establish that its self-amplifying RNA generated an immune response; it discusses that possibility as a hypothesis (page not provided). (LLM, conf=0.87)
Experimental — accuracy not yet measured - cites [14] source
The Results section ('Relative expression levels and cellular immune response'; page unavailable) reports in vivo gene expression from both mRNA and replicon RNA, with lower, shorter-lived expression from mRNA. Replicon RNA elicited immune responses, but naked mRNA produced no detectable specific immune response in this study's single-injection experiments. The Introduction does report immune responses to mRNA vaccination in other studies, so the broad claim is supported only with that qualification. (LLM, conf=0.86)
Experimental — accuracy not yet measured
May combine two claims.
PremisecitationThe F protein of RSV is a conserved target of neutralizing antibodies and a promising antigen for RSV vaccine development.
“The F protein of RSV is a conserved target of neutralizing antibodies and a promising antigen for RSV vaccine development (29).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [29] source
May combine two claims.
FindingcitationThe neutralization titers elicited by LNP/RNA are above the titer of 380 that correlates with protection in cotton rats and similar to passively acquired serum neutralizing titers that correlate with protection of human infants from severe RSV disease.
“The neutralization titers elicited by LNP/RNA are above the titer of 380 that correlates with protection in cotton rats (30) and similar to passively acquired serum neutralizing titers that correlate with protection of human infants from severe RSV disease (31,32).”
Quote in the paperSubstantial claim1 of 3 sources checkedMay combine two claims.
PremisecitationCurrently there is no vaccine for RSV and, because of a history of disease enhancement upon natural infection after immunization with an RSV vaccine candidate that elicited TH2-biased (and non-neutralizing) RSV-specific immune responses in RSV-naive infants, elicitation of a non-TH2-biased RSV-specific response is considered an essential attribute for infant RSV vaccine candidates.
“Currently there is no vaccine for RSV and, because of a history of disease enhancement upon natural infection after immunization with an RSV vaccine candidate that elicited T H 2-biased (and non-neutralizing) RSV-specific immune responses in RSV-naive infants, elicitation of a non-T H 2-biased RSV-specific response is considered an essential attribute for infant RSV vaccine candidates (33).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [33] source
The Major hurdles section says adequate RSV vaccine options were unavailable and describes enhanced disease in infants after the formalin-inactivated vaccine. The Vaccine-enhanced disease section reports poor neutralizing antibody responses in those children, and the Role of T cells in vaccine-enhanced illness section recommends avoiding Th2 responses in RSV vaccines. However, it identifies clear Th2 induction in immunized animal models, not as a directly established response in the infants; their lung eosinophilia is cited as a reason for concern. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
InterpretationcitationThe shorter duration of expression in situ after RNA immunization compared with pDNA may be related to the induction of apoptosis of the transfected cell, which could occur during RNA amplification, leading to transient gene expression.
“The shorter duration of expression in situ after RNA immunization compared with pDNA may be related to the induction of apoptosis of the transfected cell, which could occur during RNA amplification, leading to transient gene expression (12,13).”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites [12] source
- cites [13] source
The in vivo results report that replicative mRNA expression returned to baseline within 28 days, while plasmid DNA expression remained above baseline (Augmented gene expression in vivo). The Discussion proposes host-cell protein-synthesis inhibition and resulting cytopathogenicity as one possible explanation for the decline in mRNA-directed expression. It does not specifically identify apoptosis or establish that it occurs during RNA amplification. (LLM, conf=0.85)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationRecently, there has been an exponential growth in the development of clinically suitable nonviral delivery systems for siRNA.
“Recently, there has been an exponential growth in the development of clinically suitable nonviral delivery systems for siRNA (19,20); we have taken advantage of these innovations to develop the LNP/RNA formulation and have now embarked on a more extensive evaluation of other nonviral delivery systems.”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites [19] source
- cites [20] source
The Main section describes advances in synthetic nonviral siRNA carriers and reports that some delivery agents are being used in clinical trials. It does not establish exponential growth, and it says safe, effective delivery remains a barrier rather than characterizing these systems generally as clinically suitable. Page numbers were not provided in our copy. (LLM, conf=0.85)
Experimental — accuracy not yet measured
May combine two claims.
Findingown dataThe same self-amplifying RNA, when delivered using a VRP (1 × 10^6 IU), produced twice the bioluminescence at day 3 compared with LNP/RNA, but expression decayed much more rapidly and reached background levels by day 28.
“The same self-amplifying RNA, when delivered using a VRP (1 × 10 6 IU), produced twice the bioluminescence at day 3 compared with LNP/RNA, but expression decayed much more rapidly and reached background levels by day 28 (Fig. 2B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataEP delivery of pDNA (at a high dose of 20 μg) elicited anti-F IgG titers (GMTs of 6,712) on average 3.9-fold less than those elicited by 0.1 μg of LNP/RNA, although not statistically different.
“EP delivery of pDNA (at a high dose of 20 μg) elicited anti-F IgG titers (GMTs of 6,712) on average 3.9-fold less than those elicited by 0.1 μg of LNP/RNA, although not statistically different.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataMice administered a pDNA-encoding luciferase (10 μg) delivered using EP in situ displayed the highest measurable bioluminescence at all time points tested, and this level remained high for at least 63 d.
“On the other hand, mice administered a pDNA-encoding luciferase (10 μg) delivered using EP in situ displayed the highest measurable bioluminescence at all time points tested, and this level remained high for at least 63 d (Fig. 2D).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataEncapsulation of a 10-fold lower dose of RNA (0.1 μg) in LNPs increased the serum SEAP concentration and reduced the variability between animals.
“Encapsulation of a 10-fold lower dose of RNA (0.1 μg) in LNPs increased the serum SEAP concentration and reduced the variability between animals.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataSix days after intramuscular injection of 1 μg naked self-amplifying RNA encoding SEAP, gene expression was measurable but highly variable.
“Six days after intramuscular (i.m.) injection of 1 μg naked self-amplifying RNA encoding secreted alkaline phosphatase (SEAP), gene expression, as determined by serum SEAP concentration, was measurable but highly variable (Fig. 1D).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataNaked RNA required two vaccinations and microgram doses of RNA.
“This contrasted with what was observed for naked RNA, which required two vaccinations and microgram doses of RNA.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe LNP/RNA was statistically superior to naked RNA and equivalent to the 5 × 10^6 IU of VRPs [GMTs 5,861 (F-specific IgG) and 1,690 (neutralization)].
“The LNP/RNA was statistically superior to naked RNA and equivalent to the 5 ×10 6 IU of VRPs [GMTs 5,861 (F-specific IgG) and 1,690 (neutralization)].”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataAddition of empty LNPs to naked RNA decreased the serum SEAP concentration relative to LNP-encapsulated RNA, indicating that RNA encapsulation was necessary for efficient RNA delivery and reporter gene expression.
“Addition of empty LNPs (without RNA) to naked RNA (0.1 μg) decreased the serum SEAP concentration relative to LNP-encapsulated RNA, indicating that RNA encapsulation was necessary for efficient RNA delivery and reporter gene expression.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S4 (based).
Findingnone givenA recombinant F protein subunit vaccine formulated with alum was shown to be the most potent vaccine for induction of total and protective antibody responses.
“A recombinant F protein subunit vaccine formulated with alum was shown to be the most potent vaccine for induction of total and protective antibody responses.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataBioluminescence intensity induced by LNP/RNA was substantially greater than naked RNA, pDNA, and pDNA launched RNA, and was comparable to the bioluminescence produced by 1 × 10^6 IU of VRPs.
“Bioluminescence intensity induced by LNP/RNA was substantially greater than naked RNA, pDNA, and pDNA launched RNA, and was comparable to the bioluminescence produced by 1 × 10 6 infectious units (IU) of VRPs.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataRNA integrity was maintained during formulation, and the LNPs protected the RNA from degradation by RNase A.
“Agarose gel electrophoresis showed that RNA integrity was maintained during formulation (Fig. 1C, lane 4) and that the LNPs protected the RNA from degradation by RNase A (Fig. 1C, lane 5).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataMice injected with LNP/RNA (1 μg) showed measurable bioluminescence that was already high on day 3, peaked on day 7, and decreased to background by day 63.
“In an extended study (Fig. 2 B-D), mice injected with LNP/RNA (1 μg) showed measurable bioluminescence that was already high on day 3, peaked on day 7, and decreased to background by day 63 (Fig. 2C).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe potency of the LNP/RNA vaccine in mice and cotton rats was comparable to a single-cycle alphavirus vector (VRP, 1 × 10^6 IU) at a reasonable dose of RNA (1 μg) and was generally comparable to pDNA delivered using EP at higher doses.
“The potency of this unique LNP/RNA vaccine in mice and cotton rats was comparable to a single-cycle alphavirus vector (VRP, 1 × 10 6 IU) at a reasonable dose of RNA (1 μg) and was generally comparable to pDNA delivered using EP at higher doses.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataAll self-amplifying RNA vaccines provided protection from a nasal RSV challenge, reducing the lung viral load >1,000-fold compared with control animals.
“All self-amplifying RNA vaccines provided protection from a nasal RSV challenge, reducing the lung viral load >1,000-fold compared with control animals (Fig. 5C) and these data are in agreement with previous reports for naked RNA (12).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [12] source
May combine two claims.
Findingown dataThe LNP/RNA formulation boosted F-specific IgG titers approximately 8-fold (GMTs 4,355) and neutralization titers 10-fold (GMTs 1,493) relative to the same dose of naked RNA [GMTs of 526 (F-specific IgG) and 154 (neutralization)].
“The LNP/RNA formulation boosted F-specific IgG titers approximately 8-fold (GMTs 4,355) and neutralization titers 10-fold (GMTs 1,493) relative to the same dose of naked RNA [GMTs of 526 (F-specific IgG) and 154 (neutralization)].”
Quote in the paperSubstantial claimMay combine two claims.
FindingcitationThe ethanol dilution process produces small uniform lipid particles with a high RNA encapsulation efficiency.
“An ethanol dilution process (28) was used to produce small uniform lipid particles with a high RNA encapsulation efficiency.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [28] source
May combine two claims.
Findingown dataThe number-weighted mean diameters of the six LNP/RNA formulations ranged from 79 to 121 nm and Z-average diameters from 130 to 164 nm with a polydispersity index from 0.09 to 0.14.
“The number-weighted mean diameters ranged from 79 to 121 nm and Z-average diameters from 130 to 164 nm with a polydispersity index from 0.09 to 0.14.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe ratio of F-specific IgG titer to RSV neutralization titer is low for the LNP/RNA vaccine relative to the RSV-F subunit adsorbed on alum; thus it may be particularly suitable for development into a safe and effective infant RSV vaccine candidate.
“The ratio of F-specific IgG titer to RSV neutralization titer (Table S2) is low for the LNP/ RNA vaccine relative to the RSV-F subunit adsorbed on alum; thus it may be particularly suitable for development into a safe and effective infant RSV vaccine candidate.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe LNP/RNA provides the benefit of a non-TH2-biased response in addition to strong cellular immune responses.
“The LNP/RNA provides this benefit (Fig. 4) in addition to strong cellular immune responses.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S4 (based). May combine two claims.
Findingnone givenThe self-amplifying RNA vaccine platform generates a humoral and cellular immune response (both CD4 and CD8).
“(iv) the generation of a humoral and cellular immune responses (both CD4 and CD8).”
Quote in the paperNot a substantial claimMay combine two claims.
FindingreasoningFacilitated delivery of RNA, either by VRPs or LNPs, resulted in higher levels of reporter gene expression (SEAP and luciferase) than naked RNA delivery and considerably lower variability of expression between animals.
“Facilitated delivery of RNA, either by VRPs or LNPs, resulted in higher levels of reporter gene expression (SEAP and luciferase) than naked RNA delivery and considerably lower variability of expression between animals.”
Quote in the paperSubstantial claimMay combine two claims.
Premisecitation§ Nonviral delivery of self-amplifying RNA vaccinesMany of the obstacles to mRNA vaccine development have been surmounted, and recently there has been a revival in the use of nonamplifying mRNA vaccines for cancer, allergy, and gene therapy.
“However, many of the obstacles to mRNA vaccine development have been surmounted, and recently there has been a revival in the use of nonamplifying mRNA vaccines for cancer (6), allergy (7), and gene therapy (8).”
Quote in the paperNot a substantial claim0 of 3 sources checkedMay combine two claims.
Premisecitation§ Nonviral delivery of self-amplifying RNA vaccinesNaturally transient and cytosolically active mRNA can now be produced at sufficient quantity and quality for human clinical trials and is seen by many as a safer and more potent alternative to pDNA for vaccination.
“Naturally transient and cytosolically active mRNA can now be produced at sufficient quantity and quality for human clinical trials (6) and is seen by many (9) as a safer and more potent alternative to pDNA for vaccination.”
Quote in the paperSubstantial claim0 of 2 sources checkedMay combine two claims.
Premisecitation§ Nonviral delivery of self-amplifying RNA vaccinesViral vector-based technologies are generally regarded as the most efficient means to deliver nucleic acids into cells, but their utility can be restricted by preexisting or vaccine-induced antivector immunity that can decrease vaccine potency.
“Viral vector-based technologies are generally regarded as the most efficient means to deliver nucleic acids into cells, but their utility can be restricted by preexisting or vaccine-induced antivector immunity that can decrease vaccine potency (12).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [12] source
May combine two claims.
Premisecitation§ Nonviral delivery of self-amplifying RNA vaccinesRecombinant viral vector technologies have the advantage of efficient delivery of the nucleic acid payload, but their utility is often hampered by antivector immunity, production limitations, and safety concerns.
“Recombinant viral vector technologies have the advantage of efficient delivery of the nucleic acid payload, but their utility is often hampered by antivector immunity, production limitations, and safety concerns (3,4).”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [3] source
- cites [4]
May combine two claims.
PremisecitationVarious RNA vaccines have been evaluated in human clinical trials, including naked and formulated non-amplifying mRNA and self-amplifying RNA packaged in VRPs.
“Various RNA vaccines have been evaluated in human clinical trials, including naked and formulated non-amplifying mRNA (37-39) and self-amplifying RNA packaged in VRPs (25).”
Quote in the paperNot a substantial claim0 of 4 sources checkedMay combine two claims.
InterpretationreasoningRNA vaccines avoid the nuclear delivery rate-limiting step by using cytoplasmic amplification and expression, resulting in more efficient transfection of different cell types, including quiescent or slowly proliferating cells, such as vascular endothelia or myocytes.
“RNA vaccines avoid this rate-limiting step by using cytoplasmic amplification and expression, resulting in more efficient transfection of different cell types, including quiescent or slowly proliferating cells, such as vascular endothelia or myocytes.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningThe LNP formulation markedly enhances the potency of the self-amplifying RNA, thereby generating a robust and potentially generic vaccine technology.
“Therefore, the LNP formulation markedly enhances the potency of the self-amplifying RNA, thereby generating a robust and potentially generic vaccine technology.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenImage acquisition times were kept constant as bioluminescence was measured with a cooled CCD camera.
“Image acquisition times were kept constant as bioluminescence was measured with a cooled CCD camera.”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationThose studies showed RNA vaccines to be well tolerated and immunogenic.
“Those studies showed RNA vaccines to be well tolerated and immunogenic.”
Quote in the paperSubstantial claim0 of 4 sources checkedClassified as probably not a substantial claim. May combine two claims.
InterpretationreasoningBecause the replication cycle of the self-amplifying RNA is strictly cytoplasmic, the need for codon modification is diminished, and the risks of splicing and destruction of the transcript are avoided.
“In addition, because the replication cycle of the self-amplifying RNA is strictly cytoplasmic, the need for codon modification is diminished, and the risks of splicing and destruction of the transcript are avoided.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.
InterpretationreasoningFor an LNP/RNA vaccine, this sequestration might result in antigen production and/or stimulation of innate immune pathways within these immune cells.
“For an LNP/RNA vaccine, this sequestration might result in antigen production and/or stimulation of innate immune pathways within these immune cells.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S4 (based).
Interpretationnone givenProducing self-amplifying RNA from a cell-free transcription reaction eliminates the theoretical risk of generating infectious virus through recombination during production.
“(ii) elimination of theoretical risks of generating infectious virus through recombination during production”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningThe LNP delivery system may achieve these effects by a combination of increased transfection efficiency and protection of the RNA from enzymatic degradation at the site of injection.
“The LNP delivery system may achieve these effects by a combination of increased transfection efficiency and protection of the RNA from enzymatic degradation at the site of injection.”
Quote in the paperSubstantial claim
The paper relies on this without support.
There is still no commercial product for human use for nucleic acid vaccines despite more than two decades of research and development.
“Despite more than two decades of research and development on nucleic acid vaccines, there is still no commercial product for human use.”
The paper relies on this without support.
mRNA-based vaccines must match the potency of viral vectors at doses of RNA that are not cost prohibitive to be commercially competitive as a platform technology.
“However, to be commercially competitive as a platform technology, mRNA-based vaccines must match the potency of viral vectors at doses of RNA that are not cost prohibitive.”
The paper relies on this without support.
The major limitation for delivery of pDNA is the nuclear barrier, and transport across the nuclear membrane is particularly inefficient in nondividing cells such as mature myocytes.
“The major limitation for delivery of pDNA is thought to be the nuclear barrier, and transport across the nuclear membrane seems to be particularly inefficient in nondividing cells, such as mature myocytes.”
The paper relies on this without support.
The immunogenicity and protection observed in mice and cotton rats are predictive of efficacy in humans.
“Thus, this technology has potential as a platform to address multiple disease targets.”
The paper relies on this without support.
The SAM vaccine platform can be applied to multiple disease targets.
“Thus, this technology has potential as a platform to address multiple disease targets.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Self-amplifying mRNA vaccines benefited from formulation with lipid nanoparticles or cationic nanoemulsion and were able to induce protective antibody titers.
“Studies with self-amplifying mRNA vaccines investigated the effect of different formulations such as lipid-nanoparticles (LNP)10 or cationic nanoemulsion (CNE)11 on the immunogenicity of mRNA vaccines. Self-amplifying vaccines benefited from formulation with LNP or CNE and were able to induce protective antibody titers.”
From Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines (2017) · cites this paper as [10]
Geall and colleagues were the first to use LNP to encapsulate a saRNA chimera composed of the Venezuelan equine encephalitis virus (VEEV) and Sindbis virus (SINV) replicons into a vaccine which was used to immunize mice, and immunogenicity was significantly improved compared with unencapsulated group.
“Decades later, Geall and colleagues were the first to use LNP to encapsulate a saRNA chimera composed of the Venezuelan equine encephalitis virus (VEEV) and Sindbis virus (SINV) replicons into a vaccine which was used to immunize mice, and immunogenicity was significantly improved compared with unencapsulated group.199”
From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [199]
The success of the first approved siRNA-LNP therapeutic and mRNA COVID-19 vaccines originates only from their delivery using ionizable lipid-containing LNPs.
“Based on the potential toxicity of liposomes in clinical application,25,26 the success of the first approved small interfering RNA-lipid nanoparticle (LNP) therapeutic27 and mRNA COVID-19 vaccines originates only from their delivery using ionizable lipid-containing LNPs,28, 29, 30”
From COVID-19 mRNA vaccines: Platforms and current developments (2022) · cites this paper as [30]
In preclinical studies, mRNA vaccines encoding either the native RSV F protein or the stabilized prefusion conformation were successfully delivered using cationic nanoemulsions and lipid nanoparticles without any observed instances of VAED.
“In preclinical studies, mRNA vaccines encoding either the native RSV F protein or the stabilized prefusion conformation were successfully delivered using cationic nanoemulsions109 and LNPs179,180 without any observed instances of VAED.”
From mRNA vaccines for infectious diseases: principles, delivery and clinical translation (2021) · cites this paper as [180]
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BackgroundmRNA-based therapeutics: powerful and versatile tools to combat diseases
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“overall higher immunogenicity than the non-encapsulated variant.[134] However, it was not reported that SAM is more immunogenic than transient mRNA.”
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“Decades later, Geall and colleagues were the first to use LNP to encapsulate a saRNA chimera composed of the Venezuelan equine encephalitis virus (VEEV) and Sindbis virus (SINV) replicons into a vaccine which was used to immunize mice, and immunogenicity was significantly improved compared with unencapsulated group.(1…”
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“Upon injection in mice, LNP-formulated selfamplifying mRNA encoding firefly luciferase induced protein expression lasting almost two months upon IM delivery (130), while luciferase expression from protamine-formulated, nonreplicating mRNA administered ID was usually only detected for several days (133).”
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Recent advances in mRNA vaccine technology
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The 60-year evolution of lipid nanoparticles for nucleic acid delivery
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“This was first demonstrated in 2012 by the finding that LNP developed for siRNA delivery (containing DLinDMA) could also be used to encapsulate self-amplifying RNA (saRNA) coding for the F-protein of respiratory syncytial virus 134 .”
Innate immune mechanisms of mRNA vaccines
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Self-amplifying RNA vaccines for infectious diseases
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Surface-Engineered Dendrimers in Gene Delivery
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“RNA vaccines can also be engineered RNA replicons derived from certain RNA viruses lacking viral structural proteins which are capable of self-replication on delivery to the cytoplasm (Geall et al , 2012, 2013).”
Self-citationThe challenge and prospect of mRNA therapeutics landscape
Weng, Y., Li, C., Yang, T., Hu, B., Zhang, M., Guo, S., Xiao, H., Liang, X.-J., & Huang, Y. (2020) · Biotechnology Advances · doi:10.1016/j.biotechadv.2020.107534 · Cited by 417 · see citing works
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“Cationic lipids, efficacious in some applications (29, 84), can be toxic when used at higher doses and if incompletely complexed (85–87).”
BackgroundMethodMaterials for non-viral intracellular delivery of messenger RNA therapeutics
Kauffman, K. J., Webber, M. J., & Anderson, D. G. (2015) · Journal of Controlled Release · doi:10.1016/j.jconrel.2015.12.032 · Cited by 400 · see citing works
Nanotechnology for COVID-19: Therapeutics and Vaccine Research
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Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems
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mRNA Vaccine Era—Mechanisms, Drug Platform and Clinical Prospection
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“LNP Intramuscular Mice, rat Respiratory syncytial virus infection [17]”
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Novel approaches for vaccine development
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“When a viral replicon genome is delivered with an LNP and compared with a viral particle containing the same genome both generated similar immune responses in rodents (Geall et al., 2012).”
Self-citationCOVID-19 Vaccine Frontrunners and Their Nanotechnology Design
Chung, Y. H., Beiss, V., Fiering, S., & Steinmetz, N. F. (2020) · ACS Nano · doi:10.1021/acsnano.0c07197 · Cited by 341 · see citing works
A Cationic Nanoemulsion for the Delivery of Next-generation RNA Vaccines
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Self-citationNucleic acid delivery for therapeutic applications
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Respiratory syncytial virus infection in adults
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Manufacturing Considerations for the Development of Lipid Nanoparticles Using Microfluidics
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“Microfluidics has previously been shown to be an effective tool for the manufacture of LNPs [15,16,19,20], liposomes [21–23] and polymer-based nanoparticles [24–26].”
MethodResultCOVID-19 mRNA vaccines: Platforms and current developments
Szabó, G. T., Mahiny, A. J., & Vlatkovic, I. (2022) · Molecular Therapy · doi:10.1016/j.ymthe.2022.02.016 · Cited by 308 · see citing works
“…nanoparticle (LNP) therapeutic 27 and mRNA COVID-19 vaccines originates only from their delivery using ionizable lipid-containing LNPs, 28 – 30 which are known to have signi fi cantly higher delivery ef fi ciency in hepatocytes after intravenous (i.v.) injection 31 or in muscle cells after…”
Cell specific delivery of modified mRNA expressing therapeutic proteins to leukocytes
Veiga, N., Goldsmith, M., Granot, Y., Rosenblum, D., Dammes, N., Kedmi, R., Ramishetti, S., & Peer, D. (2018) · Nature Communications · doi:10.1038/s41467-018-06936-1 · Cited by 308 · see citing works
An Update on Self-Amplifying mRNA Vaccine Development
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“Other in-line mixing methods have also been employed for saRNA-LNP formulations including alternative microfluidic architectures[82] and in-line macro-mixing in a T-tube.[35, 51, 52, 78, 79] To remove solvent and adjust the final concentration, tangential flow filtration has been employed as a high-throughput method.”
BackgroundMethodInfluentialSelf-citationDNA vaccines against COVID-19: Perspectives and challenges
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“The encapsulation efficiency was > 85% [96, 97].”
Treating Disease at the RNA Level with Oligonucleotides
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“Inhalation may not suit patients with respiratory complications, and the transient nature of mRNA prompts the exploration of payloads such as self-amplifying RNA 14,54 for persistent cytokine expression.”
From influenza to COVID-19: Lipid nanoparticle mRNA vaccines at the frontiers of infectious diseases
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An Orthogonal Array Optimization of Lipid-like Nanoparticles for mRNA Delivery in Vivo
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Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines
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Controlling timing and location in vaccines
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“mRNA is inherently unstable in vivo and antigen expression levels are significantly more transient than observed with DNA vaccination, typically only persisting for several days [183,275].”
Lipid Nanoparticles as Delivery Systems for RNA-Based Vaccines
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“CNE, LNP Self-amplifying DSPC, cholesterol, DMG PEG 2000, DLinDMA F (RSV) Mice, cotton rats IM Humoral, cellular, protection [38,41]”
BackgroundMechanism of action of mRNA-based vaccines
Iavarone, C., O’hagan, D. T., Yu, D., Delahaye, N. F., & Ulmer, J. B. (2017) · Expert Review of Vaccines · doi:10.1080/14760584.2017.1355245 · Cited by 234 · see citing works
“mediates and later positive-sense genomic and subgenomic (+)RNAs [14,15].”
BackgroundSelf-citationmRNA vaccines for infectious diseases — advances, challenges and opportunities
Pardi, N., & Krammer, F. (2024) · Nature Reviews Drug Discovery · doi:10.1038/s41573-024-01042-y · Cited by 246 · see citing works
“The delivery problem was solved in the 2010s by the development of LNPs containing ‘ionizable’ lipids 48,59,60 , which are usually composed of four components: phospholipid (helper lipid), cholesterol, polyethylene glycol (PEG)-conjugated lipid and a cationic ionizable aminolipid (with a pKa of…”
Rapidly produced SAM <sup>®</sup> vaccine against H7N9 influenza is immunogenic in mice
Hekele, A., Bertholet, S., Archer, J., Gibson, D. G., Palladino, G., Brito, L. A., Otten, G. R., Brazzoli, M., Buccato, S., Bonci, A., et al. (2013) · Emerging Microbes & Infections · doi:10.1038/emi.2013.54 · Cited by 226 · see citing works
Self-citationWeissman, D. (2015) · Expert Review of Vaccines · doi:10.1586/14760584.2015.973859 · Cited by 228 · see citing works
“Routy JP, Boulassel MR, Yassine-Diab B, et al. Immunologic activity and safety of autologous HIV RNA-electroporated dendritic cells in HIV-1 infected patients receiving antiretroviral therapy.”
BackgroundInfluentialNanomedicines to Deliver mRNA: State of the Art and Future Perspectives
Gómez-Aguado, I., Rodríguez-Castejón, J., Vicente-Pascual, M., Rodríguez‐Gascón, A., Solinís, M. Á., & del Pozo‐Rodríguez, A. (2020) · Nanomaterials · doi:10.3390/nano10020364 · Cited by 224 · see citing works
Formulation and Delivery Technologies for mRNA Vaccines
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A trans-amplifying RNA vaccine strategy for induction of potent protective immunity
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“Accordingly, lower dose levels of saRNA than nrRNA may suffice to induce comparable immune responses.(7,8)”
BackgroundPepini, T., Pulichino, A.-M., Carsillo, T., Carlson, A. L., Sari-Sarraf, F., Ramsauer, K., Debasitis, J. C., Maruggi, G., Otten, G. R., Geall, A. J., et al. (2017) · The Journal of Immunology · doi:10.4049/jimmunol.1601877 · Cited by 208 · see citing works
Lipid-based mRNA vaccine delivery systems
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“The resulting emulsion had a size of 86 nm. Protective RSV vaccination was obtained in mice after two intramuscular injections of 15 mg self-amplifying mRNA encoding the fusion (F) glycoprotein of RSV. High level of HIV neutralizing antibody titers was also generated in rabbits after two intramuscular injections of 25…”
BackgroundMethodInfluentialHow mRNA therapeutics are entering the monoclonal antibody field
Van Hoecke, L., & Roose, K. (2019) · Journal of Translational Medicine · doi:10.1186/s12967-019-1804-8 · Cited by 205 · see citing works
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From empiricism to rational design: a personal perspective of the evolution of vaccine development
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Self-citationBiomaterials for vaccine-based cancer immunotherapy
Zhang, R., Billingsley, M. M., & Mitchell, M. J. (2018) · Journal of Controlled Release · doi:10.1016/j.jconrel.2018.10.008 · Cited by 201 · see citing works
Theoretical basis for stabilizing messenger RNA through secondary structure design
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Vaccine development for respiratory syncytial virus
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“Other approaches that deliver vaccine antigens through genebased vectors [43–45] or nucleic acid [46,47] are another possible avenue towards the goal of infant immunization.”
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“Adequate delivery of mRNA with lipid nanoparticles (LNPs) has been demonstrated for mRNA-based vaccines, where intramuscular injection of low doses of mRNA formulated in either LNPs or nanoemulsion induced immune protection from influenza and respiratory syncytial virus in mice, as well as cytomegalovirus and respirat…”
BackgroundCoolen, A.-L., Lacroix, C., Mercier‐Gouy, P., Delaune, E. A., Monge, C., Exposito, J., & Verrier, B. (2018) · Biomaterials · doi:10.1016/j.biomaterials.2018.12.019 · Cited by 192 · see citing works
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“Formulation of the mRNA antigen within an LNP improves the immune response to the vaccine, presumably by both protecting the mRNA from enzymatic degradation and facilitating efficient uptake and intracellular release of the mRNA in target cells.(28) For effective mRNA immunization, the vaccine mRNA may be modified to…”
BackgroundSelf-citationRNA: The new revolution in nucleic acid vaccines
Geall, A. J., Mandl, C. W., & Ulmer, J. B. (2013) · Seminars in Immunology · doi:10.1016/j.smim.2013.05.001 · Cited by 187 · see citing works
Self-citationClinical and immunological effects of mRNA vaccines in malignant diseases
Heine, A., Juranek, S., & Brossart, P. (2021) · Molecular Cancer · doi:10.1186/s12943-021-01339-1 · Cited by 185 · see citing works
Bogers, W. M., Oostermeijer, H., Mooij, P., Koopman, G., Verschoor, E. J., Davis, D., Ulmer, J. B., Brito, L. A., Cu, Y., Banerjee, K., et al. (2015) · Journal of Infectious Diseases · doi:10.1093/infdis/jiu522 · Cited by 181 · see citing works
“the higher amount of immunostimulatory RNA contained in SAM vaccine versus VRP vaccine and/or a differential antigen expression profile in situ [19].”
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Self-citationNucleic acid vaccines: prospects for non-viral delivery of mRNA vaccines
Deering, R. P., Kommareddy, S., Ulmer, J. B., Brito, L. A., & Geall, A. J. (2014) · Expert Opinion on Drug Delivery · doi:10.1517/17425247.2014.901308 · Cited by 177 · see citing works
“We and others have demonstrated that lipid-complexed mRNA vaccines adequately achieve adaptive immunity, presumably in part through the self-adjuvanting effect of activating type I IFN [22].”
BackgroundMethodInfluentialSelf-citationLou, G., Anderluzzi, G., Schmidt, S., Woods, S., Gallorini, S., Brazzoli, M., Giusti, F., Ferlenghi, I., Johnson, R. N., Roberts, C. W., et al. (2020) · Journal of Controlled Release · doi:10.1016/j.jconrel.2020.06.027 · Cited by 172 · see citing works
Self-citationDrug delivery systems for CRISPR-based genome editors
Madigan, V. J., Zhang, F., & Dahlman, J. E. (2023) · Nature Reviews Drug Discovery · doi:10.1038/s41573-023-00762-x · Cited by 180 · see citing works
Chemically Modified Platforms for Better RNA Therapeutics
Shi, Y., Zhen, X., Zhang, Y., Li, Y., Koo, S., Saiding, Q., Kong, N., Liu, G., Chen, W., & Tao, W. (2024) · Chemical Reviews · doi:10.1021/acs.chemrev.3c00611 · Cited by 184 · see citing works
Nanoscale platforms for messenger RNA delivery
Li, B., Zhang, X., & Dong, Y. (2019) · WIREs Nanomedicine and Nanobiotechnology · doi:10.1002/wnan.1530 · Cited by 170 · see citing works
“After intramuscular injection, the lipid nanoparticles (LNP/saRNA) induced Respiratory Syncytial Virus Fusion Glycoprotien-specific antibody and T-cell responses in both mice and rats (Geall et al., 2012).”
BackgroundKasiewicz, L. N., Biswas, S., Beach, A., Ren, H., Dutta, C., Mazzola, A. M., Rohde, E., Chadwick, A. C., Cheng, C., Garcia, S. P., et al. (2023) · Nature Communications · doi:10.1038/s41467-023-37465-1 · Cited by 172 · see citing works
Coronavirus Disease (COVID-19): Current Status and Prospects for Drug and Vaccine Development
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Erasmus, J. H., Khandhar, A. P., Guderian, J., Granger, B., Archer, J., Archer, M., Gage, E., Fuerte-Stone, J., Larson, E., Lin, S., et al. (2018) · Molecular Therapy · doi:10.1016/j.ymthe.2018.07.010 · Cited by 161 · see citing works
Immune Responses Induced by mRNA Vaccination in Mice, Monkeys and Humans
Cagigi, A., & Loré, K. (2021) · Vaccines · doi:10.3390/vaccines9010061 · Cited by 153 · see citing works
“The biodistribution of self-amplifying RNA vaccines has been reported to be similar to conventional mRNA vaccines, but showed delayed kinetics and stronger expression of the encoded antigen [33,34].”
BackgroundKis, Z., Kontoravdi, C., Shattock, R. J., & Shah, N. (2020) · Vaccines · doi:10.3390/vaccines9010003 · Cited by 161 · see citing works
Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system
DeRosa, F., Guild, B., Karve, S., Smith, L., Love, K., Dorkin, J. R., Kauffman, K. J., Zhang, J., Yahalom, B., Anderson, D. G., et al. (2016) · Gene Therapy · doi:10.1038/gt.2016.46 · Cited by 159 · see citing works
Blakney, A. K., Zhu, Y., McKay, P. F., Bouton, C. R., Yeow, J., Tang, J., Hu, K., Samnuan, K., Grigsby, C. L., Shattock, R. J., et al. (2020) · ACS Nano · doi:10.1021/acsnano.0c00326 · Cited by 155 · see citing works
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“S1A in the supplemental material) (48).”
MethodSelf-citationA novel receptor-binding domain (RBD)-based mRNA vaccine against SARS-CoV-2
Tai, W., Zhang, X., Drelich, A., Shi, J., Hsu, J. C., Luchsinger, L., Hillyer, C. D., Tseng, C.-T. K., Jiang, S., & Du, L. (2020) · Cell Research · doi:10.1038/s41422-020-0387-5 · Cited by 151 · see citing works
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Self-citationPorcine epidemic diarrhea virus: Molecular mechanisms of attenuation and vaccines
Li, Z., Ma, Z., Li, Y., Gao, S., & Xiao, S. (2020) · Microbial Pathogenesis · doi:10.1016/j.micpath.2020.104553 · Cited by 150 · see citing works
Nanovaccines for cancer immunotherapy
Zhang, Y. X., Lin, S., Wang, X., & Zhu, G. (2019) · Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology · doi:10.1002/wnan.1559 · Cited by 146 · see citing works
The use of RNA-based treatments in the field of cancer immunotherapy
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da Silva, J. R., Rodrigues, K. B., Pelegrin, G. F., Sales, N. S., Muramatsu, H., Silva, M. D. O., Porchia, B. F. M. M., Moreno, A. C. R., Aps, L. R. M. M., Venceslau-Carvalho, A. A., et al. (2023) · Science Translational Medicine · doi:10.1126/scitranslmed.abn3464 · Cited by 148 · see citing works
“Although multiple studies have demonstrated that both nr-mRNA–based (nucleoside-unmodified or nucleoside-modified) (16, 21, 25, 27, 35, 47) and sa-mRNA–based (24, 48, 49) vaccines can induce robust immune responses, comparative studies evaluating all three platforms side by side have been lacking.”
BackgroundAhmed, S., Schur, P. H., MacDonald, N. E., & Steinman, L. (2014) · Journal of Autoimmunity · doi:10.1016/j.jaut.2014.01.033 · Cited by 142 · see citing works
“, DNA [49] or RNA [49,50]) that are taken up by the vaccine recipient’s cells for synthesis of vaccine antigen that results in an immune response by the host.”
BackgroundmRNA in the Context of Protein Replacement Therapy
Vavilis, T., Stamoula, Ε., Ainatzoglou, A., Sachinidis, A., Lamprinou, M., Dardalas, I., & Vizirianakis, I. S. (2023) · Pharmaceutics · doi:10.3390/pharmaceutics15010166 · Cited by 143 · see citing works
Designing liposomal adjuvants for the next generation of vaccines
Perrie, Y., Crofts, F. J., Devitt, A., Griffiths, H. R., Kastner, E., & Nadella, V. (2015) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2015.11.005 · Cited by 139 · see citing works
Self-replicating alphavirus RNA vaccines
Ljungberg, K., & Liljeström, P. (2014) · Expert Review of Vaccines · doi:10.1586/14760584.2015.965690 · Cited by 137 · see citing works
“Indeed, it was shown that RREP-SAM expresses the antigen as long as up to 63 days post-immunization, which probably prolongs the initial response [68].”
BackgroundInfluential
- Authors
- Geall, A. J., Verma, A., Otten, G. R., Shaw, C. A., Hekele, A., Banerjee, K., Cu, Y., Beard, C. W., Brito, L. A., Krucker, T., O’Hagan, D. T., Singh, M., Mason, P. W., Valiante, N. M., Dormitzer, P. R., Barnett, S. W., Rappuoli, R., Ulmer, J. B., & Mandl, C. W.
- Affiliations
- Novartis Vaccines and Diagnostics, Cambridge, MA 02139; and
- Novartis Institutes for BioMedical Research, Cambridge MA 02139
- ORCID
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2012-08-20
- Volume
- 109
- Issue
- 36
- Pages
- 14604-14609
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC3437863
- OpenAlex
- W2051665875
- Cited by
- 675
- References
- 41
- Citations by year
- 2026: 34 · 2025: 63 · 2024: 66 · 2023: 66 · 2022: 79 · 2021: 78 · 2020: 74 · 2019: 41 · 2018: 31 · 2017: 28 · 2016: 31 · 2015: 27 · 2014: 31 · 2013: 24 · 2012: 2
- FWCI
- 9.79
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (submittedVersion)
- PubMed Central (submittedVersion)
- Funders
- National Institute of Allergy and Infectious Diseases (5P01 AI066287, P01AI066287)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmaceutical Science › Advancements in Transdermal Drug Delivery
- Keywords
- self-amplifying RNA vaccine, lipid nanoparticles, siRNA delivery, nucleic acid vaccines, immunogenicity, protective immune response
- MeSH terms
- Alphavirus, Analysis of Variance, Animals, Electrophoresis, Agar Gel, Escherichia coli, Female, Fluorescent Antibody Technique, Humans, Lipids, Drug Delivery Systems, Statistics, Nonparametric, Vaccines, DNA, RNA, Small Interfering, Rats, Nanoparticles
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-11.
Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-11.
Cited sources are checked by gpt-6-sol.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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