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The Mechanism of Adverse Immune Reactions of LNP-mRNA

Introduction

May 7, 2024

Many clinical trials of mRNA-based drugs or vaccines have failed to successfully pass Phase I or Phase II. The reasons behind this are various, including low efficacy of candidate drugs and lower-than-expected clinical risk/treatment benefit profiles. Preclinical safety assessments aim to identify well-tolerated and efficacious LNP-mRNA formulations, and when toxicity is observed, in vivo, in vitro and ex vivo experiments aim to understand the underlying mechanisms and, ideally, improve the formulations design under development.

Figure 1. Adverse effects of LNPs.

The main safety issues of LNP-mRNA preparations in preclinical development can be divided into immunopathogenicity and liver and spleen toxicity (only studies on modified and/or dsRNA-purified mRNA are considered). Understand the different formulations of LNP-mRNA preparations and mechanisms of adverse effects help us better design and optimize drug candidates that are well tolerated and effective.

Liver and Spleen Toxicity

Since LNP-mRNA has significant liver and spleen biodistribution, microscopy and histopathology of the liver and spleen are standard practice during preclinical development. Acute drug-induced liver injury is routinely assessed by measuring plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (APT). Most current published studies of LNP-mRNA therapeutic applications report only mild pathological findings.

In one such study, modified mRNA encoding human methylmalonyl-CoA mutase (hMUT) was synthesized to treat methylmalonic acidemia/aciduria (MMA), a serious A hypomorphic mouse model of a rare metabolic disorder. At the highest intravenous dose of the LNP-mRNA formulation, there were no clinical chemistry findings, but 80% of mice had mild paracentral splenic artery lymphopenia. This effect, which was attributed to LNPs rather than hMUT mRNA or its expression, was also observed in LNPs carrying mRNA for green fluorescent protein (eGFP).

In another study, modified mRNA encoding human arginase was used to treat a mouse model of arginase deficiency. Although there were no biochemical or histopathological findings, electron microscopy of liver sections from the group receiving luciferase mRNA single control preparation revealed the presence of submicron-sized lipid droplets.

Furthermore, a single intramuscular injection of an LNP-mRNA preparation encoding the influenza hemagglutinin H3 antigen showed increased levels of AST, ALT, and C-reactive protein in a rabbit model. Histopathological findings of the liver included focal subscapular vacuoles, inflammatory cell and erythroid infiltrates, and pleocytosis (lymphocyte expansion) was observed in the germinal center of the spleen.

Immune Response

The human body’s adverse immune responses to nanomedicines include reactogenicity, hypersensitivity reactions, systemic complement immune responses, and cytokine-mediated reactions after vaccination. For LNP-mRNA-based therapies in preclinical development, such events may compromise their safety and reduce therapeutic efficiency. However, due to the inherent compositional complexity of LNP vectors, it is not easy to determine which components of a given LNP-mRNA complex elicit unwanted innate immune responses and which conditions may exacerbate them (dose, route of administration, pre-existing inflammation, etc.) thing.

• TLR Activation and Cytokine Secretion

Stimulation of TLRs by LNP-mRNA is considered to be the upstream pathway of cytokine production. In a related study, lipopolysaccharide (LPS) was used to treat mice to induce inflammation. The inflamed mice then received a single intravenous injection of LNP-mRNA and exhibited IL-6, a C-C motif chemokine ligand 2 (CCL2) and other proinflammatory cytokines (in serum) and C-X-C motif chemokine ligand 2 (CXCL2, in liver) are elevated. Similar immune responses were observed when mice were given empty LNP. Research points out that the ionizable cationic lipid ALC-0315 is the most important immune-stimulating ingredient. The inflammatory phenotype is ablated in macrophage-depleted mice as well as Tlr4−/− mouse models. Interestingly, similar findings were observed with mRNA preparations using two other ionizable cationic lipids (DLin-MC3-DMA or C12-200).

In an independent study, colocalization of TLR4 and LNP-mRNA in endosomes following in vitro LPS activation of mouse macrophages corroborates the hypothesis that LNP-mRNA preparations trigger innate immune system responses via TLR4 in response to pre-existing inflammation. Under these conditions, LNP endosomal escape using cKK-E12 ionizable cationic lipids is impaired, while phosphorylation of protein kinase R (PKR) downstream of TLR4 results in reduced translation of intracytoplasmic mRNA. Although LNP alone was not introduced for control, these TLR4-related findings point to lipid-mediated biological effects on delivery efficiency.

Overall, TLR activation and pro-inflammatory cytokine release are common innate immune system effects induced by LNP-mRNA, sometimes causing strong adverse reactions and affecting protein translation. Activation of TLR4 by ionizable cationic lipids is a possible upstream initiating event, although the exact molecular mechanism has not yet been elucidated. Other factors may also be necessary to cause or exacerbate the inflammatory effects, which may depend on the payload, dose, or route of administration, etc.

• Inflammasome Activation

Inflammasome activation was recently identified as a unique innate immune system effect induced by LNP-mRNA. The canonical pathway of pyroptosis requires an initiation signal to activate NF-κB and initiate the transcription of NOD-like receptor pyrin domain protein 3 (NLRP3) and pro-IL-1β, the latter of which may originate from TLR activation. Secondary signals initiate the assembly and activation of the NLRP3 inflammasome.

In an in vitro study, NLRP3 inflammasome activation resulted in reduced mRNA transfection efficiency after administration of DLin-MC3-DMA-based LNP-mRNA to bone marrow-derived macrophages in vitro. LPS serves to provide the initiation signal, but mRNA may also promote initiation through TLR activation. Lysosomal disruption and damage-associated molecular patterns serve as secondary signals during LNP-mRNA escape. NLRP3 activation can be inferred from IL-1β release, cleaved gasdermin D and caspase 1 expression, and cathepsin B maturation.

When human peripheral blood mononuclear cells (PBMC) are treated with LNP-eGFP mRNA configured with DLin-MC3-DMA or SM-102, IL-1β and other pro-inflammatory cytokines such as IL-6, CCL2, CCL4 and TNF, etc.) release increases dramatically. Notably, the SM-102-based empty formulation also triggered strong IL-1β secretion, suggesting that these lipids provide priming and activation signals for inflammasome activation.

• Complement Activation and Hypersensitivity Reactions

Complement proteins in plasma and cell surfaces are essential components of the innate immune system. Complement activation, a series of proteolytic events, supports the phagocytic clearance of pathogenic substances or particles. The emergence of biologics and nucleic acid therapies has rapidly elevated immunotoxicological studies related to complement activation into routine practice in drug development. Identification of pathogenicity is the upstream pathway that initiates complement activation, of which there are three pathways: the classical pathway, initiated by pattern recognition of IgG or IgM; the alternative pathway, initiated by the hydrolysis of the thioester bond in complement protein C3.

LNP-mRNA preparations have been shown to activate the complement pathway. In an in vivo study in a cynomolgus monkey model, intravenous administration of LNP-mRNA expressing hEPO resulted in a mild and reversible increase in plasma complement protein C3a and C5b-9 levels. In another study, C3b/c and soluble C5b-9 were also increased after incubation of CD40L-expressing LNP-mRNA in complement-active human serum. Anti-PEG IgM is required for complement activation and is associated with loss of LNP integrity. These findings are consistent with previous reports that immune cells mediate accelerated clearance of nanomedicine formulations due to immunoglobulin opsonization and complement activation

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Here’s a clean, structured breakdown of the illnesses, pathological findings, and immunological phenomena mentioned in your snippet — written in a way that first‑year Yale immunology students can use as a compact reference.

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🧬 Compendium of Illnesses & Immunopathological Phenomena Mentioned in the Snippet

Below is an enumerated list of every illness, pathological condition, or immune‑related adverse effect referenced in the text, along with concise explanations and immunology‑relevant notes.

1. Methylmalonic Acidemia / Methylmalonic Aciduria (MMA)

Type: Rare inherited metabolic disorder
Cause: Deficiency of methylmalonyl‑CoA mutase or defects in cobalamin metabolism
Key Features:

• Accumulation of methylmalonic acid

• Metabolic acidosis

• Failure to thrive, neurologic impairment
  Immunology relevance:

• Not primarily an immune disease, but metabolic stress can influence immune cell function.

• Used in the snippet as a disease model for testing LNP‑mRNA delivery of the hMUT enzyme.

2. Arginase Deficiency

Type: Urea cycle disorder
Cause: Deficiency of arginase‑1, leading to impaired ammonia detoxification
Key Features:

• Hyperammonemia

• Neurological symptoms

• Growth delay
  Immunology relevance:

• Arginine metabolism is tightly linked to macrophage polarization (M1 vs. M2).

• Again, used as a model system for LNP‑mRNA therapy.

3. Lymphopenia (specifically splenic artery lymphopenia)

Type: Reduction in lymphocyte count
Cause in snippet: LNP exposure
Key Features:

• Decreased lymphocyte density in splenic periarteriolar regions
  Immunology relevance:

• Indicates immune cell depletion or redistribution

• Suggests LNP‑mediated immunomodulation independent of mRNA payload

4. Hepatocellular Injury (elevated ALT, AST, ALP)

Type: Drug‑induced liver injury (DILI)
Cause in snippet: High‑dose LNP‑mRNA or immune activation
Key Features:

• Elevated liver enzymes

• Histopathological changes (vacuoles, infiltrates)
  Immunology relevance:

• Liver is a major site of nanoparticle uptake by Kupffer cells

• Cytokine‑mediated inflammation can amplify hepatotoxicity

5. Lipid Droplet Accumulation in Hepatocytes

Type: Steatosis‑like phenotype
Cause in snippet: LNP exposure (luciferase mRNA control)
Key Features:

• Submicron lipid droplets visible on EM
  Immunology relevance:

• Suggests lipid‑handling stress in hepatocytes

• Lipid metabolism intersects with innate immune signaling (e.g., NLRP3 activation)

6. Pleocytosis (lymphocyte expansion in spleen germinal centers)

Type: Increased cellularity in lymphoid tissue
Cause in snippet: LNP‑mRNA vaccination
Key Features:

• Expansion of lymphocytes in germinal centers
  Immunology relevance:

• Indicates active adaptive immune response

• Expected in vaccine contexts, but can be exaggerated

7. Reactogenicity

Type: Acute inflammatory response to vaccination
Cause: Innate immune activation by LNPs
Key Features:

• Fever, malaise, injection‑site inflammation
  Immunology relevance:

• Driven by cytokines (IL‑6, TNF, chemokines)

• Often mediated by TLRs and inflammasomes

8. Hypersensitivity Reactions (including complement‑mediated)

Type: Immune overreaction to foreign substances
Cause: LNP components (e.g., PEGylated lipids)
Key Features:

• Complement activation

• Potential pseudoallergic reactions
  Immunology relevance:

• PEG‑specific IgM can trigger complement activation‑related pseudoallergy (CARPA)

9. Cytokine‑Mediated Inflammatory Responses

Type: Innate immune activation
Cause: TLR stimulation by LNPs or mRNA
Key Features:

• Elevated IL‑6, CCL2, CXCL2, TNF
  Immunology relevance:

• TLR4 activation is highlighted as a key upstream event

• Can impair mRNA translation via PKR activation

10. TLR4‑Mediated Inflammation

Type: Pattern‑recognition receptor activation
Cause: Ionizable cationic lipids (e.g., ALC‑0315, DLin‑MC3‑DMA, C12‑200)
Key Features:

• Cytokine release

• Reduced mRNA translation efficiency
  Immunology relevance:

• TLR4 is a central innate immune sensor

• Links LNP chemistry to immune activation

11. NLRP3 Inflammasome Activation

Type: Innate immune danger‑signal pathway
Cause: LNP‑mRNA exposure + lysosomal stress
Key Features:

• IL‑1β release

• Gasdermin D cleavage

• Caspase‑1 activation
  Immunology relevance:

• Leads to pyroptosis

• Can reduce transfection efficiency

• Triggered by both payload‑containing and empty LNPs

12. Complement Activation (C3a, C5b‑9 elevation)

Type: Innate immune cascade activation
Cause: LNP‑mRNA formulations, especially PEGylated ones
Key Features:

• Increased C3a, C5b‑9

• Potential for accelerated blood clearance
  Immunology relevance:

• Complement opsonization affects nanoparticle stability

• Can lead to hypersensitivity‑like reactions

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