LNP-Based Lung Gene Delivery Shows Rapid Protein Expression and a Risk of Lung Injury
Background: A nonviral carrier for immune modulation in donor lungs
Lipid nanoparticles (LNPs) developed to modulate immunity in donor lungs showed high gene-transfer rates and rapid protein expression in cells and lung tissue. However, significant lung injury was observed after intratracheal administration in rats. The findings indicate that safety must be evaluated alongside formulation optimization to improve delivery efficiency.
This study evaluated LNPs as a nonviral gene-delivery platform. After screening candidates in cell lines, the researchers compared protein expression with an adenoviral vector in human lung tissue slices and examined the relationship between dose, expression, and lung injury in rats. It was not a clinical trial assessing transplant rejection or patient survival.
Key findings: ST-1/DOTAP composition and expression speed
The researchers loaded messenger RNA (mRNA) encoding the reporter protein mCherry into 10 LNP candidates made by combining five ionizable lipids with two helper lipids. Gene-transfer rates in cell lines were measured by flow cytometry. A formulation combining the ionizable lipid ST-1 with the helper lipid DOTAP achieved transfer rates of 91.3 ± 6.9% in human lung epithelial cells and 88.4 ± 6.3% in human monocyte-lineage cells.
Reducing the DOTAP content from 39% to 7.8% further increased the transfer rate. However, the abstract does not specify the magnitude of the improvement or the basis for calculating the content, making it difficult to generalize this proportion as the optimal composition for all lung-delivery conditions.
In human precision-cut lung slices (PCLS), mCherry expression from the ST-1/DOTAP 7.8% formulation peaked two days after administration. This peak occurred earlier than with the adenoviral vector, but the abstract does not give the comparator vector’s exact time to peak or duration of expression. These results alone do not establish an immune-modulating effect after transplantation.
Implications and outlook: Reduced inflammation and established safety must be distinguished
Intratracheal administration of the ST-1/DOTAP 7.8% formulation carrying mCherry mRNA at 500 μg/kg into the left lung of rats caused significant lung injury. By contrast, administration of human interleukin-10 (hIL-10) mRNA at 50 μg/kg reduced LNP-induced lung inflammation, and the researchers reported their target hIL-10 concentration of 59 pg per 1 mg of protein in the left lung.
The two conditions differed in both dose and mRNA type. Therefore, the reduction in inflammation should not be attributed to dose reduction alone, nor should it be concluded that hIL-10 completely eliminated toxicity. There is also no basis for claiming that a nonviral carrier avoids immune responses simply because it is nonviral.
The study shows that rapid expression and the risk of tissue injury both need to be evaluated in lung mRNA delivery. Before clinical application, expression and inflammation must be further assessed across formulations, doses, and mRNA types. The provided abstract reports no results demonstrating successful genome editing or long-term safety.
Source: Research abstract and bibliographic information indexed in Europe PMC.
OBJECTIVE: To evaluate the efficiency and protein expression profiles of newly synthesized lipid nanoparticles (LNPs) as a nonviral vector gene delivery platform for gene editing of donor lungs for immunomodulation. METHODS: mCherry mRNA was formulated with 10 LNP candidates (5 ionizable lipids × 2 helper lipids), and gene delivery efficiency in cell lines was assessed using flow cytometry. The most promising LNP was optimized, and its protein expression was compared with that of adenoviral vectors (ADVs) using an mCherry enzyme-linked immunosorbent assay ELISA in a human precision-cut lung slice (PCLS) model. Additionally, intratracheal administration in a rat in vivo model was used to evaluate the relationship between LNP dose and protein expression using mCherry and human interleukin-10 (hIL-10) mRNA. RESULTS: An ionizable lipid ST-1 (formulated with 1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) achieved high transfection rates of 91.3 ± 6.9% in human lung epithelial cells and 88.4 ± 6.3% in human monocytic cells. Reducing the DOTAP content from 39% to 7.8% further improved the transfection rate. Peak mCherry expression in human PCLS with ST-1 DOTAP (7.8%) occurred at 2 days postadministration, which was faster than with adenoviral vectors. Intratracheal administration of 500 μg/kg of ST-1 DOTAP (7.8%) with mCherry to rat left lungs caused significant lung injury; however, 50 μg/kg of hIL-10 mRNA mitigated the LNP-induced lung inflammation, achieving the target concentration of 59 pg/mg protein in the left lung. CONCLUSIONS: LNPs achieved rapid and efficient protein expression in vitro and in vivo that exceeded the performance of viral vectors; however, significant lung injury was observed with intratracheal LNP administration. This findings underscores the need to further optimize LNP-mediated gene delivery to the lung before translation to the clinic.
When developing technology to deliver immune-modulating mRNA to donor lungs, both the speed of protein production and the extent of lung tissue injury must be assessed. The ST-1/DOTAP formulation showed rapid expression in human lung tissue slices, but intratracheal administration in rats also revealed lung injury. In follow-up preclinical studies, varying the dose of the same mRNA or comparing mRNA types at the same dose could help disentangle the relationship between expression and inflammation. Reduced inflammation under the hIL-10 condition provides a basis for further optimization, but it is not evidence that the approach is safe for lung transplant recipients or reduces rejection.