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. 2020 Apr 27;10(1):7052.
doi: 10.1038/s41598-020-64017-0.

Systemic Modified Messenger RNA for Replacement Therapy in Alpha 1-antitrypsin Deficiency

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Free PMC article

Systemic Modified Messenger RNA for Replacement Therapy in Alpha 1-antitrypsin Deficiency

Ahmad Karadagi et al. Sci Rep. .
Free PMC article

Abstract

Alpha 1-antitrypsin (AAT) deficiency arises from an inherited mutation in the SERPINA1 gene. The disease causes damage in the liver where the majority of the AAT protein is produced. Lack of functioning circulating AAT protein also causes uninhibited elastolytic activity in the lungs leading to AAT deficiency-related emphysema. The only therapy apart from liver transplantation is augmentation with human AAT protein pooled from sera, which is only reserved for patients with advanced lung disease caused by severe AAT deficiency. We tested modified mRNA encoding human AAT in primary human hepatocytes in culture, including hepatocytes from AAT deficient patients. Both expression and functional activity were investigated. Secreted AAT protein increased from 1,14 to 3,43 µg/ml in media from primary human hepatocytes following mRNA treatment as investigated by ELISA and western blot. The translated protein showed activity and protease inhibitory function as measured by elastase activity assay. Also, mRNA formulation in lipid nanoparticles was assessed for systemic delivery in both wild type mice and the NSG-PiZ transgenic mouse model of AAT deficiency. Systemic intravenous delivery of modified mRNA led to hepatic uptake and translation into a functioning protein in mice. These data support the use of systemic mRNA therapy as a potential treatment for AAT deficiency.

Conflict of interest statement

A.G.C, M.E.E., X.Z, E.G., R.A.W., L.M.R., A.L.F., and P.G.V.M. are employees of, and receive salary and stock options from, Moderna Inc. A.K., H.Z., S.S., C.J., G.N., and E.E. declare no competing interests.

Figures

Figure 1
Figure 1
eGFP transfection time course. Primary human hepatocytes were transfected with eGFP encoding mRNA by lipofection in culture. Protein expression peaked after 1 day and continued robust expression was seen 4 days as assessed by immunofluorescence imaging. Corrected total fluorescence was measured and plotted.
Figure 2
Figure 2
AAT encoding mRNA translation into protein by hepatocytes and secretion into the extracellular space. (a) An increase in AAT production was observed in hepatocytes exposed to modified mRNA. Gel separation and immunoblotting revealed increased AAT in the treated group (n = 16). (b) ELISA of secreted AAT protein in culture media showed an increase in total amount of secreted AAT protein (n = 14).
Figure 3
Figure 3
Elastase activity assay shows retained AAT protease inhibition and protein function. (a) Elastase activity was measured in n = 14 cases. A majority of cases display a decreased elastase activity when incubated with conditioned media from cell cultures. (b) Median elastase activity of all cases showed a robust inhibition of elastase activity in the presence of conditioned media displaying similar kinetics as control elastase inhibitor. (c) End-point (60 minutes) analysis showed significant (Wilcoxon matched-pairs signed rank test) decrease in elastase activity in conditioned media from cells treated with AAT modified mRNA.
Figure 4
Figure 4
Expression of human AAT protein following intravenous injection of modified mRNA in wild type mouse. Modified mRNA in LNP formulation was administered as a one-time intravenous (tail-vein) injection at 0.5 mg/kg into C57BL/6 mice (n = 3) and liver tissue was collected 1 hour; 24 hours and 48 hours post injection. (a) Liver tissue was stained for human AAT protein, which revealed a robust intracellular expression in hepatocytes followed by accumulation in the sinusoidal space, scalebar 100 µm. Tissues were collected and stained at 1, 2, and 24 hours post injection and was compared to untreated control animal were no human AAT protein was expressed. (b) DAB staining changes were compared between time points (Kruskal-Wallis test, median with interquartile range). (c) RT-qPCR was used to confirm the delivery of AAT encoding mRNA into liver tissue, where AAT signal passed threshold at 27 cycles and no signal was detected in untreated samples.
Figure 5
Figure 5
In vivo delivery and analysis of modified mRNA encoding human AAT into the NSG-PiZ mouse model of AATD. (a) Whole liver was sectioned and in situ hybridization was used to show global distribution of mRNA in the liver following intravenous delivery by tail-vein injection. (b) Biochemical analysis did not reveal any sign of cellular stress or damage caused by mRNA delivery. (c) Most of the mRNA can be detected by in situ hybridization in the liver sinusoids 2 hours after injection. The mRNA subsequently relocated into hepatocytes by 24 and 48 hours after injection. Hepatocytes with and without globules contained mRNA. (d) A large amount of mRNA could be detected by qPCR in analysed liver tissue. (e) Elastase activity assay showing inhibition in serum from animals receiving AAT mRNA and PBS treated animals.

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