RNA ImmunoGenic分析：一种检测人全血体外转录mRNA免疫原性的方法

AKM Ashiqul Haque; Petra Weinmann; Sumit Biswas; Rupert Handgretinger; Markus Mezger; Michael S. D. Kormann; Justin S Antony

doi:10.21769/BioProtoc.3850

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Peer-reviewed

RNA ImmunoGenic Assay: A Method to Detect Immunogenicity of in vitro Transcribed mRNA in Human Whole Blood

RNA ImmunoGenic分析：一种检测人全血体外转录mRNA免疫原性的方法

AH AKM Ashiqul Haque ^*

PW Petra Weinmann ^*

SB Sumit Biswas

RH Rupert Handgretinger

MM Markus Mezger

MK Michael S. D. Kormann ^†

Justin S Antony ^† email

(*contributed equally to this work, † Shared senior authors) 发布: 2020年12月20日第10卷第24期 DOI: 10.21769/BioProtoc.3850 浏览次数: 5462

评审: Alessandro DidonnaAbhinit NagarAnonymous reviewer(s)

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Abstract

The mRNA therapeutics is a new class of medicine to treat many various diseases. However, in vitro transcribed (IVT) mRNA triggers immune responses due to recognition by human endosomal and cytoplasmic RNA sensors, but incorporation of modified nucleosides have been shown to reduce such responses. Therefore, an assay signifying important aspects of the human immune system is still required. Here, we present a simple ex vivo method called ‘RNA ImmunoGenic Assay’ to measure immunogenicity of IVT-mRNAs in human whole blood. Chemically modified and unmodified mRNA are complexed with a transfection reagent (TransIT), and co-incubated in human whole blood. Specific cytokines are measured (TNF-α, INF-α, INF-γ, IL-6 and IL-12p70) using ELISAs. The qPCR analysis is performed to reveal the activation of specific immune pathways. The RNA ImmunoGenic Assay provides a simple and fast method to detect donor specific - immune response against mRNA therapeutics.

Graphic abstract:

Schematic representation of RNA ImmunoGenic Assay

Keywords: cmRNA (cmRNA)

IVT-mRNA (IVT-mRNA)

Immunogenicity (免疫原性)

Cytokines (细胞因子)

Whole blood assay (全血检测)

Background

The mRNA therapeutics is an important class of gene therapy (Sahin et al., 2014; Antony et al., 2015). Although no mRNA drugs are marketed, the efficacy demonstrated in ongoing clinical trials is remarkable. The in vitro transcribed mRNA induces immune responses due to its recognition by human endosomal and cytoplasmic RNA sensors including Toll-like receptors (TLRs), RIG-I like receptors (RLRs), and NOD-like receptors (NLRs). Therefore, the assessment of immunogenicity of mRNA therapeutics is pivotal for their possible use in humans.

Existing cell-based methods have been reported to study immunogenicity of IVT-mRNA including peripheral blood mononuclear cells (PBMCs) based assays, dendritic cells-based assays and THP-1 cell-based assays (Kariko et al., 2011; Vaidyanathan et al., 2018). However, these assays are associated with laborious ficoll isolation together with isolation related immune cell activation, and artificial mono-immune cell systems that do not imitate the complexity of human immune cells. Therefore, a fast method that mimics the human immune system is desirable. Here, we report an adapted version of whole blood assay (WBA) as an ‘RNA ImmunoGenic Assay’ which is simple and fast method and eliminates the hurdles associated with PBMCs isolation and DCs enrichment (Haque et al., 2020). Interestingly, the assay utilizes human whole blood and it provides the most physiologically relevant immune cell types, especially platelets, and white and red blood cells (RBCs) in an easily accessible ex vivo context.

The proposed assay is capable of sensing different immune responses caused by IVT-mRNA administration such as NF-kB activation and interferon stimulation. Notably, results of ‘RNA ImmunoGenic assay’ using whole blood can be compared to PBMC based assay. Since the assay requires small volumes of whole blood (< 10 ml), it is suitable for pediatric patients and other patients with severe illnesses. Moreover, the RNA ImmunoGenic assay is capable to detect personalized immune responses to specific mRNA compounds with or without formulation. Together, the presented RNA ImmunoGenic assay is easy to complete, fast to perform, and provides measurable immunogenicity results to be used in a clinical setting.

Materials and Reagents

IVT mRNA production
1. Petri dish (Greiner Bio-One, catalog number: 633161 )
2. Rattler plating beads (5 mm) (Zymo Research, catalog number: S1001 , store at RT)
3. One Shot^TM TOP10 chemical competent E. coli (Thermo Fisher, catalog number: C404003 , store at -80 °C)
4. pVAX1.A120 vector based on pVAX1 (Life Technologies, catalog number: V26020 , store at -20 °C)
5. XhoI (NEB, catalog number: R0146L , store at -20 °C)
6. KpnI (NEB, catalog number: R0142L , store at -20 °C)
7. NheI (NEB, catalog number: R0131L , store at -20 °C)
8. NEBuffer^TM
9. 1x CutSmart^® Buffer (provided with the appropriate enzyme, store at -20 °C)
10. 1x NEBuffer^TM 1.1 (provided with the appropriate enzyme, store at -20 °C)
11. 1x NEBuffer^TM 2.1 (provided with the appropriate enzyme)
12. T4 DNA ligase (NEB, catalog number: M0202L, store at -20 °C )
13. 1x T4 DNA Ligase Reaction Buffer (provided with the appropriate enzyme, store at -20 °C)
14. S.O.C. medium (Thermo Fisher, catalog number: 15544034 ), store at RT
15. MEGAscript^TM T7 Transcription kit (Invitrogen, catalog number: AM1334 ), store at -20 °C
16. m27,3'-OGP3G (ARCA Cap Analog)–Solution (Jena Bioscience, catalog number: NU-855L ), store at -20 °C
17. Modified Base 1, e.g., N1-Methylpseudo-UTP (Jena Bioscience, catalog number: NU-890L ), store at -20 °C
18. Modified Base 1, e.g., 2-Thio-UTP (Jena Bioscience, catalog number: NU-1151L ), store at -20 °C
19. MEGAclear^TM Kit (Invitrogen, catalog number: AM1908 ), store at 4 °C
20. TURBO^TM DNase (Thermo Fisher, catalog number: AM2239 ), store at -20 °C
21. Agilent RNA 6000 Nano Kit (Agilent, catalog number: 5067-1511 ), store reagents at 4 °C, chips at RT
22. Luria Broth Base (Miller's LB Broth Base) (Thermo Fisher, catalog number: 12795027 ), store at RT
23. Select Agar (Thermo Fisher, catalog number: 30391023 ), store at RT
24. Kanamycin Sulfate (Thermo Fisher, catalog number: 11815024 ), store at RT
25. GeneJET PCR Purification kit (Thermo Fisher, catalog number: K0702 ), store at RT
Blood Collection and ex vivo stimulation
1. 12-well plate (Corning, catalog number: 3512 )
2. EDTA containing vacutainer tubes (Sarstedt, catalog number: 02.1066.001 )
3. Precellys bacterial/fungal RNA kit (PeqLab, catalog number: 12-7611-01 ), store at RT
4. Precellys Tissue homogenizing CKMix–2 ml (Bertin Instruments, catalog number: P000918-LYSK0-A ), store at RT
5. Resiquimod (R848) (Sigma-Aldrich, catalog number: SML0196 ), store at -20 °C
6. TransIT^®-mRNA Transfection Kit (Mirus, catalog number: MIR 2225 ), store at 4 °C
Measurement of cytokines by ELISA
1. 96-well plate (provided with the ELISA kit)
2. Absorbent paper
3. Acetat Foil for Microtesting (Sarstedt, catalog number: 82.1586 )
4. Capture Antibody (provided with the ELISA kit)
5. Biotinylated Detection Ab (provided with the ELISA kit)
6. Washing buffer
7. DPBS, no calcium, no magnesium (Thermo Fisher, catalog number: 14190094 ), store at RT
8. TWEEN^® 20 (Sigma-Aldrich, catalog number: P9416-50ML ), store at RT
9. HRP Conjugate working solution (provided with the ELISA kit)
10. Substrate Reagent (provided with the ELISA kit)
11. TNF-alpha Human Uncoated ELISA Kit with Plates (Thermo Fisher, catalog number: 88-7346-22 ), store at 4 °C
12. Invitrogen^TM eBioscience^TM Platin-ELISA-Kit for human IFN-alpha (Fisher Scientific, catalog number: 15561697 ), store at 4 °C
13. IFN gamma Human Uncoated ELISA Kit with Plates (Thermo Fisher, catalog number: 88-7316-22 ), store at 4 °C
14. IL-6 Human Uncoated ELISA Kit with Plates (Thermo Fisher, catalog number: 88-7066-22 ), store at 4 °C
15. IL-12 p70 Human Uncoated ELISA Kit with Plates (Thermo Fisher, catalog number: 88-7126-22 ), store at 4 °C
16. 2 N HCl (Stop solution) (Carl Roth, catalog number: T134.1 ), store at RT
Real-time RT-PCR
1. PAXgene 96 Blood RNA Kit (Qiagen, catalog number: 762331), store at RT
2. iScript^TM cDNA Synthesis Kit (Bio-Rad, catalog number: 1708891), store at -20 °C
3. Power SYBR Green PCR Master mix (Thermo Fisher, catalog number: 4367659), store at -20 °C
4. Erythrocyte Lysis Buffer (PAN Biotech, catalog number: P10-90100 ), store at RT
5. Primers:
  1. hTNFAlpha forward: 5′- GGA GAA GGG TGA CCG ACT CA-3′
  2. hTNFAlpha reverse: 5′-CTG CCC AGA CTC GGC AA-3′
  1. hIFNAlpha forward: 5′-GAC TCC ATC TTG GCT GTG A-3′
  2. hIFNAlpha reverse: 5′-TGA TTT CTG CTC TGA CAA CCT-3′
  3. hIFNgamma forward: 5′-CCA ACG CAA AGC AAT ACA TGA-3′
  4. hIFNgamma reverse: 5′-CCT TTT TCG CTT CCC TGT TTT A-3′
  1. hIL-12 forward: 5′- CAA GAA CTT GCA GCT GAA G-3′
  2. hIL-12 reverse: 5′- TGG GTC TAT TCC GTT GTG TC-3′
  3. IL‐6 forward: 5′-AGACAGCCACTCACCTCTTCAG-3′
  4. IL-6 reverse: 5′-TTCTGCCAGTGCCTCTTTGCTG-3′
  5. IRF7 forward: 5′-CCACGCTATACCATCTACCTGG-3′
  6. IRF7 reverse: 5′-GCTGCTATCCAGGGAAGACACA-3′
  7. IRF3 forward: 5′-TCTGCCCTCAACCGCAAAGAAG-3′
  8. IRF3 reverse: 5′-TACTGCCTCCACCATTGGTGTC-3′
  9. eIF 2A forward: 5′-CTGGACCTCATGCAGCTTTAGCm-3′
  10. eIF 2A reverse: 5′-CTCCATAGTAGGAAGCTCCTGTC-3′
  11. RNase L forward: 5′-AAGGCTGTTCAAGAACTACACTTG-3′
  12. RNase L reverse: 5′-TGGATCTCCAGCCCACTTGATG-3′
Flow cytometry analysis
1. DBPS, no calcium, no magnesium (Thermo Fisher, catalog number: 14190094 ), store at RT
2. 10% FBS; FBS Superior (Sigma-Aldrich, catalog number: S0615 ), store at -20 °C
3. Erythrocyte Lysis Buffer (PAN Biotech, Catalog number: P10-90100 ), store at RT

Equipment

ThermoShaker Univortemp MT 100 (Universal Labortechnik)
Centrifuge 5430R (Eppendorf, catalog number: 5427000015 )
2100 Bioanalyzer Instrument (Agilent, catalog number: G2939BA )
2100 Bioanalyzer Laptop (Agilent Original Bundle PC, catalog number: G2953CA )
Cell culture hood HeraSafe KS (Thermo Fisher, catalog number: 10110910 )
CO₂ incubator HeraCell VIOS 160i (Thermo Fisher, catalog number: 15381075 )
Precellys evolution homogenizer (Bertin Technologies, catalog number: P000062-PEVO0-A )
Ensight^TM multimode plate reader (Perkin Elmer, catalog number: HH34000000 )
Flow Cytometer, BD LSRFortessa^TM X20 (BD Biosciences)
ViiA 7 Real-Time PCR System (Thermo Fisher, catalog number: 4453545)

Software

2100 Expert for Bioanalyzer (Agilent)
Graphpad Prism v.6.0d software (www.graphpad.com)
Kaleido^TM2.0 Data Acquisition and Analysis software for Ensight^TM plate reader (PerkinElmer)
BD FACSDiva^TM Software for Flow Cytometry Analysis (BD Biosciences)
ViiA7 software v.1.2 (applied Biosystems by life technologies)
NEBioCalculator v.1.12.0 (New England Biolabs)

Procedure

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Haque, A. A., Weinmann, P., Biswas, S., Handgretinger, R., Mezger, M., Kormann, M. S. D. and Antony, J. S. (2020). RNA ImmunoGenic Assay: A Method to Detect Immunogenicity of in vitro Transcribed mRNA in Human Whole Blood. Bio-protocol 10(24): e3850. DOI: 10.21769/BioProtoc.3850.

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