ACE mRNA (Additional Chimeric Element incorporated IVT mRNA) for Enhancing Protein Expression by Modulating Immunogenicity

Abstract

The development of in vitro transcribed mRNA (IVT mRNA)-based therapeutics/vaccines depends on the management of IVT mRNA immunogenicity. IVT mRNA, which is used for intracellular protein translation, often triggers unwanted immune responses, interfering with protein expression and leading to reduced therapeutic efficacy. Currently, the predominant approach for mitigating immune responses involves the incorporation of costly chemically modified nucleotides like pseudouridine (Ψ) or N1-methylpseudouridine (m1Ψ) into IVT mRNA, raising concerns about expense and the potential misincorporation of amino acids into chemically modified codon sequences. Here, an Additional Chimeric Element incorporated mRNA (ACE mRNA), a novel approach incorporating two segments within a single IVT mRNA structure, is introduced. The first segment retains conventional IVT mRNA components prepared with unmodified nucleotides, while the second, comprised of RNA/DNA chimeric elements, aims to modulate immunogenicity. Notably, ACE mRNA demonstrates a noteworthy reduction in immunogenicity of unmodified IVT mRNA, concurrently demonstrating enhanced protein expression efficiency. The reduced immune responses are based on the ability of RNA/DNA chimeric elements to restrict retinoic acid-inducible gene I (RIG-I) and stimulator of interferon genes (STING)-mediated immune activation. The developed ACE mRNA shows great potential in modulating the immunogenicity of IVT mRNA without the need for chemically modified nucleotides, thereby advancing the safety and efficacy of mRNA-based therapeutics/vaccines.

Keywords: RNA structure; immunogenicity; mRNA therapeutics; protein expression.

Figure 1

Detection and quantification of dsRNA by‐products in IVT mRNAs with various chemical modifications (unmodified, Ψ‐, 5mC‐, or Ψ, 5mC‐modified) before and after cellulose purification, as assessed using the J2 antibody. A) The presence of dsRNA by‐products in unmodified and chemically modified (Ψ‐, 5mC‐, or a Ψ, 5mC‐) IVT mRNA was detected using J2 antibodies before and after cellulose purification. Dot bands in the figure represent the detected dsRNA by‐products, and the bar graph illustrates the quantification of these dsRNA dot bands, presented as adjusted band volumes. B) Type I IFN responses in THP1‐Dual cells were assessed before and after cellulose purification of unmodified and chemically modified IVT mRNA (Ψ‐, 5mC‐, or a Ψ, 5mC‐) by measuring Lucia luciferase expression, indicating ISG54 activity. C) Protein expression efficiency before and after cellulose purification of unmodified or chemically modified IVT mRNA (Ψ‐, 5mC‐, or a Ψ, 5mC‐), verified by measuring firefly luciferase levels 24 h post‐transfection in HEK 293T cell line. Statistical significance was determined using a Student's t‐test. (ns: not significant and **p < 0.01).

Figure 2

Additional Chimeric Element incorporated IVT mRNA (ACE mRNA). A) Schematic illustration of design and preparation of Additional Chimeric Element incorporated mRNA (ACE mRNA), incorporating RNA/DNA chimeric elements downstream of the poly(A) tail at the 3′ ends of IVT mRNA. PCR products containing the T7 promoter, 5′ UTR, ORF, 3′ UTR, poly(dA:dT), and additional sequences (sequence B or C) served as the DNA template for IVT mRNA synthesis. DNA oligos with sequences complementary to the introduced additional sequences (sequence B or C) in IVT mRNA were slowly annealed into the IVT mRNA, resulting in the creation of RNA/DNA chimeric element incorporated mRNA (ACE mRNA). B) Confirmation of the successful introduction of RNA/DNA chimeric elements into ACE mRNA. In the polyacrylamide gel electrophoresis assay, the bands corresponding to DNA oligos (DNA25: complementary to sequence B + 6 dT, DNA60: complementary to sequence C + 11 dT) disappeared after the slow annealing process with IVT mRNA containing sequence B or C. The mRNA integrity of ACE mRNA was further verified by 1% agarose electrophoresis assay. C) Verification of successful introduction of RNA/DNA chimeric elements into ACE mRNA. The presence of RNA/DNA chimeric elements within the IVT mRNA was detected using the S9.6 antibody, which can differentiate RNA/DNA structures. D) Evaluation of type I IFN responses of ACE mRNA compared to various mRNA constructs, assessed in THP1‐Dual cells. The levels of type I IFN induction by two ACE mRNA variants (mRNA/DNA25B, mRNA/DNA60C) were compared with Ψ‐modified IVT mRNA, unmodified IVT mRNA, short RNA/DNA with the same sequence and length as the RNA/DNA chimeric elements introduced into either mRNA/DNA25B or mRNA/DNA60C (RNA/DNA25B, RNA/DNA60C), and a simple mixture of unmodified IVT mRNA and short RNA/DNA chimeric elements (mRNA+RNA/DNA25B, mRNA+RNA/DNA60C). E–G) Evaluation of protein expression efficiency for ACE mRNA variants (mRNA/DNA25B, mRNA/DNA60C) encoding RFP compared to Ψ‐modified IVT mRNA and unmodified IVT mRNA. All mRNA samples were delivered to E) HeLa cells, F) HEK 293T, or G) Raw264.7 cells using LNP as the delivery method, and RFP expression was quantified 24 h post‐transfection. Statistical significance was determined using Student's t‐test (ns: not significant, *p < 0.05, **p < 0.01, and ***p < 0.001)

Figure 2

Additional Chimeric Element incorporated IVT mRNA (ACE mRNA). A) Schematic illustration of design and preparation of Additional Chimeric Element incorporated mRNA (ACE mRNA), incorporating RNA/DNA chimeric elements downstream of the poly(A) tail at the 3′ ends of IVT mRNA. PCR products containing the T7 promoter, 5′ UTR, ORF, 3′ UTR, poly(dA:dT), and additional sequences (sequence B or C) served as the DNA template for IVT mRNA synthesis. DNA oligos with sequences complementary to the introduced additional sequences (sequence B or C) in IVT mRNA were slowly annealed into the IVT mRNA, resulting in the creation of RNA/DNA chimeric element incorporated mRNA (ACE mRNA). B) Confirmation of the successful introduction of RNA/DNA chimeric elements into ACE mRNA. In the polyacrylamide gel electrophoresis assay, the bands corresponding to DNA oligos (DNA25: complementary to sequence B + 6 dT, DNA60: complementary to sequence C + 11 dT) disappeared after the slow annealing process with IVT mRNA containing sequence B or C. The mRNA integrity of ACE mRNA was further verified by 1% agarose electrophoresis assay. C) Verification of successful introduction of RNA/DNA chimeric elements into ACE mRNA. The presence of RNA/DNA chimeric elements within the IVT mRNA was detected using the S9.6 antibody, which can differentiate RNA/DNA structures. D) Evaluation of type I IFN responses of ACE mRNA compared to various mRNA constructs, assessed in THP1‐Dual cells. The levels of type I IFN induction by two ACE mRNA variants (mRNA/DNA25B, mRNA/DNA60C) were compared with Ψ‐modified IVT mRNA, unmodified IVT mRNA, short RNA/DNA with the same sequence and length as the RNA/DNA chimeric elements introduced into either mRNA/DNA25B or mRNA/DNA60C (RNA/DNA25B, RNA/DNA60C), and a simple mixture of unmodified IVT mRNA and short RNA/DNA chimeric elements (mRNA+RNA/DNA25B, mRNA+RNA/DNA60C). E–G) Evaluation of protein expression efficiency for ACE mRNA variants (mRNA/DNA25B, mRNA/DNA60C) encoding RFP compared to Ψ‐modified IVT mRNA and unmodified IVT mRNA. All mRNA samples were delivered to E) HeLa cells, F) HEK 293T, or G) Raw264.7 cells using LNP as the delivery method, and RFP expression was quantified 24 h post‐transfection. Statistical significance was determined using Student's t‐test (ns: not significant, *p < 0.05, **p < 0.01, and ***p < 0.001)

Figure 3

Type I IFN responses of ACE mRNA in wild‐type THP1‐Dual™ and various knock‐out (KO) cell lines of THP1‐Dual cells. To identify the specific PRRs responsible for decreasing the immune response of ACE mRNA, various knock‐out (KO) cell lines of THP1‐Dual cells were used. A) Wild‐type THP1‐Dual cells, B) RIG‐I KO THP1‐Dual cells, C) MAVS KO THP1‐Dual cells, D) MDA5 KO THP1‐Dual cells, E) cGAS KO THP1‐Dual cells, or F) STING KO THP1‐Dual cells were treated with various mRNA constructs, including ACE mRNA (unmodified IVT mRNA, ACE mRNA (mRNA/DNA25B), or Ψ‐modified IVT mRNAs) using Lipofectamine 2000. The type I IFN responses were assessed by quantifying Lucia luciferase expression in either wild‐type or various KO cell lines of THP1‐Dual cells. G) The extent of reduction in ISG54 activity on ACE mRNA in cGAS KO or STING KO THP1‐Dual cells compared to wild‐type THP1‐Dual cells H) Proposed mechanism of the ACE mRNA immunosensing pathway distinct from that of unmodified IVT mRNA. The overall capacity of ACE mRNA to reduce type I IFN responses is attributed to its potential to 1) inhibit the ATPase activity required for RIG‐I activation, thereby limiting RIG‐I‐mediated type I IFN responses, 2) engage in cGAS‐mediated immune activation, leading to a slight increase in type I IFN responses, and 3) restrict interactions between STING, RIG‐I, and MAVS, resulting in the inhibition of type I IFN responses. Statistical significance was ascertained using Student's t‐test (ns: not significant, *p < 0.05 and **p < 0.01).

Figure 4

Therapeutic protein expression (hEPO) of ACE mRNA in vivo. The ACE mRNA variants (mRNA/DNA25B, mRNA/DNA65C) and IVT mRNAs (unmodified IVT mRNA and Ψ‐modified IVT mRNA encoding hEPO) were formulated into LNP to verify the efficiency of therapeutic protein expression in vivo. A) The encapsulation efficiency (%) of all mRNA constructs was measured by a ribogreen assay. B) The hydrodynamic sizes and PDI of the formulated LNPs were measured using dynamic light scattering (DLS). C) Schematic representation illustrating the in vivo administration of LNP encapsulating various mRNA samples encoding hEPO. A total of 2 micrograms of each mRNA variant were administered retro‐orbitally (n = 3). Blood collection was performed at 3, 6, 9, and 18 h post‐injection using a Lancet, and the collected blood was immediately mixed with 3.8% sodium citrate buffer to prevent coagulation. Subsequently, the collected blood was subjected to centrifugation for plasma separation. D) The levels of produced hEPO in the collected plasma samples were examined using an enzyme‐linked immunosorbent assay (ELISA). In addition, the total protein expression was verified by calculating the area under the curve (AUC) using detected hEPO concentration in serum samples collected at 4 time points (3, 6, 9, and 18 h post‐injection). Statistical significance was determined through a Student's t‐test (ns: not significant, *p < 0.05, **p < 0.01, and ***p < 0.001)

Figure 5

Type I IFN response and protein expression of Ψ‐modified IVT mRNA incorporating RNA/DNA chimeric element (Ψ‐modified ACE mRNA). A) THP1‐Dual cells were treated with Ψ‐modified IVT mRNA and Ψ‐modified ACE mRNA (mRNA/DNA25B, mRNA/DNA65C) to measure ISG54 activity. B) HEK 293T cells and Raw 264.7 cells were transfected with Ψ‐modified IVT mRNA or Ψ‐modified ACE mRNA variants (mRNA/DNA25B, mRNA/DNA65C) encoding firefly luciferase, and the efficiency of protein expression was determined by quantifying luciferase expression levels.