Patent Grant
11970700
The Sequence Listing submitted Dec. 13, 2022 as a text file named “mRNA_E3_ST26.xml,” created on Dec. 12, 2022, and having a size of 18,087 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).
The present invention relates to the enhancing genetic element (“E3” or genetic element “E3” hereinafter) for the intracellular expression of target protein encoded in RNA therapeutics such as mRNA vaccines and RNA medicines. More specifically, the present invention relates to the RNA molecule having the genetic element “E3” capable of enhancing the intracellular expression of target protein encoded in RNA molecule; the plasmid to be used to synthesize the RNA molecule having the genetic element “E3”; the method for synthesizing RNA molecule having the genetic element “E3” using the said plasmid and utilizing the synthesized RNA molecule as RNA therapeutics.
RNA therapeutics constitute a rapidly expanding category of drugs. RNA therapeutics comprise two types: one type is RNA that RNA itself acts on targets such as siRNA (short interfering RNA) or miRNA (microRNA), and the other type is RNA that the protein encoded in it acts on targets such as mRNA vaccine.
In the latter type of RNA therapeutics, since the intracellular expression levels of the encoded target proteins are directly linked to the efficacy thereof, high expression levels of the encoded target proteins are desired in many cases. If the expression efficiency of the encoded target protein is high, a more excellent effect can be expected when the same amount of RNA drug is administered. In addition, since a relatively low dosage is required for the same effect, the dosage can be lowered, which can reduce drug manufacturing costs and side effects.
Particularly, as for mRNA vaccines that have been drawing increasing attention recently, the high efficiency in the intracellular expression of target proteins (i.e., antigens) is also desired. mRNA vaccines have some advantages compared to conventional vaccines such as DNA vaccines, subunit vaccines, inactivated vaccines and attenuated vaccines. First, mRNA vaccines provide relatively high safety. Because mRNA vaccines are a non-infectious, non-integrating platform distinct from DNA vaccines, there is no risk of integration into the genome of the host cell, so there is no potential risk of mutagenesis. For these reasons, the U.S. FDA does not include mRNA vaccines in the category of gene therapies unlike DNA vaccines. Also, the mRNA molecules of mRNA vaccines are decomposed by normal cellular processes. The antigen protein encoded in mRNA is only transiently produced, and foreigner mRNA molecules are normally decomposed in cytoplasm and removed shortly. Second, mRNA vaccines are beneficial in terms of efficacy. mRNA is versatile enough for easy modifications, which reinforces its stability while simultaneously improving the efficiency of translation. Also, the encapsulation of mRNA in delivery carriers enables an efficient in vivo delivery, whereby the rapid uptake into cytoplasm of target cells and the expression of target protein encoded in it is accelerated. mRNA is the minimal genetic vector; therefore, anti-vector immunity is avoided, and mRNA vaccines can be administered repeatedly. Third, mRNA vaccines are beneficial in terms of production. The mRNA molecules of mRNA vaccines are synthesized by means of in vitro transcription reaction, wherein the synthesis efficiency is high. Therefore, manufacture of mRNA vaccines is rapid, cost effective and highly scalable.
The said advantages explain the rapid advancement of mRNA vaccines. However, mRNA vaccines still need improvement in many aspects. Particularly, it is necessary to develop the technology for enhancing the efficiency of intracellular expression of target protein encoded in mRNA.
Hence, as a way of enhancing the intracellular expression of target proteins encoded in RNA therapeutics, the present inventors intend to provide the genetic element “E3”, which is placed adjacent to the 5′-end of the target protein-coding sequence and capable of enhancing the intracellular expression of target proteins, and also a method for synthesizing the RNA molecules having the genetic element “E3” and using the synthesized RNA molecules as RNA therapeutics.
Thus, the first object of the present invention is to provide the DNA sequence set forth in SEQ ID NO: 1 of the genetic element “E3”, which is placed adjacent to the 5′-end of the target protein-coding sequence and capable of enhancing the intracellular expression of target proteins encoded in RNA molecules. The DNA sequence set forth in SEQ ID NO: 1 for the said genetic element “E3” is presented based on the sequence of plasmid to be used for the synthesis of RNA molecules having the genetic element “E3”, and
The other object of the present invention is to provide a method for synthesizing RNA molecules having the genetic element “E3” using the plasmid having nucleotide sequence set forth in SEQ ID NO: 1 and utilizing the synthesized RNA molecules as RNA therapeutics including mRNA vaccines.
To achieve the said objects, the inventors of the present invention have discovered the genetic element “E3” that is placed adjacent to the 5′-end of the target protein sequence encoded in RNA molecules and capable of enhancing the intracellular expression of the encoded target protein, developed the plasmid having the said genetic element “E3”, and synthesized the RNA molecules using the said plasmid, introduced the synthesized RNA molecules into cells, and demonstrated the effect of genetic element “E3” in the enhanced intracellular expression of the target protein in comparison to that when the genetic element “E3” of the present invention is not applied, thereby completing the present invention.
Thus, according to a first aspect of the present invention, the present invention provides the RNA sequence of the genetic element “E3” which is placed adjacent to the 5′-end of the target protein sequence encoded in RNA molecules and capable of enhancing the intracellular expression of the target protein. To be specific, the present invention provides the nucleotide sequence set forth in SEQ ID NO: 1, which is presented as the sequence on the plasmid (see
It is obvious that the nucleotide sequence set forth in SEQ ID NO: 1 can be partially modified by a person skilled in the art using existing technology. The said modification includes the partial substitution of the nucleotide sequence, partial addition to the nucleotide sequence, and partial deletion of the nucleotide sequence. Nevertheless, it is most desirable to use the nucleotide sequence set forth in SEQ ID NO: 1 disclosed in the present invention.
According to a second aspect of the present invention, the present invention provides the RNA molecules, which functionally enhance the intracellular expression of the target protein encoded in RNA molecules, comprising the following genetic factors and arrangement:
Wherein, the said Poly U sequence is 20-200 nucleotides in length.
According to a third aspect of the present invention, the present invention provides a method for placing the genetic element “E3” DNA sequence set forth in SEQ ID NO: 1 into the plasmid to be used for RNA synthesis, synthesizing the RNA molecules having the enhancing genetic element “E3” RNA sequence set forth in SEQ ID NO: 2, using the said plasmid, and utilizing the synthesized RNA molecules having the genetic element “E3” as RNA therapeutics. The present invention provides an example of an application as mRNA vaccines. However, the present invention may well be applied to other RNA therapeutics without limit.
According to a fourth aspect of the present invention, the plasmid of the present invention comprises a) 5′-untranslated region (UTR) sequence, b) translation initiation codon ATG, c) genetic element “E3” DNA sequence set forth in SEQ ID NO: 1, d) target-protein coding sequence placed adjacent to the 3′-end of the said genetic element “E3” DNA sequence set forth in SEQ ID NO: 1, e) 3′-untranslated region (UTR) sequence, and f) Poly A sequence, which are placed in the said order.
Wherein, the said Poly A sequence is 20-200 nucleotides in length.
According to a fifth aspect of the present invention, the present invention provides a method for synthesizing the RNA molecules having the genetic element “E3” using the plasmid comprising a) 5′-untranslated region (UTR) sequence, b) translation initiation codon ATG, c) genetic element “E3” DNA sequence set forth in SEQ ID NO: 1, d) target protein-coding sequence placed adjacent to the 3′-end of the said genetic element “E3” DNA sequence set forth in SEQ ID NO: 1, e) 3′-untranslated region (UTR) sequence, and f) Poly A sequence, which are placed in the said order, and utilizing the synthesized RNA molecules as RNA therapeutics.
Wherein, the said Poly A sequence is 20-200 nucleotides in length.
If using the RNA molecule having the RNA sequence set forth in SEQ ID NO: 2 which is synthesized with the plasmid having the DNA sequence set forth in SEQ ID NO: 1, as an RNA therapeutics, the expression level of target protein encoded in the RNA molecule can be enhanced, making it possible to obtain a higher intracellular expression level of the target protein without increasing the doses of RNA therapeutics, thereby enhancing the effects from same amount of RNA therapeutics, and reducing drug manufacturing costs and side effects since a relatively low dosage is required for the same effect.
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention.
In the drawings:
Hereinafter, the present invention is described in more detail on the basis of the Embodiments, which however constitute only some examples of the present invention, not limiting the applicability thereof.
First, the plasmid SARS-CoV-2-Spike-E3_pUC-GW-Kan shown in
Specifically, the constructed plasmid SARS-CoV-2-Spike-E3_pUC-GW-Kan has in order the 5′-UTR (untranslated region) sequence, the genetic element “E3” for enhancing the target protein carrying the DNA sequence set forth in SEQ ID NO: 1 right behind the initiation codon of coronavirus spike protein gene, the sequence encoding the coronavirus spike protein as the target protein-coding sequence, 3′-UTR sequence and Poly A sequence.
Large amount of the constructed plasmid was prepared by introducing the constructed plasmid into E. coli. Specifically, the plasmid SARS-CoV-2-Spike-E3_pUC-GW-Kan was introduced into E. coli (DH5α strain) and the transformant was cultivated in a Luria-Bertani broth containing 50 μg/ml kanamycin. After overnight cultivation, a conventional plasmid large-preparation was conducted.
The circular plasmid SARS-CoV-2-Spike-E3_pUC-GW-Kan prepared in the said process underwent the restriction enzyme treatment for its linearization and then the linearized plasmid was purified. Specifically, the prepared circular plasmid SARS-CoV-2-Spike-E3_pUC-GW-Kan was treated with the restriction enzyme NotI (37° C., 16 hours) to cleave the site right behind the end of Poly A tail sequence, and the linearized plasmid was purified using a conventional phenol/chloroform extraction. The linearized plasmid prepared was used as a DNA template for the in vitro transcription reaction for RNA synthesis.
Similarly, the linearized control plasmid SARS-CoV-2-Spike_pUC-GW-Kan was prepared, and used as a template for the in vitro transcription reaction for the synthesizing the control RNA. The plasmid map for the control plasmid is shown in
The test mRNA having the genetic element “E3” set forth in SEQ ID NO: 2 and the control mRNA not containing the genetic element “E3” set forth in SEQ ID NO: 2 were synthesized via the in vitro transcription reaction using the linearized plasmid prepared in Example 1 as a DNA template. In the synthesis reaction of the test mRNA, the linearized plasmid SARS-CoV-2-Spike-E3_pUC-GW-Kan was used as a DNA template, whereas the linearized plasmid SARS-CoV-2-Spike_pUC-GW-Kan was used as a DNA template in the synthesis reaction of the control mRNA. Specifically, this experiment comprises the following steps.
In the in vitro transcription reaction, a mixture of nucleotide triphosphates (NTP) was used, wherein the NTP mixture may contain chemically modified NTP, natural NTP, or a mixture of natural NTP and chemically modified NTP.
To synthesize the test mRNA and the control mRNA via the in vitro transcription reaction, the HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs) was used. The reaction components for the in vitro transcription reaction are shown in Table 1 below.
The in vitro transcription reaction was performed at 37° C. for 2 hours. Following the in vitro transcription reaction, the reaction mixture was stored overnight at 4° C. for the next-day purification or immediately used for the RNA purification. For the RNA purification, firstly to decompose the DNA template included in the reaction mixture, 5 units of RNase-free DNase I was added to the reaction mixture and then the reaction mixture was incubated at 37° C. for 30 minutes. Following the treatment with DNase I, the RNA was purified using the Monarch® RNA Cleanup Kit (New England Biolabs) according to the manufacturer's instruction. Quantification of the purified RNA was performed using the NanoDrop (Thermo Fisher Scientific). Also, the proper size and integrity of RNA was confirmed by agarose gel electrophoresis.
Next, the purified RNA underwent the capping and 2′-O-methylation. For the reaction, the Vaccinia Capping Enzyme and 2′-O-Methyltransferase were used according to the manufacturer's (New England Biolabs) instruction. Specifically, firstly, the purified uncapped RNA was mixed with the DEPC-treated water until the final volume reached 13.5 μl. The mixture was kept at 65° C. for 5 minutes to denature the RNA molecules, and immediately chilled in an ice-bath. Then, the denatured RNA was mixed with 10× capping buffer (2.0 μl), 10 mM GTP (1.0 μl), 4 mM S-adenosylmethionine (SAM, 1.0 μl), RNase inhibitor (20 units, 0.5 Vaccinia Capping Enzyme (10 units, 1.0 μl) and 2′-O-Methyltransferase (50 units, 1.0 μl), and then the mixture was incubated at 37° C. for 1 hour. The capped RNA was purified using the Monarch® RNA Cleanup Kit (New England Biolabs) according to the manufacturer's instruction. Following the purification, quantification of RNA was performed using the NanoDrop (Thermo Fisher Scientific). Lastly, the proper size and integrity of RNA was confirmed by agarose gel electrophoresis.
The intracellular expression of the target protein (antigen in this Example) was compared using the test mRNA which has the genetic element “E3” placed adjacent to the 5′-end of the target protein according to the present invention, and also the corresponding control mRNA which does not have the genetic element “E3”. Both the test and control mRNA were prepared in Example 2.
Specifically, firstly, HeLa cells were cultured in a cell culture dish (900) to be used for the introduction of RNA into the cells. The cells were harvested by treating with trypsin. Then, cells were seeded per well on a 24-well plate to be 2×105 cells/well. Following the cell seeding, the 2 μg RNA prepared in Example 2 was introduced into the HeLa cells using the commercial transfection reagent (Lipofectamine™ MessengerMAX™, Invitrogen) according to the manufacturer's instruction. At 12 hours after the intracellular introduction, the cell lysate was secured to examine the intracellular expression of the target protein using a conventional western blotting. The 1:1000 dilution of rabbit anti-SARS Spike c-terminal (Abcam) and the mouse anti-rabbit IgG-HRP were used as the primary and secondary antibodies for the western blotting, respectively.
As results of triplicate experiments to compare the efficiency of the intracellular expression of the target protein between the test and control mRNA, the test mRNA exhibited improved efficiency of the intracellular expression of the target protein by approximately 300% compared with the control mRNA (
The said experimental result demonstrated the effects of the genetic element “E3” in enhancing the efficiency of the intracellular expression of the target protein according to the present invention. That is, the present invention was found to be effectively applicable to some cases wherein high intracellular expressions of target proteins are required.
The target protein of the RNA prepared in Example 2 is SARS-CoV-2 antigen. Therefore, the RNA prepared in Example 2 is applicable as mRNA vaccines.
In this Example, the effects of the present invention when applied to mRNA vaccine was investigated using the RNA molecules prepared in Example 2 with the technology held by the inventors of the present invention.
As the test and control mRNA, the mRNA used as the test and control mRNA in Example 3 were used, respectively. The control and test vaccine samples for the animal test were prepared in the same method. For reference, only the manufacture of the test vaccine sample is given below.
The test vaccine sample (E3_LNP-P-1/2, hereinafter) was manufactured by encapsulating the test mRNA in peptide-conjugated lipid nanoparticles (LNP). The peptides P1 and P2 conjugated to lipid nanoparticles here were synthesized by a specialized company.
The terminal residue of the peptides P1 and P2 is cysteine, which was coupled to the maleimide group of the PEG-lipid [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] to manufacture the peptide-conjugated PEG-lipid for use in the peptide-conjugated LNP manufacturing. To briefly describe the coupling of the peptides with the maleimide group of PEG-lipid, the peptides P1 and P2 were dissolved in water (1:1 mole ratio), while the PEG-lipid was dissolved in dimethylformamide (DMF). Here, the mole ratio between the total peptide and PEG-lipid was 1.2:1. The prepared peptide solution and the prepared PEG-lipid solution were mixed together, and the mixture was agitated at 4° C. for 18 hours. To remove any uncoupled peptides, the reaction mixture was dialyzed against 2 L water for at least 18 hours. Upon completion of the dialysis, the peptide-conjugated PEG-lipid was recovered via ultrafiltration using Amicon ultra centrifugal filters (Merck Millipore, MWCO: 3K) and then the filtrate was subjected to diafiltration using 70% ethanol. The prepared peptide-conjugated PEG-lipid was kept at −20° C. until use.
Using the peptide-conjugated PEG-lipid prepared in the said process, the E3_LNP-P-1/2 was manufactured as follows. Using the NanoAssemblr microfluidic device (Precision Nanosystems), the lipid components dissolved in ethanol (including the peptide-conjugated PEG-lipid) were mixed with the test mRNA dissolved in 50 mM sodium citrate buffer solution (pH 4.0). The mole percentages of the lipid components comprised 50% DLin-MC3-DMA (ionizable cationic lipid), 10% DSPC, 38.5% cholesterol and 1.5% peptide-conjugated PEG-lipid. The lipid component solution (ethanol phase) and the test mRNA solution (aqueous phase) were assembled in the microfluidic device at 1:3 flow rate. The total assembled flow rate was 12 ml/minutes per microfluidic cartridge. The manufactured peptide-conjugated LNP sample was dialyzed against phosphate-buffered saline (PBS, pH 7.2) for at least 18 hours using a Slide-A-Lyzer™ dialysis cassettes (Thermo Fisher Scientific). The E3_LNP-P-1/2 sample obtained through the dialysis was concentrated using Amicon ultra centrifugal filters (Merck Millipore), and then finally filtrated using 0.22 μm filters. The final obtained E3_LNP-P-1/2 solution was kept at −20° C. until use.
The control vaccine sample was manufactured according to the same method as described above using the control mRNA instead of the test mRNA. The control vaccine sample manufactured was named LNP-P-1/2.
Using the prepared test and control vaccine sample, we conducted the animal test as follows. 5-week-old female C57BL/6 mice were used as the test animal. All mice were bred and raised in a specific pathogen free (SPF) facility. The mice were assigned the following groups and underwent the nasal administration of the vaccine sample. The control vaccine sample LNP-P-1/2 was administered to the control group (C) nasally (10 μg mRNA), whereas the test vaccine sample E3_LNP-P-1/2 was administered to the test group (T) nasally (10 μg mRNA). The nasal administration was conducted twice, in Week 0 and Week 3. For the nasal administration, each mouse was placed in a gas chamber and anesthetized with isoflurane. After a short while, the anesthetized mouse was taken out of the gas chamber and placed in supine position. The nasal administration of the vaccine sample was performed using a P20 pipette fitted with a gel loading tip. After nasal administration, the mouse was laid back inside the gas chamber in supine position. This procedure was repeated for the next animal in sequence.
15 days after the second nasal administration, the sera, nasal wash and bronchoalveolar lavage fluid (BALF) were collected to evaluate the effectiveness of the vaccination. Then, protease inhibitor was added to the collected sera, nasal wash and BALF, which were subsequently kept at −80° C.
The titer of anti-spike protein antibody included in the collected sera, nasal wash and BALF was analyzed using the ELISA. Specifically, 1 μg/ml spike protein diluted in PBS was coated onto ELISA plates overnight at 4° C. or for 2 hours at 37° C. Then, the ELISA plate was blocked at room temperature for 2 hours with the blocking buffer (PBS+1% BSA+0.1% Tween-20). After washing of ELISA plate, the collected sera, nasal wash and BALF samples were added at different dilutions. The detection was performed using commercially available ELISA kit. For detection, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:5,000 dilution) and HRP-conjugated anti-mouse IgA (1:10,000 dilution) were used. The results are presented in
Based on the said result, the mRNA vaccine manufactured according to the present invention provides the superior efficacy of the conventional mRNA vaccine.
Notwithstanding the detailed description of the specific aspects of the present invention, those skilled in the art will recognize, or be able to ascertain the description involves no more than desirable Examples of the present invention. Hence, the scope of the present invention is not intended to be limited to the description above, but rather is as set forth in the following claims and their equivalents.
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|---|
| Alike W. van der Velden et al. Vector Design for Optimal Protein Expression (2001), vol. 31:1,7 (Year: 2001). |
| EMBL Database, Accession No. LN055459. Retrieved from the internet: <URL:https://www.ebi.ac.uk/Tools/dbfetch/dbfetch?db=embl&id=LN055459&style=raw> (Year: 2014). |
| EMBL Database, Accession No. HG993169. Retrieved from the internet :<URL:https://www.ebi.ac.uk/Tools/dbfetch/dbfetch?db=embl&id=HG993169&style=raw> (Year: 2021). |
