Patent Application
20240200042
The instant application contains a Sequence Listing which has been submitted electronically in ST.26 format and is hereby incorporated by reference in its entirety. Said ST.26 copy, created on Mar. 6, 2024 is named SEQUENCE LISTING.xml and is 114 Kilobytes in size.
The application relates to the technical field of biomedicine, in particular to a limited self-replicating mRNA molecular system, producing method and use.
Messenger RNA (mRNA) therapy is a novel treatment approach with broad clinical potential, including vaccines targeting infectious sources, treatments for cancer or genetic diseases, regenerative therapy, and immunotherapy. Compared to protein based biological agents, the advantages of messenger RNA therapy include that messenger RNA can synthesize proteins through the body's own cells, without the need for complex protein synthesis and purification processes or production lines; Intracellular and membrane binding proteins can be used as therapeutic targets; It can be rapidly industrialized under cell-free GMP conditions, with a short cycle from research and development to product development.
However, the application of messenger RNA therapy is limited by factors such as structural instability, innate immunogenicity, and inefficient in vivo delivery, the development direction of this technology lies in: Firstly, it must avoid rejection by the innate immune system, which may mistake therapeutic messenger RNA for non-self-nucleic acids, resulting in rejection. This is particularly important for repeated administration of messenger RNA therapy drugs, as immune memory may limit the effectiveness of drug products. Present research suggests that chemical modification of the nucleoside base of messenger RNA can reduce innate immune rejection and improve the efficiency of translating messenger RNA into proteins. However, it is still unclear how to carry out nucleoside modification, the proportion of modifications, and how to combine nucleotide modifications. Secondly, ordinary messenger RNA is unstable, easily degradable, and has a short expression duration. Studies have shown that ordinary messenger RNA can only be expressed in cells for 24 hours. Self-replicating messenger RNA, as it can self-replicate, can amplify the protein translation instructions of messenger RNA to enhance and prolong the expression of messenger RNA to proteins. The self-replicating messenger RNA molecular system used in existing technology originates from the genome skeleton of alphavirus, where the partial skeleton encoding viral RNA replicase is intact, and the skeleton encoding the virus structural protein is replaced by the target protein coding sequence. The messenger RNA molecular system has the following defects: firstly, compared to non-self-replicating messenger RNA, the nucleotide sequence of self-replicating messenger RNA is much longer, the cell burden is heavy, and in vitro transcription synthesis of messenger RNA is technically difficult, resulting in high industrial production costs; Secondly, the self-replicating messenger RNA molecular system is essentially an RNA pseudo virus that can self-replicate, and its viral properties are obvious, for example, it is impossible to predict the number of times it replicates, and there is a possibility of unlimited replication (pseudo virus reproduction in vivo), for another example, when vesicular stomatitis virus antigen and rabies virus antigen are packaged as self-replicating RNA mentioned above, there is a possibility of amplifying their toxicity. Thirdly, the messenger RNA molecular system mentioned above has high cytotoxicity, and due to the inability to be nucleoside modified, the immune response of cells or the body greatly exceeds that of non-self-replicating messenger RNA.
Therefore, it is necessary to provide a limited self-replicating messenger RNA molecular system.
The purpose of this application is to provide a limited self-replicating mRNA molecular system, producing method and use, to solve the technical problem of mRNA being unable to achieve limited self-replication in existing technologies.
The first aspect, the application provides a limited self-replicating mRNA molecular system, including:
Optionally, the said mutated alphavirus replicase includes sequentially connected nsP1 region, nsP2 region, nsP3 region, and nsP4 region, the amino acid sequence of the said mutated alphavirus replicase is shown in SEQ ID NO: 1, the said mutated alphavirus replicase generated the mutant serine S to proline P in position 796 and the mutant arginine R to aspartate D in position 1187 of SEQ ID NO:1.
Optionally, the said first mRNA includes a mutated replicase coding sequence, the said mutated replicase coding sequence includes a RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.2;
Optionally, any of the said first mRNA and the said second mRNA further includes a 5′ cap structure, a 5′UTR sequence, a 3′UTR sequence, and a polyadenylate sequence;
The said 5′UTR sequence includes a RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.9, the said 3′UTR sequence includes a RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.10, the said 5′ cap structure is selected from 3′-O-Me-m7G, m7GpppG, m2 7,3′-O GpppG, m7Gppp (5′) N1 and m7Gppp (m 2′-O) N1.
Optionally, part or all of the uracil in the said first mRNA or the said second mRNA has been chemically modified to enhance the stability of the first mRNA or second mRNA in the organism, the first mRNA or second mRNA has been chemically modified by replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of uracil in the first mRNA or second mRNA with N1-methyl-pseudouridine.
Optionally, the said first mRNA and the said second mRNA are treated with RNase III, the said first mRNA and the said second mRNA are purified by fast protein liquid chromatography.
Optionally, the said target protein includes SARS-COV-2 antigenic peptides;
The second aspect, the application provides a method for producing the limited self-replicating mRNA molecular system, including:
Optionally, also including:
Optionally, synthesize the first mRNA, including:
Optionally, synthesize the second mRNA, including:
The third aspect, the application provides a biological material, the said biological material is any one of A1) to A6):
The fourth aspect, the application provides a drug combination, the said drug combination includes any mRNA molecular system, and a delivery agent.
The fifth aspect, the application provides a use of the first mRNA encoding a mutated alphavirus replicase as immunological adjuvant to regulate the immune system.
The sixth aspect, the application provides a use of any mRNA molecular system or biological material or drug combination in preparing cell re editing reagents, gene editing reagents, therapeutic drugs for Barth syndrome, vaccines for infectious diseases or tumor vaccines.
The beneficial effects of the present application are as follows: in this application the limited self-replicating mRNA molecular system including: a first mRNA encoding a mutated alphavirus replicase; and at least one second mRNA encoding a target protein; by generating specific mutation adjustments in the nsP2 subunit of mutated replicase, this limited self-replicating mRNA molecular system can achieve limited self-replication and avoid cytotoxicity; by constructing different mRNA with mutated alphavirus replicase and different target proteins, the mutated alphavirus replicase encoded by the first mRNA can simultaneously replicate multiple different target proteins, achieving sustained expression of multiple target proteins.
The following will provide a clear and complete description of the technical solution in the embodiments of the invention, in conjunction with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technical personnel in this field without creative labor fall within the scope of protection of this application.
Referring to “embodiments” in this article means that specific features, structures, or features described in conjunction with the embodiments may be included in at least one embodiment of the present application. The phrase appearing in various positions in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. Technicians in this field explicitly and implicitly understand that the embodiments described in this article can be combined with other embodiments.
The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.
Positive stranded RNA virus genome serves as a template for translation and replication, it leads to multi-level interactions between host translation factors and RNA replication. All known positive stranded RNA viruses carry genes for RNA dependent RNA polymerase (RdRp) used for genome replication. However, unlike other RNA viruses, positive stranded RNA viruses do not shell the RNA polymerase. Therefore, when new cells are infected, viral RNA replication begins only when the genomic RNA is translated to produce RNA polymerase (which is also a replication factor for most positive stranded RNA viruses). All characterized positive stranded RNA viruses assemble their RNA replication complexes onto the cell membrane. Positive stranded RNA viruses produce negative stranded RNA, positive stranded RNA, double stranded RNA (dsRNA), and sub-genomic mRNA during replication, which themselves are strong inducers of innate immune response pathways.
The positive stranded RNA virus genome has the same polarity with cellular mRNA, and the positive stranded RNA virus genome RNA can be directly translated using the cellular translation system. Firstly, non-structural proteins are synthesized as precursor proteins and cleaved into mature non-structural proteins using viral proteases. Then, after translation and multi protein processing, a complex consisting of RNA polymerase (RdRp), additional non-structural proteins, viral RNA, and host cytokines is assembled. The assembled replication complex (RC) is used for viral RNA synthesis.
“RNA dependent RNA polymerase” or “RdRp” is known as an enzyme, protein, or peptide that catalyzes the de novo synthesis of RNA from RNA templates. A replicase is a complex of viral multi proteins or multi protein processing products that have RdRp activity and catalyze the replication of specific viral RNA. RdRp and the replicase are typically encoded by viruses with RNA genomes. Therefore, replicase not only provides the function of RNA dependent RNA polymerase, but also further includes additional viral non-structural multiprotein subunits that provide functions other than RdRp activity.
“Recombinant vector” refers to DNA or RNA based vector or plasmid that carries genetic information in the form of nucleic acid sequences. The terms “plasmid”, “vector”, “recombinant vector”, and/or “expression vector” may be used interchangeably in this article.
An example of the application provides a limited self-replicating mRNA molecular system, including: a first mRNA and at least one second mRNA; wherein the first mRNA encoding a mutated alphavirus replicase, each of the second mRNA encoding a target protein, limited replication of at least one target protein is achieved through mutated alphavirus replicase.
Wherein the said mutated alphavirus replicase is mutated in the nsP2 region generating the mutant in position 259 (serine S is mutated to proline P) and the mutant in position 650 (arginine R is mutated to aspartate D). Specifically, the mutated alphavirus replicase includes sequentially connected nsP1 region (537 amino acids), nsP2 region (799 amino acids), nsP3 region (482 amino acids), and nsP4 region (1254 amino acids), the amino acid sequence of the said mutated alphavirus replicase is shown in SEQ ID NO: 1, the mutated alphavirus replicase generated the mutant serine S to proline P in position 796 of SEQ ID NO:1 and the mutant arginine R to aspartate D in position 1187 of SEQ ID NO:1.
In this example, as shown in
In an optional implementation, the first mRNA includes a mutated replicase coding sequence, the said mutated replicase coding sequence includes a RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.2. The nucleic acid sequence shown in SEQ ID NO.2 has high GC content, and the codon with high GC content is selected without changing the corresponding amino acid sequence, which is 7-20% higher than the GC content of the wild-type replicase DNA sequence. Specifically, as shown in SEQ ID NO.2, the high GC content DNA sequence at positions 1-1611 corresponds to the nsP1 region, the high GC content DNA sequence at positions 1612-4008 corresponds to the nsP2 region, the high GC content DNA sequence at positions 4009-5454 corresponds to the nsP3 region, the high GC content DNA sequence at positions 5455-9216 corresponds to the nsP4 region. The nucleic acid sequence shown in SEQ ID NO.11 is the replicase DNA sequence of wild alphavirus, where positions 1-1611 correspond to the original DNA sequence of nsP1 region, positions 1612-4008 correspond to the original DNA sequence of nsP2 region, positions 4009-5454 correspond to the original DNA sequence of nsP3 region, and positions 5455-9216 correspond to the original DNA sequence of nsP4 region.
In an optional implementation, each second mRNA includes sequentially connected a 5′ end specific sequence for the replicase, a target protein coding sequence, and a 3′ end specific sequence for the replicase, at both ends of the target protein coding sequence, specific sequences recognized of the alphavirus replicase are connected to improve the translation level of the target protein coding sequence, achieving the same effect without retaining the entire alphavirus RNA framework system, specifically, the 5′ end specific sequence is a RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.7, the 5′ end specific sequence is a RNA sequence is derived from the first to 221 st positions of the original DNA sequence corresponding to the nsP1 region of the replicase, the 3′ end specific sequence is a RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.8, the 3′ end specific sequence is derived from the second to 985th positions of the original DNA sequence corresponding to the nsP4 region of the replicase. The mRNA combination in this embodiment has limited replication, completely removing the virus attribute and completely eliminating the possibility of virus proliferation in vivo using the current alphavirus vector.
Furthermore, any of the said first mRNA and the said second mRNA further includes a 5′ cap structure, a 5′UTR sequence, a 3′UTR sequence, and a polyadenylate sequence; wherein the said first mRNA includes the following elements sequentially connected in the 5′ to 3′ direction: the 5′ cap structure, the 5′UTR sequence, the mutated replicase coding sequence, the 3′UTR sequence and the polyadenylate sequence. Similarly, each of the second mRNA includes the following elements sequentially connected in the 5′ to 3′ direction: the 5′ cap structure, the 5′UTR sequence, the 5′ end specific sequence for the replicase, the target protein coding sequence, the 3′ end specific sequence for the replicase, the 3′UTR sequence and the polyadenylate sequence. Specifically, the preferred target protein coding sequence is the RNA sequence corresponding to the open reading frame (ORF) in the target protein coding gene, the 5′UTR sequence includes a RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.9, the 3′UTR sequence includes a RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.10, the 5′ cap structure is selected from 3′-O-Me-m7G, m7GpppG, m2 7,3′-O GpppG, m7Gppp(5′)N1 and m7Gppp(m 2′-O)N1, preferably 3′-O-Me-m7G. The polyadenylate sequence is a sequence containing 60-200 adenylates; preferably, the polyadenylate sequence is a sequence containing 120 adenylates.
In the example, part or all of the uracil in the said first mRNA or the said second mRNA has been chemically modified to enhance the stability of the first mRNA or second mRNA in the organism, the first mRNA or second mRNA has been chemically modified by replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of uracil in the first mRNA or second mRNA with N1-methyl-pseudouridine. Furthermore, in the example, 100% of uracil in the first mRNA or second mRNA has been replaced with N1-methyl-pseudouridine, to reduce innate immune rejection and to improve the efficiency of mRNA translation into proteins.
In the example, the first mRNA and second mRNA obtained by in vitro transcription of the recombinant vector are first treated with RNase III and then purified by fast protein liquid chromatography, which can further improve the efficiency of mRNA translation into protein.
In the example, theoretically, the target protein can be any acceptable protein or peptide, such as:
The limited self-replicating mRNA molecular system includes one second mRNA, encoding SARS-COV-2 antigenic peptides, the antigenic peptide can be selected from the receptor binding domain RBD of SARS-COV-2, the spike protein S1 subunit of SARS-COV-2, or the full-length sequence of spike protein S of SARS-COV-2; the above spike proteins are derived from SARS-COV-2 Delta mutant strains or SARS-COV-2 original strains.
The limited self-replicating mRNA molecular system includes two second mRNA, one encodes IL-2 and the other encodes Alpha-fetoprotein with no amino group.
The limited self-replicating mRNA molecular system includes five second mRNA, encode separately L1 protein of HPV6, L1 protein of HPV11, L1 protein of HPV16, L1 protein of HPV18, and E6 protein of HPV.
The limited self-replicating mRNA molecular system includes two second mRNA, encode separately envelope glycoprotein E of HSV and envelope glycoprotein D of HSV.
The limited self-replicating mRNA molecular system includes one second mRNA, encodes Influenza virus HA antigen.
The limited self-replicating mRNA molecular system includes five second mRNA, encode separately HIV Gag antigen, HIV EnV antigen, and HIV CD40L;
The limited self-replicating mRNA molecular system includes five second mRNA, encode separately C-Myc protein, Klf4 protein, Sox2 protein, OCT4 protein, and Lin28 protein.
The limited self-replicating mRNA molecular system includes two second mRNA, encode separately Cas9 protein and DNAJC19 protein;
An example of the application provides a biological material, the biological material including: (i) a nucleic acid molecule encoding the first mRNA; and (ii) a nucleic acid molecule encoding the second mRNA.
Wherein the nucleic acid molecule encoding the first mRNA include the nucleic acid sequence shown in SEQ ID NO.2, the nucleic acid molecule encoding the second mRNA include sequentially the nucleic acid sequence shown in SEQ ID NO.7, target protein DNA coding sequence, and the nucleic acid sequence shown in SEQ ID NO.8.
In an optional implementation, the nucleic acid molecule encoding the first mRNA include sequentially the nucleic acid sequence shown in SEQ ID NO.9, the nucleic acid sequence shown in SEQ ID NO.2, the nucleic acid sequence shown in SEQ ID NO.10, and polyadenylate sequence. The nucleic acid molecule encoding the second mRNA include sequentially the nucleic acid sequence shown in SEQ ID NO.9, the nucleic acid sequence shown in SEQ ID NO.7, target protein DNA coding sequence, the nucleic acid sequence shown in SEQ ID NO.8, the nucleic acid sequence shown in SEQ ID NO.10, and polyadenylate sequence.
An example of the application provides a biological material, the biological material including: a first recombinant vector containing the nucleic acid molecule encoding the first mRNA; and a second recombinant vector containing the nucleic acid molecules encoding the second mRNA.
An example of the application provides a biological material, the biological material including: a transgenic animal cell line containing the first recombinant vector; and a transgenic animal cell line containing the second recombinant vector.
Step 1: Using GeneArt™ Gibson Assembly® HiFi reaction (Thermo Fisher, USA, A46624) synthesized the mutated replicase DNA coding sequence (the nucleic acid molecule encoding the first mRNA does not contain a polyadenylate sequence). After successful synthesis, the mutated replicase DNA coding sequence was cloned into the pcDNA3.3 vector plasmid for industrial production.
1.1 Mutant replicase DNA coding sequence: 5′UTR sequence (SEQ ID NO.9), mutant replicase coding sequence (SEQ ID NO.2), and 3′UTR sequence (SEQ ID NO.10). Among them, the mutant replicase coding sequence (SEQ ID NO.2) is divided into four DNA fragments: nsP1 region fragment (SEQ ID NO.3), nsP2 region fragment (SEQ ID NO.4), nsP3 region fragment (SEQ ID NO.5), and nsP4 region fragment (SEQ ID NO.6), All four DNA fragments are modified high GC content fragments. Four DNA fragments were directly ordered in gblock form from IDT Corporation in the United States.
The specific steps include assembling the Gibson reaction according to Table 1, reacting at 50° C. for 60 minutes in a PCR instrument to obtain the PCR product.
1.2 Transform PCR Products into One Shot™ TOP10 Chemically Responsive Escherichia coli Cells, Including the Following Steps:
Among them, the poly-(a) tail contains 120 adenylates.
Prepare PCR premix according to Table 2 (total volume 200u L, eight reactions with 25 μL for each);
1. Assemble the mRNA cap structure and nucleotide mixture according to table 4:
Cap structure 3′-O-Me-m7G (5′) ppp (5′) G RNA cap analog (New England Biolabs, cat. no. S1411S), Methylcytidine-5′-triphosphate (Me-CTP; Trilink, cat. no. N1014),N1-methyl-pseudo-UTP (Trilink, cat. no. N1019), other components were from megascript T7 Kit (Ambion, cat. no. AM1334).
2. Assemble the in vitro transcription system of the first mRNA according to table 5:
3. The reaction was incubated in the PCR instrument at 37° C. for 3˜6 h.
4. Add 2 μL turbo DNase (from MEGAscript T7 kit, Ambion, cat. No. am1334) to each sample.
5. Gently mix and incubate at 37 º C for 15 min.
6. The reactions treated with DNase and RNAa seIII were purified using MEGAclear Kit (Ambion, cat. No. am1908); eluted the modified mRNA with a total of 100 μL of elution buffer (50 μL of elution buffer eluted twice).
7. Purified modified mRNA was treated by phosphatase (Antarctic phosphatase (New England Biolabs, cat. No. m0289s)).
8. Add 11 μL 10× phosphatase buffer to each sample (about 100μ), followed by the addition of 2 μL phosphatase; gently mix the samples and then incubate them at 37° C. for 0.5˜1 h.
9. After elution, the concentration of the modified first mRNA was measured in a nanodrop spectrophotometer. The expected total production should be about 50 ug (range of 30˜70 ug; 100 μL elution volume for 40 μL of IVT response at a time was 300-700 ng/μL). The concentration was adjusted to 100 ng/μL by adding elution buffer or TE buffer (pH 7.0), or FPLC purification.
The synthesis steps of the second mRNA are similar to those of the first mRNA, including the following steps:
Step 1: Using GeneArt™ Gibson Assembly® HiFi reaction (Thermo Fisher, USA, A46624) synthesized the specifically modified target protein DNA coding sequence (the nucleic acid molecule encoding the second mRNA does not contain a polyadenylate sequence);
Wherein specifically modified target protein DNA coding sequence: 5′UTR sequence (SEQ ID No.9), 5′ end specific sequence for the replicase (SEQ ID No.7), target protein DNA coding sequence (shown in Table 6), 3′ end specific sequence for the replicase (SEQ ID No.8), 3′UTR sequence (SEQ ID No.10).
Step 2: Add the poly-(a) tail mRNA through PCR to obtain the DNA synthesis template for the second mRNA;
Step 3: in vitro transcription for synthesis of the second mRNA.
The 27 second mRNAs shown in Table 6 were synthesized according to the above methods.
The above SEQ ID No.14 to SEQ ID No.39 and SEQ ID No.47 were modified with high GC content on the basis of the corresponding original sequence without changing the original amino acid sequence.
An example of the application provides a drug combination, the drug combination includes multiplexed molecular messenger RNA and a delivery agent, wherein multiplexed molecular messenger RNA includes the first mRNA prepared in example 1 and the second mRNA-1 prepared in example 2.
Doxycycline was introduced into the mouse genome to induce the knock down of taffazin protein, and Barth syndrome model mouse was established. The genotyping DNA was determined by PCR analysis, primers were as follows:
In this case, only males were used. Doxycycline was placed in the drinking water of mice at a concentration of 2 mg/l, and 10% sucrose was also contained.
10 μL protamine (protamine ipex5000, MEDA pharmaceutical company, 5000 IU/m) was added to 280 μL water, as 280 μL water+10 μL protamine 5000, prepare 0.5 mg/ml protamine solution, and 0.5 mg/ml multiple molecular messenger RNA (the molar ratio of multiple molecules is 1:1 in solution). Add an equal amount of protamine solution to the RNA solution, and quickly purge it up and down for at least 10 times. Leave it at room temperature for 10 minutes to prepare 130 nm protamine RNA nanoparticles, which are placed in the mouse subcutaneous pump (Alzet pump, https://www.alzet.com/guide-to-use/scid/) for continuous dosing.
Barth syndrome mice (TG) were divided into six groups: TG1, TG2, TG3, TG4, tg5, TG6;
The mouse was operated on a closed electric treadmill with adjustable speed and inclination (slope angle) and also with an electric shock transmission network of an electric shock intensity of 1 mA. Initially, the animals were allowed to adapt to the environment by resting on the treadmill for 30 min, and the test started with a 10% slope and a speed of 5 m/min. Gradually increase 5 m/min every 5 minutes to a final speed of 25m/min.
After multiple molecular messenger RNA therapy for Barth syndrome, cardiac ejection fraction ultrasound results suggest that multiple molecular messenger RNA therapy for Barth syndrome can improve its cardiac function.
As shown in
The forced exercise results of Barth syndrome treated with multiple molecular messenger RNA suggest that multiple molecular messenger RNA treatment of Barth syndrome can improve its exercise ability. The animals initially rested on the treadmill for 30 min to allow the animals to adapt to the environment, and the test started with a 10% slope and a speed of 5 m/min. Gradually increase 5 m/min every 5 minutes to the final speed of 25 m/min. Therefore, the duration of exercise was 36.8 minutes and the distance traveled was 507.4m. The results suggested that before the treatment, the model mice failed to maintain 15m/min and 10% inclination on the belt, and none of the model mice could maintain running when treadmill speed was more than 20 meters/minute, while after 6 weeks of multiple molecular messenger RNA treatment, the model mice could maintain running, indicating that multiple molecular messenger RNA treatment can significantly improve the exercise ability of the model mice.
As shown in
The function of taffazin protein in Barth syndrome mice (TG) was lost under doxycycline induction. Barth syndrome symptoms appeared and cardiomyopathy phenotype appeared. Cardiac pathology suggested cardiac fibrosis. Multiple molecular messenger RNA treatment for 8 weeks significantly improved the degree of cardiac fibrosis, and was superior to the effect of common messenger RNA treatment.
An example of the application provides a limited self-replicating mRNA molecular system, the limited self-replicating mRNA molecular system includes the first mRNA prepared in example 1 and the second mRNA-2 prepared in example 2, the target protein is Hydrolyzed GFP protein.
Cells were transfected with common mRNA encoding hydrolyzed GFP protein (Group 1), bimolecular mRNA from Example 4 (Group 2), and full-length self-replicating mRNA encoding hydrolyzed GFP (Group 3), respectively. The steps are shown in i-vii. the following expression of hydrolyzed GFP report gene (expressed GFP is rapidly degraded by its own hydrolytic enzyme, which can instantly reflect the duration and expression level of multiple messenger RNA molecules).
The steps for transfecting cells with a limited self-replicating mRNA molecular system in Example 4 are as follows:
(i) After thawing, 10 μL of the limited self-replicating mRNA molecular system from Example 4 (molar ratio of first mRNA and second mRNA-2 is 6:4) was added in 40 μL OPTI-MEM, then gently mix.
(ii) In another test tube, add 45 μL OPTI-MEM, and then add 5 μL Lipofectamine RNAiMax, then gently mix.
Repeat pipetting.
(iii) Add the diluted Lipofectamine RNAiMax to the diluted limited self-replicating mRNA molecular system of Example 4, and gently mix repeatedly.
(iv) Incubate the mixture at room temperature for 15 minutes.
(v) Using 100 μL the limited self-replicating mRNA molecular system/transfection reagent complex from Example 4 was uniformly added to one well of the six well plate.
(vi) Gently shake the board left and right to ensure the transfected complex can diffuse uniformly.
(vii) Put it to cell culture incubator with the 37° C., 5% CO2, and 5% O2.
By detecting the fluorescence intensity of GFP protein, the mRNA half-life in the cells of Group 1, the mRNA half-life in the cells of Group 2, and the mRNA half-life in the cells of Group 3 were detected, and cellular innate immune responses were performed on Group 1, Group 2, and third Group 3.
Detect the cells number after transfection in Group 1, Group 2, and third Group 3.
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An example of the application provides a limited self-replicating mRNA molecular system, the limited self-replicating mRNA molecular system includes the first mRNA prepared in example 1 and the second mRNA-22, the second mRNA-23, the second mRNA-24, the second mRNA-25, the second mRNA-26 prepared in example 2, the target proteins include C-Myc protein, Klf4 protein, Sox2 protein, OCT4 protein, and Lin28 protein.
1. Add 1 ml of 0.1% (wt/vol) gelatin to each well of the six well plate;
Leave at room temperature for at least 1 hour. Alternatively, overnight at 4 º C, one day before inoculating human NuFF feeder cells, remove gelatin by suction and let the plate dry at room temperature.
2. NuFF feeder cells (Newborn human foreskin fibroblasts, GlobalStem, cat. no. GSC-3001G), thaw a bottle of mitotic inactivated NuFFs and inoculate the cells onto a gelatin cell plate.
3. 6-12 hours after inoculating reprogrammed target fibroblasts, replace the fibroblast culture medium (Pluriton containing B18R (eBioscience, cat. no. 34-8185-85, 200 ng/ml)) with Pluriton (stemgent) completely reprogrammed medium, use 2 ml per well, and then incubate the cells overnight at 37° C., 5% CO2, and 5% 02 medium.
4. Use Lipofectamine RNAiMax (Invitrogen, cat. no. 56532) for transfection;
(i) After thawing, 10 μL of the limited self-replicating mRNA molecular system from Example 4 (molar ratio of double mutated replicase, OCT4, KLF4, c-MYC, SOX2, and LIN28A is 6:1:1:1:1:1 respectively, 100 ng/μL) was added in 40 μL OPTI-MEM, then gently mix.
(ii) In another test tube, add 45 μL OPTI-MEM, and then add 5 μL Lipofectamine RNAiMax, then gently mix.
Repeat pipetting.
(iii) Add the diluted Lipofectamine RNAiMax to the diluted limited self-replicating mRNA molecular system of Example 4, and gently mix repeatedly.
(iv) Incubate the mixture at room temperature for 15 minutes.
(v) Using 100 μL the limited self-replicating mRNA molecular system/transfection reagent complex from Example 4 was uniformly added to one well of the six well plate.
(vi) Gently shake the board left and right to ensure the transfected complex can diffuse uniformly.
(vii) Put it to cell culture incubator with the 37° C., 5% CO2, and 5% 02.
5. Repeat the i-vii steps every 72 hours until reprogrammed cell clones appear.
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An example of the application provides a limited self-replicating mRNA molecular system, the limited self-replicating mRNA molecular system includes the first mRNA prepared in example 1 and the second mRNA-3 prepared in example 2, the target protein is Cas9 protein.
The limited self-replicating mRNA molecular system (multiple messenger RNA) from Example 6 was subjected to DNA JC19 gene editing or Taffazin gene editing in induced pluripotent stem cells.
1. Electro-transfection of induced pluripotent stem cells: assemble the gene editing reaction system according to Table 7, including Taffazin gene gRNA sequence (SEQ ID NO.40, directly ordered from IDT company) and DNAJC19 gRNA sequence (SEQ ID NO.17, directly ordered from IDT company).
PCR amplification of Taffazin gene fragments;
PCR amplification of DNAJC19 gene fragment.
3. Surveyor enzyme evaluation of human induced stem cell Taffazin gene editing or DNAJC19 gene editing (IDT, cat. no. 706025), assembly of reaction system as shown in Table 8,
4. Mix evenly and incubate at 42° C. for 60 minutes.
5. Add 1/10 volume termination solution of the Surveyor Mutation Detection Kit to terminate the reaction and ⅙ volume of DNA.
6. The products of Surveyor nuclease digestion were analyzed by electrophoresis of 4-20% TBE gel at 200 V for about 60 minutes.
7. Dye the gel with 0.5 g/ml ethidium bromide in 1×TBE for 10 minutes. Wash the gel in water for 10 minutes.
8. The gel was imaged with an ultraviolet transmission apparatus.
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The example provides a mRNA vaccine, includes the first mRNA prepared in example 1, the second mRNA-5 prepared in example 2, and protamine, form 130 nanometer protamine RNA particles for delivery. The target protein is SARS-COV-2 antigenic peptides (wild type spike protein S).
The example provides a mRNA vaccine, includes the first mRNA prepared in example 1, the second mRNA-28 prepared in example 2, and protamine, form 130 nanometer protamine RNA particles for delivery. The target protein is SARS-COV-2 antigenic peptides (Delta strain spike protein S).
The example provides a mRNA vaccine, includes the first mRNA prepared in example 1, the second mRNA-8, the second mRNA-9, the second mRNA-10, the second mRNA-11, the second mRNA-12 prepared in example 2, and protamine, form 130 nanometer protamine RNA particles for delivery. The target proteins include L1 protein of HPV6, L1 protein of HPV11, L1 protein of HPV16, L1 protein of HPV18, and E6 protein of HPV.
The example provides a mRNA vaccine, includes the first mRNA prepared in example 1, the second mRNA-13, and the second mRNA-14 prepared in example 2, and protamine, form 130 nanometer protamine RNA particles for delivery. The target proteins include envelope glycoprotein E of HSV and envelope glycoprotein D of HSV.
The example provides a mRNA vaccine, includes the first mRNA prepared in example 1, the second mRNA-15 prepared in example 2, and protamine, form 130 nanometer protamine RNA particles for delivery. The target protein is Influenza virus HA antigen.
The example provides a mRNA vaccine, includes the first mRNA prepared in example 1, the second mRNA-16, the second mRNA-17, and the second mRNA-18 prepared in example 2, and protamine, form 130 nanometer protamine RNA particles for delivery. The target proteins include HIV Gag antigen, HIV EnV antigen, and HIV CD40L.
The example provides a mRNA vaccine, includes the first mRNA prepared in example 1, the second mRNA-19, the second mRNA-20, and the second mRNA-21 prepared in example 2, and protamine, form 130 nanometer protamine RNA particles for delivery. The target proteins include the NL-S protein of African swine fever virus, the cd2v ep402r protein of African swine fever virus, and the TK protein of African swine fever virus.
The example provides a drug combination, used for treating colon cancer, includes the first mRNA prepared in example 1, the second mRNA-6, the second mRNA-7 prepared in example 2, and protamine, form 130 nanometer protamine RNA particles for delivery. The target proteins include IL-2 and Alpha-fetoprotein with no amino group.
The example provides a mRNA vaccine, includes the first mRNA prepared in example 1, the second mRNA-27 prepared in example 2, and protamine, form 130 nanometer protamine RNA particles for delivery. The target protein is rabies antigen (rabies glycoprotein).
The above is only the embodiment of the application. It should be pointed out here that ordinary technicians in the art can make improvements without departing from the creative idea of the application, but these belong to the protection scope of the application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111054809.9 | Sep 2021 | CN | national |
| 202111080355.2 | Sep 2021 | CN | national |
This application is a continuation of International Application No. PCT/CN2021/126076 filed on Oct. 25, 2021, which claims priority to Chinese Patent Application No. 202111054809.9, filed on Sep. 9, 2021 and No. 202111080355.2, filed on Sep. 15, 2021, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2021/126076 | Oct 2021 | WO |
| Child | 18598120 | US |
