Patent Application
20240401076
A Sequence Listing associated with this application is being filed concurrently herewith and is hereby incorporated by reference into the present specification. The text file containing the Sequence listing is titled “Sequence_Listing.xml”, was created on Aug. 23, 2024, and is 109 kilobytes in size.
The present disclosure relates to the field of biomedicine, and particularly, to the design of self-replicating RNA molecules and uses thereof.
The development of mRNA-based vaccines and therapeutics has become one of the hot spots in recent years. The mechanism of mRNA, when serving as vaccine or using for therapy, is to stimulate the immune system to respond, or to express proteins for treating. Therefore, the protein expression level is closely related to the therapeutic effect. However, as one of the disadvantages, mRNA is not very stable and may degrade within a few days in cells, resulting in an unsustainable protein expression level. If it is used for disease treatment in a long period, patients have to be injected with a large amount of mRNA, which may increase the toxic side effects of mRNA therapy.
Therefore, at present, mRNA-based vaccines or therapeutics still need to be improved.
The present disclosure aims to solve one of the technical problems in the related art at least to some extent.
Through in-depth study on the mechanism of translation and self-amplification of RNA of various RNA viruses in cells, the Applicant found that: by using RNA molecules encoding N, P and L proteins derived from Rhabdovirus as the core region, RNA can be self-replicated and translated in animal cells, and the core region, as a powerful “engine”, can provide “kinetic energy” for high-efficient transcription amplification and for activating macromolecular proteins. The core region can further carry a “loading region” to replicate or translate the target molecules, which cover almost all protein medicaments available on the market. According to the embodiments of the present disclosure, the “loading region” can be designed with different protein coding boxes to enable the organism to produce various peptides, enzymes, antibodies, channel proteins, receptor proteins and the like in cells, thereby achieving different therapeutic purposes, covering tumor pipelines, vaccine pipelines, rare disease pipelines and prospective general product pipelines. Therefore, the Applicant provides a novel self-replicating RNA molecule, which is referred to as reRNA™. As mentioned above, reRNA™ includes an element coding sequence capable of implementing RNA translation and self-amplification, and a carried gene sequence encoding the target molecule.
Based on the above, in a first aspect of the present disclosure, the present disclosure provides a self-replicating RNA molecule. The self-replicating RNA molecule includes a first RNA sequence, encoding an N protein or a functional fragment of the N protein, a second RNA sequence, encoding a P protein or a functional fragment of the P protein, and a third RNA sequence, encoding an L protein or a functional fragment of the L protein. The self-replicating RNA molecule is suitable for producing the N protein, the P protein, the L protein, or the functional fragments thereof in animal cells. Referring to
In a second aspect of the present disclosure, the present disclosure provides a DNA molecule, encoding the self-replicating RNA molecule described in any one of the preceding items.
In a third aspect of the present disclosure, the present disclosure provides an expression vector, carrying the DNA molecule as mentioned above.
In a fourth aspect of the present disclosure, the present disclosure provides a method for preparing the self-replicating RNA molecule as mentioned above. The method includes: expressing, using the DNA molecule or expression vector as mentioned above, the self-replicating RNA molecule in host cells, and collecting the self-replicating RNA molecule.
In a fifth aspect, the present disclosure provides an RNA-protein complex. The RNA-protein complex includes the self-replicating RNA molecule as mentioned above, and a protein including the N protein or functional fragment thereof, the P protein or functional fragment thereof, and the L protein or functional fragment thereof.
In a sixth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition. The pharmaceutical composition includes the self-replicating RNA molecule, DNA molecule, expression vector, or RNA-protein complex as mentioned above.
In a seventh aspect, the present disclosure provides a method for expressing a target molecule in an individual. The method includes administering to the individual the self-replicating RNA molecule, DNA molecule, expression vector, RNA-protein complex, or pharmaceutical composition as mentioned above.
Additional aspects and advantages of the present disclosure will be set forth in part in the following description, and in part will be obvious from the following description, or may be learned by practice of the present disclosure.
The above and/or additional aspects and advantages of the present disclosure will be apparent and easily understood from the description of the embodiments taken in conjunction with the following figures, in which:
FIG.4 shows RNA self-replication results of green fluorescent protein according to Example 2 of the present disclosure,
Embodiments of the present disclosure are described in detail below. Examples of which are illustrated in the accompanying figures. The embodiments described below by referring to the accompanying figures are exemplary and are intended to explain the present disclosure, but not to be construed as limitations of the present disclosure.
As used herein, the terms “include”, “including”, “comprise”, “comprising” are open expressions, that is, indicating the inclusion of the contents specified in the present disclosure, while not excluding other aspects.
As used herein, the term “optionally” or “optional” generally means that the event or situation described subsequently may but not necessarily occur, and the description includes the situation in which the event or situation occurs and the situation in which the event or situation does not occur.
As used herein, the term “self-replicating RNA molecule” may also be referred to as “self-amplifying RNA”. The self-replicating RNA molecule differs from common mRNA mainly in that the self-replicating RNA molecule uses its own RNA sequence as a template for self-replication. According to the embodiments of the present disclosure, the self-replicating RNA molecules can be translated and replicated in cytoplasm even without entering the nucleus, thereby avoiding the potential risks caused by integration with the genome. Generally, mRNA encodes the protein to be expressed, and the ribosomes in cells are used to complete the translation and protein production. According to the embodiments of the present disclosure, the self-replicating RNA molecule carries a sequence capable of expressing RNA polymerase (RNA-dependent RNA polymerase), and after the RNA molecule is translated into RNA polymerase in cytoplasm, more self-replicating RNA molecules can be generated by using the self-replicating RNA molecule as a template.
Through in-depth study on the mechanism of translation and self-amplification of RNA of various RNA viruses in cells, the Applicant found that, when the RNA molecule encoding N protein, P protein and L protein derived from Rhabdovirus is used as a core region, or when the RNA molecule encoding NSP1, NSP2, NSP3 and NP4 proteins derived from positive-strand RNA viruses as the core region, self-replication and translation of RNA can be achieved in animal cells. The core region, as a powerful “engine”, can provide “kinetic energy” for high-efficient transcription amplification and for activating macromolecular proteins. The core region can further carry a “loading region” to replicate or translate the target molecules, which cover almost all protein medicaments available on the market. According to the embodiments of the present disclosure, the “loading region” can be designed with different protein coding boxes to enable an organism to produce various peptides, enzymes, antibodies, channel proteins, receptor proteins and the like in cells, thereby achieving different therapeutic purposes, covering tumor pipelines, vaccine pipelines, rare disease pipelines and prospective general product pipelines. Therefore, the Applicant provides a novel self-replicating RNA molecule, which is referred to as reRNA™ As mentioned above, reRNA™ includes an element coding sequence capable of implementing RNA translation and self-amplification, and a carried gene sequence encoding the target molecule. According to the embodiments of the present disclosure, reRNA™, by taking the characteristics of RNA virus into consideration, can achieve the in vivo amplification from RNA to mRNA and then from mRNA to protein, thereby expressing therapeutic medicaments with high efficiency and long-term effect to achieve better clinical efficacy.
The mechanism of mRNA for treatment is to stimulate a respond of the immune system or express proteins. Therefore, the protein expression level is closely related to the therapeutic effect. However, as mentioned above, the conventional mRNA is not stable enough, and it may degrade within a few days in cells, resulting in unsustainable protein expression level. If mRNA is used for long-period disease treatment, patients may be required to be injected with a large amount of mRNA, which may intensify the toxic side effects of mRNA therapy. The self-amplify RNA at a very low dose can achieve the same protein expression level as conventional mRNA, as it can self-replicate in the cytoplasm. For example, the amount of self-replicating RNA molecules can be hundreds or even thousands of times smaller than that of conventional messenger RNA, while exerting the same immune stimulating effect and protein biological activity. The dosage and times of injection in drug therapy or immune stimulation can be reduced, thereby prolonging the therapeutic effect and reducing the potential toxic side effects of medicaments and drug delivery carriers. In addition, the self-replicating RNA molecules may form double-stranded RNA during replication, which is very similar to viral RNA during the replication process, and thus it may stimulate the innate immune response of cells and further enhance the effect of the vaccines.
The Applicant found in the research the following fact. At least the sequence encoding RNA polymerase is linked to the sequence expressing the target protein by the self-replicating RNA molecule, and thus the molecular weight of the whole mRNA molecule is much greater than that of the conventional mRNA. The excessively great molecular weight may lead to a significant decrease in delivery efficiency, translation efficiency, and replication efficiency. In order to improve these efficiencies, the Applicant conducted in-depth research, in order to find the shortest nucleic acid fragment capable of normally exerting the functions of self-replication and translation.
In an aspect of the present disclosure, the present disclosure provides a self-replicating RNA molecule. The self-replicating RNA molecule includes a first RNA sequence, a second RNA sequence, and a third RNA sequence. The first RNA sequence encodes an N protein or a functional fragment thereof. The second RNA sequence encodes a P protein or a functional fragment thereof. The third RNA sequence encodes an L protein or a functional fragment thereof. Alternatively, the self-replicating RNA molecule includes a fourth RNA sequence, and the fourth RNA sequence encodes an Nsp protein or a functional fragment thereof. The self-replicating RNA molecule is suitable for producing the N protein, the P protein, the L protein, or the functional fragments thereof in animal cells, or the self-replicating RNA molecule is suitable for producing the Nsp protein or the functional fragment thereof in animal cells. Therefore, the first, second and third RNA sequences, or the fourth RNA sequence, constitute the “core region” of the self-replicating RNA molecule, also referred to as the “core life region”. The core region can serve as the smallest region capable of implementing self-replication and translation in the animal cells. The core region can further carry other encoding sequences to express or replicate the target molecules, thereby exerting the functions of medicaments or vaccines.
The term “functional fragment” as used herein refers to a part of the full-length sequence of a protein, but this part can still play a function related to the self-replication of RNA molecules. For example, the functional fragment may be a truncated protein of the full-length sequence, or a protein of a full-length sequence in which the amino acid sequence has undergone changes such as substitutions, mutations, or deletions. According to the embodiments of the present disclosure, the functional fragment of the N protein can bind to RNA molecules to protect RNA from being affected by nuclease. The functional fragment of the P protein can bind to the N protein to locate L polymerase on the template, and it can also be used as a basic component of RNA polymerase transcription and replication complex. Further, the functional fragment of L protein can play the role of RNA polymerase, which is related to RNA transcription and replication.
According to the embodiments of the present disclosure, the biggest technical difficulty how is to simultaneously achieve the translation of RNA and self-amplification in cells. With the existing mRNA technology, the translation of RNA can be achieved in cells, but the self-amplification of RNA cannot be implemented. In order to solve this problem, the Applicant investigated the mechanism of RNA translation and self-amplification of various RNA viruses in cells to find a protein or protein complex capable of implementing the translation and self-amplification of RNA in cells. Through a lot of research work, the Applicant finally confirmed that nucleocapsid protein complex of Rhabdovirus was the optimal candidate combination. The nucleocapsid protein complex of Rhabdovirus contains N protein, P protein, and L protein. In order to determine the smallest functional unit, the Applicant used the nucleocapsid protein complex protein of vesicular stomatitis virus (VSV, Indiana) for screening. The results reveal that the N protein, P protein and L protein cannot individually implement the translation and self-amplification of reRNA™ in cells, and the combination of two proteins of NP, NL and PL also cannot implement the translation and self-amplification of reRNA™ in cells. A complex of three proteins NPL can achieve the translation and self-amplification of reRNA™ in cells. It should be noted that such a combination includes, but is not limited to, the NPL protein combinations of Vesiculorius, Lyssavirus, Ephemeroviru, non-virulent Rhabdovirus, etc. of Rhabdoviridae, and the cross-combination. In this combination, the N protein functions as an RNA-binding protein for protecting RNA from being affected by nuclease. The P protein functions as a linker protein to bind to the N protein, and locates L polymerase on the template. The P protein is also a basic component of RNA polymerase transcription and replication complex. The L protein functions as an RNA polymerase and is related to RNA transcription and replication. Those skilled in the art can understand that the N protein, P protein and L protein may be derived from different types of viruses.
According to the embodiments of the present disclosure, at least one of the N protein, the P protein, and the L protein is independently derived from Rhabdoviridae virus. According to the embodiments of the present disclosure, the Rhabdoviridae virus includes at least one virus selected from Vesiculorius, Lyssavirus, and Ephemerovirus. Vesiculorius includes, but is not limited to, Vesicular stomatitis New Jersey virus, Maraba virus, Vesicular stomatitis Alagoas virus Indiana 3, Cocal virus Indiana 2, Isfahan virus, Chandipura virus, Spring viraemia of carp virus, American bat vesiculovirus, Carajas vesiculovirus, Jurona vesiculovirus, Malpais Spring vesiculovirus, Morreton vesiculovirus, Perinet vesiculovirus, Piry vesiculovirus, Radi vesiculovirus, and Yug Bogdanovac vesiculovirus. Lyssavirus includes, but is not limited to, West Caucasian bat virus. Aravan lyssavirus, Khujand lyssavirus, Rabies virus, Australian bat lyssavirus, European bat lyssavirus 1, European bat lyssavirus 2, Irkut virus, Shimoni bat virus, Lagos bat virus, Mokola virus, Lleida bat lyssavirus, Ikoma lyssavirus, Gannoruwa bat lyssavirus, Bokeloh bat lyssavirus, Duvenhage lyssavirus. Ephemerovirus includes, but is not limited to, Bovine fever ephemerovirus, Adelaide River ephemerovirus, Berrimah ephemerovirus, and Kimberley ephemerovirus.
Thus, according to the embodiments of the present disclosure, Rhabdoviridae virus includes at least one virus selected from Vesicular stomatitis New Jersey virus, Maraba virus, Vesicular stomatitis Alagoas virus Indiana 3, Cocal virus Indiana 2, Isfahan virus, Chandipura virus, Spring viraemia of carp virus, American bat vesiculovirus, Carajas vesiculovirus, Jurona vesiculovirus, Malpais Spring vesiculovirus, Morreton vesiculovirus, Perinet vesiculovirus,
Piry vesiculovirus, Radi vesiculovirus, Yug Bogdanovac vesiculovirus, West Caucasian bat virus. Aravan lyssavirus, Khujand lyssavirus, Rabies virus, Australian bat lyssavirus, European bat lyssavirus 1, European bat lyssavirus 2, Irkut virus, Shimoni bat virus, Lagos bat virus, Mokola virus, Lleida bat lyssavirus, Ikoma lyssavirus, Gannoruwa bat lyssavirus, Bokeloh bat lyssavirus, Duvenhage lyssavirus, Bovine fever ephemerovirus, Adelaide River ephemerovirus, Berrimah ephemerovirus, and Kimberley ephemerovirus
According to the embodiments of the present disclosure, the N protein, the P protein, and the L protein may also be each independently derived from different virus serotypes of the same virus species, for example, including, but not limited to, Indiana strain, New Jersey strain, and Cocal strain of vesicular stomatitis virus (VSV).
According to the embodiments of the present disclosure, the N protein has an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, or SEQ ID NO: 25.
According to the embodiments of the present disclosure, the P protein has an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, or SEQ ID NO: 26.
According to the embodiments of the present disclosure, the L protein has an amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, or SEQ ID NO: 27.
According to the embodiments of the present disclosure, the Nsp protein has at least one of amino acid sequences as set forth in SEQ ID NO: 31 to SEQ ID NO: 34. The Applicant found that the core complex of the self-replicating RNA sequence derived from positive-strand RNA virus is nonstructural protein (Nsp).
According to some specific embodiments of the present disclosure, the self-replicating core sequence includes one of the following six combinations of base sequences encoding the proteins:
the N protein has the amino acid sequence as set forth in SEQ ID NO: 1, the P protein has the amino acid sequence as set forth in SEQ ID NO: 2, and the L protein has the amino acid sequence as set forth in SEQ ID NO: 3,
the N protein has the amino acid sequence as set forth in SEQ ID NO: 7, the P protein has the amino acid sequence as set forth in SEQ ID NO: 8, and the L protein has the amino acid sequence as set forth in SEQ ID NO: 9,
the N protein has the amino acid sequence as set forth in SEQ ID NO: 13, the P protein has the amino acid sequence as set forth in SEQ ID NO: 14, and the L protein has the amino acid sequence as set forth in SEQ ID NO: 15,
the N protein has the amino acid sequence as set forth in SEQ ID NO: 19, the P protein has the amino acid sequence as set forth in SEQ ID NO: 20, and the L protein has the amino acid sequence as set forth in SEQ ID NO: 21,
the N protein has the amino acid sequence as set forth in SEQ ID NO: 25, the P protein has the amino acid sequence as set forth in SEQ ID NO: 26, and the L protein has the amino acid sequence as set forth in SEQ ID NO: 27,
the Nsp protein has the amino acid sequence as set forth in SEQ ID NO: 31 to SEQ ID NO: 34. The Applicant found that the core complexes of self-replicating RNA sequences derived from positive-strand RNA viruses are NSP1 (SEQ ID NO: 31), NSP2 (SEQ ID NO: 32), NSP3 (SEQ ID NO: 33) and NSP4 (SEQ ID NO: 34).
According to the embodiments of the present disclosure, the first RNA sequence in the self-replicating RNA molecule has a base sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, or SEQ ID NO: 28.
According to the embodiments of the present disclosure, the second RNA sequence has a base sequence as set forth in SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 23, or SEQ ID NO: 29.
According to the embodiments of the present disclosure, the third RNA sequence has a base sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24 or SEQ ID NO: 30.
According to the embodiments of the present disclosure, the fourth RNA sequence has at least one of base sequences as set forth in SEQ ID NO: 35 to SEQ ID NO: 38.
According to some specific embodiments of the present disclosure, the self-replicating RNA molecule includes one of the following six base sequence combinations:
sequence combination 1: the first RNA sequence has the base sequence as set forth in SEQ ID NO: 4, the second RNA sequence has the base sequence as set forth in SEQ ID NO: 5, and the third RNA sequence has the base sequence as set forth in SEQ ID NO: 6,
sequence combination 2: the first RNA sequence has the base sequence as set forth in SEQ ID NO: 10, the second RNA sequence has the base sequence as set forth in SEQ ID NO: 11, and the third RNA sequence has the base sequence as set forth in SEQ ID NO: 12,
sequence combination 3: the first RNA sequence has the base sequence as set forth in SEQ ID NO: 16, the second RNA sequence has the base sequence as set forth in SEQ ID NO: 17, and the third RNA sequence has the base sequence as set forth in SEQ ID NO: 18,
sequence combination 4: the first RNA sequence has the base sequence as set forth in SEQ ID NO: 22, the second RNA sequence has the base sequence as set forth in SEQ ID NO: 23, and the third RNA sequence has the base sequence as set forth in SEQ ID NO: 24,
sequence combination 5: the first RNA sequence has the base sequence as set forth in SEQ ID NO: 28, the second RNA sequence has the base sequence as set forth in SEQ ID NO: 29, and the third RNA sequence has the base sequence as set forth in SEQ ID NO: 30, and
sequence combination 6: the fourth RNA sequence has the base sequence as set forth in SEQ ID NO: 35 to SEQ ID NO: 38. This sequence combination is a self-replicating RNA sequence derived from positive-strand RNA virus, and its core complexes are NSP1, NSP2, NSP3, and NSP4.
According to an embodiment of the present disclosure, the self-replicating RNA molecule further includes a target molecule encoding at least one target molecule.
According to the embodiments of the present disclosure, the target molecule is suitable to serve as at least one of nucleic acid medicaments, protein medicaments, pathogen vaccines, tumor vaccines, and therapeutic agents for rare diseases. Thus, by utilizing the self-amplification characteristics of reRNA™ (the self-replicating RNA molecules and self-amplifying RNA molecules are interchangeable with reRNA™ in the present disclosure), the protein medicaments can be delivered by using very low dose of reRNA™. In addition, the self-amplifying reRNA™ can greatly improve the amount of protein synthesis and achieve the long-term protein expression. Based on these characteristics, the reRNA™ technology can be applied to various aspects of life science and medicine, such as nucleic acid drug delivery, protein drug delivery, pathogen vaccines, tumor vaccines, treatment of rare diseases and so on.
According to the embodiments of the present disclosure, the coding region of the target molecule can encode at least one of fluorescent protein, granulocyte-macrophage colony stimulating factor and ovalbumin OVA. Optionally, the fluorescent protein includes at least one of green fluorescent protein, red fluorescent protein, and yellow fluorescent protein. For example, by expressing ovalbumin in tumor cells, the immune system of animals can be activated to eliminate tumor cells, thereby achieving the purpose of treating cancer.
The self-replicating RNA molecule of the present disclosure may also include one or more modified nucleotides, thereby having advantages such as improved stability, resistance to degradation and elimination in vivo. The types of the modified nucleotide include, but are not limited to, 5-methylcytidine (m5C), 5-methyluridine (m5U), N6-methyladenosine (m6A), 2-thiouridine (s2U), 2-O-methyluridine (Um), 1-methyladenosine (m1A), 2-methyladenosine (m2A), 2-1-O-methyladenosine (Am), 2-methylthio-N6-methyladenosine (ms2m6A), N6-isopentenyladenosine (16A), 2-methylthio-N6-isopentenyladenosine (ms216A), N6-(cis-hydroxy isopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxy isopentenyl)adenosine (ms2io6A), N6-glycyl carbamoyl adenosine (g6A), N6-threonyl carbamoyl adenosine (t6A), 2-methylthio-N6-threonyl carbamoyl adenosine (ms2t6A), N6-methyl-N6-threonyl carbamoyl adenosine (m6t6A), N6-hydroxy-norvalyl carbamoyl adenosine (hn6A), 2-methylthio-N6-hydroxy-norvalyl carbamoyl adenosine (ms2hn6A), 2-O-ribosyladenosine(phosphate) (Ar(p)), inosine (I), 1-methylinosine (m1I), 1,2-O-dimethylinosine (mIm), 3-methylcytidine (m3C), 2T-O-methylcytidine (Cm), 2-thiocyanine (s2C), N4-acetylcytidine (ac4C), 5-fonnylcytidine (f5C), 5,2-O-dimethylcytidine (m5Cm), N4 acetyl 2TO methyl cytidine (ac4Cm), lysidine (k2C), 1-methylguanosine (m1G), N2-methylguanosine (m2G), 7-methylguanosine (m7G), 2-O-methylguanosine (Gm), N2,N2-dimethylguanosine (m22G), N2,2-O-dimethylguanosine (m2Gm), N2,N2,2-O-trimethylguanosine (m22Gm), (2-O-ribosylguanosine (phosphate) (Gr(p)), wybutosine (yW), wybutosine peroxide (O2yW), hydroxyl wybutosine (OHyW), hydroxyl wybutosine with insufficient modification (OHyW*), wyosine (imG), methyl guanosine (mimG), queuosine (Q), epoxy queuosine (oQ), galactosyl-queuosine (galQ), mannose-queuosine (manQ), 7-cyano-7-deaza guanosine (preQo), 7-aminomethyl-7-deaza guanosine (preQi), gupurine glycoside (G*), dihydrouridine (D), 5,2-O-dimethyluridine (m5Um), 4-thiouridine (s4U), 5-methyl-2-thiouridine (m5s2U), 2-sulfur-2-O-methyluridine (s2Um), 3-(3-amino-3-carboxypropyl) uridine (acp3U), 5-hydroxyuridine (ho5U), 5-methoxyuridine (mo5U), uridine 5-oxoacetic acid (cmo5U), methyl uridine 5-oxoacetate (mcmo5U), 5-(carboxyhydroxymethyl) uridine (chm5U), 5-(methyl carboxyl hydroxymethyl) uridine (mchm5U), 5-methoxycarbonyl methyl uridine (mcm5U), S-methoxycarbonylmethyl-2-O-methyluridine (mcm5Um), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5-methylaminomethyl uridine (mnm5U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyl-2-seleno uridine (mnm5se2U), 5-carbamoylmethyl uridine (ncm5U), 5-carbamoylmethyl-2-O-methyluridine (ncm5Um), 5-carboxymethyl aminomethyl uridine (cmnm5U), 5-carboxymethyl aminomethyl-2-L-O methyluridine (cnmm5Um), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), N6, N6-dimethyladenosine (m62A), 2-O-methylinosine (Tm), N4-methylcytidine (m4C), N4,2-O-dimethylcytidine (m4Cm), 5-hydroxymethylcytidine (hm5C), 3-methyluridine (m3U), 5-carboxymethyluridine (cm5U), N6, T-O-dimethyladenosine (m6Am), N6,N6,O-2-trimethyladenosine (rn62Am), N2,7-dimethylguanosine (m27G), N2,N2,7-trimethylguanosine (m227G), 3,2T-O-dimethyluridine (m3Um), 5-methyldihydrouridine (m5D), 5-formyl-2-O-methyl cytidine (f5Cm), 1,2-O-dimethylguanosine (m1Gm), 1,2-O-dimethyladenosine irinomethyluridine (mAm), S-taurino-methyl-2-thiouridine (tm5s2U), 4-demethylguanosine (imG-14), isoguanosine (imG2), N6-acetyladenosine (ac6A), hypoxanthine, inosine, 8-oxo-adenine, and the 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyl uracil, 5-methyl uracil, 5-(C2-C6)-alkenyl uracil, 5-(C2-C6)-alkynyl uracil, 5-(hydroxymethyl) uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-denitrifying guanine, 8-azaguanine, 7-denitrogenation-7-substituted guanine, 7-denitrogenation-7-(C2-C6) alkynyl guanine, 7-denitrogenation-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-denitrifying purine, 7-azapurine-7-substituted purine, 7-azapurine-8-substituted purine, hydrogen (debased residue), m5C, m5U, m6A, s2U, W, or 2-O-methyl-U.
In a second aspect of the present disclosure, the present disclosure provides a DNA molecule encoding the self-replicating RNA molecule described in any one of the preceding items. Those skilled in the art can understand that nucleic acids encoding self-replicating RNA molecules can be provided to cells in the form of DNA. In this way, the corresponding self-replicating RNA molecules can be synthesized in cells, and further, the self-replicating RNA molecules can be replicated and translated into corresponding proteins and corresponding target molecules in cells.
It should be noted that, for the DNA molecule mentioned in the specification and claims of the present disclosure, those skilled in the art should understand that the DNA molecule actually includes any one or two complementary double strands. For convenience, in this specification and the claims, although only one strand is given in most cases, another strand complementary to the mentioned one is actually disclosed. In addition, the specific sequences of DNA molecules in the present disclosure can be obtained according to the sequences of RNA molecules and the principle of base complementary pairing, and disclosure of one of them means that the other is also disclosed.
In a third aspect of the present disclosure, the present disclosure provides an expression vector carrying the aforementioned DNA molecule. The expression vector here is not particularly limited to a specific type, as long as it can replicate and express the corresponding RNA molecule in the host cells. As mentioned above, the nucleic acids encoding the self-replicating RNA molecules can be provided to cells in the form of DNA, allowing the corresponding self-replicating RNA molecules to be synthesized in cells, and further, the self- replicating RNA molecules can be replicated and translated into the corresponding proteins and target molecules in cells.
When the aforementioned DNA molecule is linked to the expression vector, it can be directly or indirectly linked to the control elements on the expression vector, as long as these control elements can control the translation and expression of the DNA molecule, that is, the DNA molecule is operably linked to the control elements. Of course, these control elements can be directly from the vector itself, or they can be exogenous, that is, not from the vector itself.
Herein, “operably linked” refers to a foreign gene is linked to a vector, enabling the control elements in the vector such as transcription control sequences and translation control sequences, etc. to exert their expected functions of regulating the transcription and translation of an exogenous gene. Common vectors can be, for example, plasmids, phages and the like.
In a fourth aspect of the present disclosure, the present disclosure provides a method for preparing the aforementioned self-replicating RNA molecule. The method includes: expressing, using the aforementioned DNA molecules or expression vectors, the aforementioned self-replicating RNA molecule in host cells; and collecting the self-replicating RNA molecule.
According to the embodiments of the present disclosure, by adopting the DNA molecules and expression vectors, the self-replicating RNA molecules expressed in the host cells such as animal cells usually are present in the form of complexes. For example, referring to
In a fifth aspect of the present disclosure, referring to
In a sixth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition, which includes the aforementioned self-replicating RNA molecule, DNA molecule, expression vector, or RNA-protein complex.
In a seventh aspect of the present disclosure, the present disclosure provides a method for expressing a target molecule in an individual. The method includes: administering to the individual the aforementioned self-replicating RNA molecule, DNA molecule, expression vector, RNA-protein complex, or pharmaceutical composition.
Additional aspects and advantages of the present disclosure will be set forth in part in the following description, and in part will be obvious from the following description, or may be learned by practice of the present disclosure. The specific techniques or conditions that are not specifically indicated in the present disclosure are shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specification. The used reagents or instruments without indicating the manufacturers thereof are all conventional and commercially available products.
In the present example, according to the information in the GeneBank database, the DNA sequence (base sequence as set forth in SEQ ID NO: 39) encoding N protein of vesicular stomatitis virus (VSV) Indiana strain (IND), the DNA sequence (base sequence as set forth in SEQ ID NO: 40) encoding P protein of vesicular stomatitis virus (VSV) Indiana strain (IND), the DNA sequence (base sequence as set forth in SEQ ID NO: 41) encoding L protein of vesicular stomatitis virus (VSV) Indiana strain (IND), and the DNA sequence encoding green fluorescent protein (GFP) were obtained. The amino acid sequences of the N protein, P protein, and L protein of the vesicular stomatitis virus (VSV) Indiana strain (IND) obtained after expression and translation were as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. Specifically, reRNA™ DNA sequences carrying the following sequences were constructed respectively:
IND-P-GFP;
It should be noted that, unless otherwise specified in the present disclosure, N represents the coding sequence of the N protein, P represents the coding sequence of the P protein, L represents the coding sequence of the L protein, IND represents that N, P or L followed by is derived from the Indian strain of vesicular stomatitis virus (VSV), NJ represents that N, P or L followed by is derived from the New Jersey strain of vesicular stomatitis virus (VSV), and GFP represents the coding sequence of the green fluorescent protein.
Furthermore, 293T cells were transfected in vitro with the DNA sequences constructed above, and cultured for 24 hours after the transfection. It was determined by observing the fluorescence signal of the green fluorescent protein whether the GFP coding sequence was correctly translated and expressed. The results were shown in
In vitro delivery based on reRNA™ was performed on 293T cells in the similar manner as described in Example 1. The present example merely differed from Example 1 in that, the reRNA™ RNA sequence carrying at least one of the RNA sequence (base sequence as set forth in SEQ ID NO: 4) encoding the N protein of the Indian strain (IND) of the vesicular stomatitis virus (VSV), the RNA sequence (base sequence as set forth in SEQ ID NO: 5) encoding the P protein of the Indian strain (IND) of the vesicular stomatitis virus (VSV), and the RNA sequence (base sequence as set forth in SEQ ID NO: 6) encoding the L protein of the Indian strain (IND) of the vesicular stomatitis virus (VSV) was constructed. The specific reRNA™ RNA sequences were as follows:
The 293T cells were cultured for 24 hours to observe the fluorescence signal of green fluorescent protein. The results were shown in
According to the information in the GeneBank database, the RNA sequence (base sequence as set forth in SEQ ID NO: 16) encoding N protein of New Jersey strain (NJ) of vesicular stomatitis virus (VSV), the RNA sequence (base sequence as set forth in SEQ ID NO: 17) encoding P protein of New Jersey strain (NJ) of vesicular stomatitis virus (VSV), and the RNA sequence (base sequence as set forth in SEQ ID NO: 18) encoding L protein of New Jersey strain (NJ) of vesicular stomatitis virus (VSV) were further obtained, wherein, the amino acid sequences of N protein, P protein and L protein of the New Jersey strain (NJ) of vesicular stomatitis virus (VSV) obtained by expression of the above coding sequence were as set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively. In vitro delivery based on reRNA™ was performed on 293T cells in the similar manner as described in Example 1 In this example, reRNA™ RNA molecules loaded with GFP were prepared, and the coding genes of N, P and L proteins in the core regions were respectively derived from different combinations of the above-mentioned Indian strain (IND) of vesicular stomatitis virus in Example 2 and the above-mentioned New Jersey strain (NJ) of vesicular stomatitis virus. Specifically, the following reRNA™ RNA sequences carrying the following sequences were constructed (the following RNA sequences all contain GFP coding sequence, which is not present for convenience of description):
The same dose of the above-mentioned reRNA™ was introduced into 293T cells, and the cells were cultured for 24 hours after transfection. The expression of loaded gene GFP was confirmed by observing the fluorescence signal, and the self-replication of GFP was confirmed by qPCR.
The experimental results were shown in
In this example, the Applicant found that any combination of N, P and L proteins of the above three strains of vesicular stomatitis virus can effectively initiate the expression of loaded gene (GFP). Specifically, according to the information in GeneBank database, the coding gene (base sequence as set forth in SEQ ID NO: 23) of P protein of the Cocal strain (COC) of vesicular stomatitis virus (VSV) was further acquired, and the obtained amino acid sequence of P protein of the Cocal strain (COC) of vesicular stomatitis virus (VSV) was as set forth in SEQ ID NO: 20. In vitro delivery based on reRNA™ was performed on 293T cells in substantially the same manner as described in Example 2. In the present example, reRNA™ RNA molecules containing GFP and N protein derived from the New Jersey strain (NJ) of vesicular stomatitis virus described in Example 3, P protein derived from the Cocal strain (COC), and L protein derived from the Indiana strain (IND) described in Examples 1 and 2 were prepared as examples to carry out the experiment.
The above-mentioned reRNA™ was introduced into 293T cells, and the cells were cultured for 24 hours after transfection, and the expression of GFP was confirmed by observing the fluorescence signal.
The experimental results were shown in
In vitro delivery based on reRNA™ was performed on 293T cells in substantially the same manner as described in Example 2. In this example, the reRNA™ RNA molecules containing the loaded GFP were prepared, and the NPL genes in the core region were derived from any one of different combinations of vesicular stomatitis virus and rabies virus. The amino acid sequences of N protein, P protein, and L protein of Rabies virus were as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively. The RNA sequences encoding the N protein, P protein and L protein of Rabies virus were as set forth in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.
Different reRNA™ with the same dose were introduced into 293T cells, and the cells were cultured for 24 hours after transfection. The expression of loaded gene GFP was confirmed by observing the fluorescence signal. The results indicate that different combinations of NPL derived from different Rhabdoviruses can initiate the expression of loaded gene (GFP).
In vitro delivery based on reRNA™ was performed on 293T cells in substantially the same manner as described in Example 2. In this example, the reRNA™ molecules containing loaded GFP were prepared, and the NPL in the core region carried different conservative mutation of RNA sequences encoding N, P, and L proteins, as set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 respectively. The amino acid sequences of the obtained N, P, and L proteins were as set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 12, respectively. The 293T cells were transfected with the same dose of different reRNA™ and the cells were cultured for 24 hours after transfection. The expression of loaded gene GFP was confirmed by observing the fluorescence signal. The results indicate that all the different NPL combinations carrying conservative mutations can initiate the expression of GFP.
In vitro delivery based on reRNA™ was performed on mouse MC38 cells in substantially the same manner as described in Example 2. The present example differed from Example 2 merely in that: reRNA™ containing loaded mGM-CSF (mouse granulocyte-macrophage colony stimulating factor) was prepared, and the core region was NPL (derived from Indian strain (IND) of vesicular stomatitis virus described in Examples 1 and 2). The reRNA™ loaded with mGM-CSF was introduced into mouse MC38 cells, and the cell culture supernatant was collected at different times to detect mGM-CSF secretion. The results indicate that it can effectively initiate the expression of mGM-CSF and make it secrete to the outside of the cells.
The reRNA™ (reRNA™ OVA group) and reRNA™ (Blank group) containing loaded RNA sequences encoding OVA antigen were prepared. The core region of reRNA™ was NPL (derived from Indian strain (IND) of vesicular stomatitis virus described in Example 1 and Example 2), and the amino acid sequence of OVA antigen obtained by expression was as set forth in SEQ ID NO: 42. Specifically, reRNA™ was introduced into C57 mouse spleen cells, and the mouse spleen cells were recovered after 24 hours of culture. The antigen-presenting cells were tested for OVA antigen presentation by flow cytometry.
The experimental results were shown in
In the specification, descriptions referring to the terms “an embodiment”, “some embodiments”, “examples”, “specific examples” or “some examples” mean that specific features, structures, materials or characteristics described in connection with this embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and integrate different embodiments or examples and features of different embodiments or examples described in this specification without contradicting each other.
Although the embodiments of the present disclosure have been illustrated and described above, it can be understood that the above embodiments are exemplary and should not be understood as limitations of the present disclosure. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111178106.7 | Oct 2021 | CN | national |
This application a continuation of International Application No. PCT/CN2022/124192, filed on Oct. 9, 2022, which claims the priority and benefit of the patent application Ser. No. 20/211,1178106.7 filed with China National Intellectual Property Administration on Oct. 9, 2021. The disclosures of the aforementioned applications are hereby incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/124192 | Oct 2022 | WO |
| Child | 18630513 | US |
