TECHNICAL FIELD

The present invention relates to a method for functionalizing mRNA. More specifically, the present invention relates to a method for stabilizing an mRNA carrier, an mRNA vaccine, and the like.

BACKGROUND ART

mRNA delivery has attracted attention as a method of supplying a therapeutic protein into a body safely and continuously. On the other hand, such mRNA delivery is greatly problematic in that enzymatic degradation of mRNA promptly occurs in a living body. To solve this issue, a method of protecting mRNA from degradation by using an mRNA carrier and a method of improving an mRNA molecule itself have been studied. However, since the action of RNA-degrading enzyme is extremely strong in a living body, further technical innovation has been desired.

Regarding the method of improving an mRNA molecule itself, a method of replacing some mRNA nucleotides with chemically modified nucleotides has been studied (for example, Non Patent Literature 1). To date, various chemically modified nucleotides have been comprehensively studied. However, chemically modified nucleotides capable of significantly improving the enzyme resistance of mRNA have not yet been reported. Moreover, since the efficiency of translating a protein from mRNA is often largely decreased by performing modification on the nucleotides, it has been difficult to perform chemical modification, as desired.

Patent Literature 1 discloses a polyion complex consisting of a polycationic polymer and an mRNA, and the use of the polyion complex in mRNA delivery. In addition, Non Patent Literature 2 discloses the stability of a polymeric micelle-type mRNA carrier, evaluation of enzymatic degradation, and the effect of performing cholesterol modification on the polymer. However, these publications do not disclose a method for stabilizing an mRNA carrier, by which the enzyme resistance of an mRNA can be improved in a living body, without largely decreasing the efficiency of translating a protein from the mRNA.

The conventional live vaccine, in which the pathogenicity of a pathogen has been attenuated, is likely to cause side effects as a result of the reactivation of the pathogenicity. The conventional inactivated vaccine, in which the pathogenicity of a pathogen has been lost, is problematic in that cellular immunity is hardly induced because infected cells do not appear.

In the case of a nucleic acid vaccine such as a DNA vaccine or an mRNA vaccine, a nucleic acid is used to present an antigenic protein. That is to say, in the case of such a nucleic acid vaccine, the administered nucleic acid is transferred into the nucleus or cytoplasm of an antigen-presenting cell and an antigenic protein is then allowed to express and to be present therein, so as to activate immunity. Since the nucleic acid vaccine does not have a problem regarding the reactivation of the pathogenicity, the nucleic acid vaccine is considered to have higher safety, in comparison to the live vaccine. Moreover, since the antigen-presenting cell presents an antigenic protein in the nucleic acid vaccine, it is possible to induce cellular immunity. Thus, the nucleic acid vaccine can be applied to cancer or chronic infection. Furthermore, in the case of the nucleic acid vaccine, it is possible to relatively freely design a nucleic acid only by changing the sequence, and thus, the nucleic acid vaccine is advantageous in that it can be used in the personalized treatment of cancer, and in that it can quickly respond to virus mutation, so that it can quickly cope with pandemic, and the like.

Among these vaccines, the DNA vaccine is considered to have the risk of causing mutagenesis due to random insertion into the host genome. In contrast, the mRNA vaccine, which uses an mRNA for antigenic protein presentation, is considered not to have the risk of causing mutagenesis to the host genome, and thus, the mRNA vaccine has attracted attention in recent years.

In order to obtain immune induction effects by using an mRNA vaccine, it is necessary to allow an antigenic protein to express, and at the same time, to provoke an inflammatory response. However, since the mRNA itself has low immunogenicity, it hardly provokes the inflammatory response. Hence, it has conventionally been necessary to simultaneously administer an adjuvant for provoking the inflammatory response, as well as the mRNA (for example, Patent Literature 2).

Such a conventional mRNA vaccine has the following three problems. The first problem is that when a foreign matter is used as an adjuvant, considerable attention should be paid to the safety of the foreign matter. The second problem is that if the administered mRNA is different from the adjuvant in terms of histological distribution, it is likely that a sufficient inflammatory response is not provoked in a cell that expresses an antigenic protein. The third problem is that upon administration of the mRNA into a living body, a carrier is often necessary to protect the mRNA from enzymatic degradation, but the adjuvant is likely to affect the function of the carrier.

Non Patent Literature 3 discloses, as an mRNA vaccine, an mRNA that is integrated with protamine as an adjuvant component.

CITATION LIST

Patent Literature

Patent Literature 1: WO 2015/121924 APatent Literature 2: JP-2012-502074-A

Non Patent Literature

Non Patent Literature 1: Meis, J. E. and Chen, F. (2002) EPICENTRE Forum 9(1), 10Non Patent Literature 2: Uchida, S., et al.,Biomaterials(2016) 82, pp. 221-228.Non Patent Literature 3: Karl-Josef Kallen et al., Human Vaccines & Immunotherapeutics (2013) 9: 10, pp. 2263-2276

SUMMARY OF INVENTION

Technical Problem

Under such circumstances, it has been desired to develop a functionalized mRNA.

In a first aspect, it is an object of the present invention to provide a novel method for stabilizing an mRNA carrier.

In second and third aspects, it is an object of the present invention to provide a novel mRNA vaccine enabling efficient protein expression and immune induction.

Solution to Problem

As a result of intensive studies, the present inventors have found that an mRNA can be functionalized by allowing an RNA oligomer having a sequence complementary to an mRNA to hybridize with the mRNA, thereby completing the present invention.

In addition, the present inventors have found that an mRNA can be chemically modified by chemically modifying an RNA oligomer having a sequence complementary to the mRNA, and then allowing the RNA oligomer to hybridize with the mRNA, so that the mRNA can be stabilized, or the mRNA is incorporated into a carrier, so that the carrier can be stabilized, thereby completing a first aspect of the present invention.

Moreover, the present inventors have found that both efficient protein expression from mRNA and immune induction can be achieved by allowing an RNA oligomer having a sequence complementary to the poly A sequence of an mRNA encoding an antigen to hybridize with the mRNA, thereby completing a second aspect of the present invention.

Furthermore, the present inventors have found that both efficient protein expression from mRNA and immune induction can be achieved by allowing a first RNA oligomer having a sequence complementary to the sequence of an mRNA encoding an antigen to hybridize with the mRNA, and also by allowing a second RNA oligomer having a sequence complementary to the first RNA oligomer to hybridize with the first RNA oligomer, thereby completing a third aspect of the present invention.

The present invention is as follows.

A functionalized mRNA, which comprises a double-stranded RNA comprising an mRNA and at least one modified RNA oligomer hybridizing with the mRNA.

The first aspect of the present invention is as follows.

[1-1] An mRNA carrier, which loads therein an mRNA encoding a target gene and at least one RNA oligomer hybridizing with the mRNA, wherein

the RNA oligomer comprises:

(a) an RNA sequence consisting of a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA, or

(b) an RNA sequence having an identity of 90% or more to a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA, and hybridizing with the mRNA, and the RNA oligomer is chemically modified.

[1-1A] An mRNA carrier, which loads therein an mRNA encoding a target gene and at least one RNA oligomer hybridizing with the mRNA, wherein

the RNA oligomer comprises:

(a) an RNA sequence consisting of a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA, or

(b) an RNA sequence having an identity of 90% or more to a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA, and hybridizing with the mRNA, and the RNA oligomer is chemically unmodified or is chemically modified.

[1-2] The carrier according to the above [1-1] or [1-1A], wherein the RNA sequence consists of a sequence of 15 to 23 nucleotides.

[1-3] The carrier according to the above [1-2], wherein the RNA sequence consists of a sequence of 17 nucleotides.

[1-4] The carrier according to any one of the above [1-1] to [1-3], wherein the chemical modification is performed on the 5′-terminus or 3′-terminus of the sequence of the RNA oligomer via an overhang sequence consisting of 1 to 5 nucleotides.

[1-5] The carrier according to the above [1-4], wherein the overhang sequence is a sequence consisting of 2 nucleotides.

[1-6] The carrier according to any one of the above [1-1] to [1-5], wherein the chemical modification is modification with a hydrophobic group.

[1-7] The carrier according to the above [1-6], wherein the modification with a hydrophobic group is cholesterol modification.

[1-8] The carrier according to any one of the above [1-1] to [1-5], wherein the chemical modification is polyethylene glycol modification.

[1-9] The carrier according to any one of the above [1-1] to [1-8], wherein the carrier is a polymeric micelle or a lipidic mRNA carrier.

[1-10] A pharmaceutical composition comprising the carrier according to any one of the above [1-1] to [1-9].

[1-11] A method for stabilizing a carrier, which comprises techniques allowing a carrier to load therein an mRNA encoding a target gene and at least one RNA oligomer hybridizing with the mRNA, wherein

the RNA oligomer comprises:

(a) an RNA sequence consisting of a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA, or

(b) an RNA sequence having an identity of 90% or more to a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA, and hybridizing with the mRNA, and the RNA oligomer is chemically modified.

[1-11A] A method for stabilizing a carrier, which comprises techniques allowing a carrier to load therein an mRNA encoding a target gene and at least one RNA oligomer hybridizing with the mRNA, wherein

the RNA oligomer comprises:

(a) an RNA sequence consisting of a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA, or

(b) an RNA sequence having an identity of 90% or more to a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA, and hybridizing with the mRNA, and the RNA oligomer is chemically unmodified or is chemically modified.

[1-12] A double-stranded RNA comprising an mRNA encoding a target gene and at least one RNA oligomer hybridizing with the mRNA, wherein

the RNA oligomer comprises:

(a) an RNA sequence consisting of a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA, or

(b) an RNA sequence having an identity of 90% or more to a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA, and hybridizing with the mRNA, and the RNA oligomer is chemically unmodified or is chemically modified.

[1-13] The double-stranded RNA according to the above [1-12], wherein the RNA sequence consists of a sequence of 15 to 23 nucleotides.

[1-14] The double-stranded RNA according to the above [1-13], wherein the RNA sequence consists of a sequence of 17 nucleotides.

[1-15] The double-stranded RNA according to any one of the above [1-12] to [1-14], wherein the chemical modification is performed on the 5′-terminus or 3′-terminus of the sequence of the RNA oligomer via an overhang sequence consisting of 1 to 5 nucleotides.
[1-16] The double-stranded RNA according to the above [1-15], wherein the overhang sequence is a sequence consisting of 2 nucleotides.
[1-1.7] The double-stranded RNA according to any one of the above [1-12] to [1-16], wherein the chemical modification is modification with a hydrophobic group.
[1-18] The double-stranded RNA according to the above [1-17], wherein the modification with a hydrophobic group is cholesterol modification.
[1-19] The double-stranded RNA according to any one of the above [1-12] to [1-16], wherein the chemical modification is polyethylene glycol modification.
[1-20] An mRNA carrier loading therein the double-stranded RNA according to any one of the above [1-12] to [1-19].
[1-21] The carrier according to the above [1-20], which is a polymeric micelle or a lipidic mRNA carrier.
[1-22] A pharmaceutical composition comprising the carrier according to the above [1-20] or [1-21].

The second aspect of the present invention is as follows.

[2-1] An mRNA vaccine, which comprises a double-stranded RNA consisting of an mRNA encoding an antigen and at least one RNA oligomer hybridizing with at least the poly A sequence of the mRNA, wherein the at least one RNA oligomer is chemically unmodified.

[2-1A] An mRNA vaccine, which comprises a double-stranded RNA consisting of an mRNA encoding an antigen and at least one RNA oligomer hybridizing with at least the poly A sequence of the mRNA, wherein the at least one RNA oligomer is chemically umnodified or is chemically modified.
[2-2] The mRNA vaccine according to the above [2-1] or [2-1A], wherein the RNA oligomer consists of a nucleotide sequence of 10 to 500 nucleotides.
[2-3] The mRNA vaccine according to the above [2-1] or [2-2], wherein the RNA oligomer has a triphosphoric acid structure at the 5′-terminus thereof.
[2-4] The mRNA vaccine according to any one of the above [2-1] to [2-3], wherein the double-stranded RNA is formed by allowing one RNA oligomer to hybridize with at least the poly A sequence of the mRNA.
[2-5] The mRNA vaccine according to any one of the above [2-1] to [2-4], wherein the double-stranded RNA is in a naked form.
[2-6] The mRNA vaccine according to any one of the above [2-1] to [2-5], which is not used together with an adjuvant.
[2-7] The mRNA vaccine according to any one of the above [2-1] to [2-6], which is for use in the prevention or treatment of a disease in a subject in need thereof.
[2-8] A method for preventing or treating a disease, comprising administering the mRNA vaccine according to any one of the above [2-1] to [2-6] to a subject in need of the prevention or treatment of the disease.

The third aspect of the present invention is as follows.

[3-1] An mRNA vaccine, which comprises a double-stranded RNA consisting of an mRNA encoding an antigen, at least one first RNA oligomer hybridizing with the mRNA, and a second RNA oligomer hybridizing with the first RNA oligomer, wherein

the first RNA oligomer comprises:

(a) an RNA sequence comprising a first RNA sequence consisting of a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA, and a second RNA sequence consisting of a sequence of 10 to 200 nucleotides complementary to the sequence of the second RNA oligomer, in this order from the 5′-terminus thereof,

(b) an RNA sequence comprising a first RNA sequence having an identity of 90% or more to a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA and hybridizing with the mRNA, and a second RNA sequence having an identity of 90% or more to a sequence of 10 to 200 nucleotides complementary to the sequence of the second RNA oligomer and hybridizing with the second RNA oligomer, in this order from the 5′-terminus thereof,

(c) an RNA sequence comprising a second RNA sequence consisting of a sequence of 10 to 200 nucleotides complementary to the second RNA oligomer, and a first RNA sequence consisting of a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA, in this order from the 5′-terminus thereof, or

(d) an RNA sequence comprising a second RNA sequence having an identity of 90% or more to a sequence of 10 to 200 nucleotides complementary to the sequence of the second RNA oligomer and hybridizing with the second RNA oligomer, and a first RNA sequence having an identity of 90% or more to a sequence of 12 to 40 nucleotides complementary to the sequence of the mRNA and hybridizing with the mRNA, in this order from the 5′-terminus thereof.

[3-2] The mRNA vaccine according to the above [3-1], wherein the first RNA oligomer consists of a sequence of 22 to 240 nucleotides.

[3-3] The mRNA vaccine according to the above [3-1] or [3-2], wherein the number of the first RNA oligomers to be allowed to hybridize with one mRNA is 1 to 50.

[3-4] The mRNA vaccine according to any one of the above [3-1] to [3-3], wherein the first RNA oligomer comprises the RNA sequence (a) or the RNA sequence (b), and the second RNA oligomer has a triphosphoric acid structure at the 5′-terminus thereof.

[3-5] The mRNA vaccine according to any one of the above [3-1] to [3-3], wherein the first RNA oligomer comprises the RNA sequence (c) or the RNA sequence (d), and the first RNA oligomer has a triphosphoric acid structure at the 5′-terminus thereof.

[3-6] The mRNA vaccine according to any one of the above [3-1] to [3-5], wherein the terminus of the double-stranded RNA on the side at which the second RNA oligomer hybridizes with the first RNA oligomer is a blunt end.

[3-7] The mRNA vaccine according to any one of the above [3-1] to [3-6], wherein the second RNA oligomer comprises a sequence of 10 to 200 nucleotides.

[3-8] The mRNA vaccine according to any one of the above [3-1] to [3-7], wherein the double-stranded RNA is in a naked form.

[3-9] The mRNA vaccine according to any one of the above [3-1] to [3-8], which is not used together with an adjuvant.

[3-10] The mRNA vaccine according to any one of the above [3-1] to [3-9], which is for use in the prevention or treatment of a disease in a subject in need thereof.

Advantageous Effects of Invention

The present invention can provide a functionalized mRNA.

The first aspect of the present invention provides a novel method for stabilizing an mRNA or an mRNA carrier. Preferably, the first aspect of the present invention can realize relatively flexible mRNA modification, while maintaining the efficiency of translating a protein from the mRNA in a living body. More preferably, the first aspect of the present invention can suppress enzymatic degradation of the mRNA in a living body.

The second and third aspects of the present invention provide a novel mRNA vaccine capable of efficient protein expression and immune induction. Preferably, the mRNA vaccine according to the second and third aspects of the present invention enables antigen presentation and immune induction, without simultaneous administration of an adjuvant. More preferably, the mRNA vaccine according to the second and third aspects of the present invention can induce stronger cellular immunity. Further preferably, the mRNA vaccine according to the second and third aspects of the present invention can induce stronger humoral immunity than in the case of single administration of the mRNA.

