CN114717229B

CN114717229B - Cell-free and vector-free in vitro RNA transcription of therapeutic mRNA and nucleic acid molecules

Info

Publication number: CN114717229B
Application number: CN202110008455.8A
Authority: CN
Inventors: 邝纬阳; 林庭匡
Original assignee: Massena Therapy Hong Kong Ltd
Current assignee: Massena Therapy Hong Kong Ltd
Priority date: 2021-01-05
Filing date: 2021-01-05
Publication date: 2024-09-10
Anticipated expiration: 2041-01-05
Also published as: WO2022148131A1; CN114717229A

Abstract

The present invention relates to cell-free and vector-free in vitro RNA transcription methods and nucleic acid molecules for therapeutic mRNA. More specifically, the invention provides a nucleic acid molecule comprising, from the 5 'end to the 3' end, a 5 '-cap structure, a 5' -untranslated region (5 '-UTR) of human β -globin, at least one coding region, a 3' -untranslated region (3 '-UTR) of human α -globin, and a 3' -polyadenylation tail, said at least one coding region being operably linked to said 5'-UTR and said 3' -UTR, and an in vitro transcription method. The in vitro transcription methods and nucleic acid molecules of the invention not only enable maximum yields of mRNA of up to about 2.3mg/mL per hour or even higher to be obtained, but also the resulting mRNA exhibits enhanced gene expression stability and translation efficiency.

Description

Cell-free and vector-free in vitro RNA transcription of therapeutic mRNA and nucleic acid molecules

Technical Field

The present disclosure relates generally to the field of molecular biology, and more particularly to cell-free and vector-free in vitro RNA transcription methods of mRNA and nucleic acid molecules.

Background

Messenger RNA (mRNA) is a relatively new therapeutic molecule that has broad clinical application potential, including cancer treatment, vaccine, and regenerative therapies. Advantages of mRNA-based drugs compared to recombinant protein-based drugs include: 1. the production is cost-effective; 2. longer treatment effect; 3. rapid synthesis and purification; 4. no endotoxin and infectious agent; 5. post-translational modification is advantageous. mRNA is also a very beneficial alternative to gene therapy because it does not integrate the gene into the genome, does not require entry into the nucleus, and expression is directly controllable. In general, mRNA-based therapies are safe and cost-effective.

Although mRNA-based therapies are promising, mRNA stability and the ability to deliver mRNA to cells are poor. Thus, there remains a need in the art for improved methods of in vitro RNA transcription as well as more stable mRNA.

Disclosure of Invention

In one aspect, the present disclosure provides an RNA nucleic acid molecule comprising or consisting of, from the 5 'end to the 3' end: a 5 '-cap structure, a human β -globin 5' untranslated region (5 '-UTR), at least one coding region, a human α -globin 3' untranslated region (3 '-UTR), and a 3' -poly a tail, said at least one coding region operably linked to said 5'-UTR and said 3' -UTR.

In one embodiment, the coding region encodes at least one polypeptide or protein of interest, optionally selected from a growth factor, an antigenic polypeptide or protein, an allergenic polypeptide or protein, a therapeutic polypeptide or protein, or a fragment, variant or derivative of the foregoing.

In one embodiment, the coding region further encodes at least one selected from the group consisting of: signal peptide, peptide tag or protein tag, localization signal or localization sequence, and peptide linker.

In one embodiment, the 5' -UTR comprises or consists of a sequence according to SEQ ID NO:1, or with an RNA sequence according to SEQ ID NO:1, has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

In one embodiment, the 3' -UTR comprises or consists of a sequence according to SEQ ID NO:2, or with an RNA sequence according to SEQ ID NO:2, has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

In one embodiment, the 5' -cap structure is a 5' -anti-reverse cap analogue (5 ' -ARCA), preferably the 5' -ARCA is 7m G (3 ' -O-Me) pppG.

In one embodiment, the 3' -poly a tail comprises 10 to 200, 20 to 100, 40 to 80, or 50 to 70 adenine nucleotides.

In another aspect, the present disclosure provides a DNA nucleic acid molecule comprising or consisting of, from the 5 'end to the 3' end: a promoter, a human β -globin 5 '-untranslated region (5' -UTR), at least one coding region, a human α -globin 3 '-untranslated region (3' -UTR), and a transcription terminator, said at least one coding region being operably linked to said 5'-UTR and said 3' -UTR.

In one embodiment, the coding region encodes at least one polypeptide or protein of interest, optionally selected from a peptide growth factor, an antigenic polypeptide or protein, an allergenic polypeptide or protein, a therapeutic polypeptide or protein, or a fragment, variant or derivative of the foregoing.

In one embodiment, the promoter is selected from the group consisting of T3, T7, sny, or SP6 promoters.

In one embodiment, the promoter is selected from the group consisting of a T3 promoter and the transcription terminator is selected from the group consisting of a T7 transcription terminator.

In one embodiment, the 5' -UTR comprises or consists of a sequence according to SEQ ID NO:3, or with a DNA sequence according to SEQ ID NO:3, has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

In one embodiment, the 3' -UTR comprises or consists of a sequence according to SEQ ID NO:4, or with a DNA sequence according to SEQ ID NO:4, has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

In one embodiment, the DNA nucleic acid molecule further comprises a first restriction site located upstream of the promoter and a second restriction site located downstream of the transcription terminator.

In one embodiment, the DNA nucleic acid molecule further comprises a third restriction site located between the promoter and the 5'-UTR and a fourth restriction site located between the 3' -UTR and the transcription terminator.

In yet another aspect, the present disclosure provides an in vitro transcription method comprising the steps of:

(a) Providing a DNA nucleic acid molecule according to the present disclosure as a transcription template;

(b) Optionally, amplifying the DNA nucleic acid molecule;

(c) The DNA nucleic acid molecule is amplified in 5' -anti-reverse cap analogue (5 ' -ARCA), preferably 7m G (3 ' -O-Me) pppG

Performing in vitro transcription in the presence of a reagent to obtain a reaction mixture comprising 5' -ARCA-terminated mRNA;

(d) Optionally, removing the transcription template from the reaction mixture comprising 5' -ARCA-capped mRNA by adding dnase; and

(E) Adding a polyadenylic acid polymerase reaction mixture to the reaction mixture comprising 5 '-ARCA-capped mRNA for 3' -polyadenylic acid tail addition to obtain a 5 '-ARCA-capped mRNA having a 3' -polyadenylic acid tail.

In yet another aspect, the present disclosure provides a composition comprising an RNA nucleic acid molecule according to the present disclosure and/or an RNA nucleic acid molecule obtained according to the in vitro transcription method of the present disclosure, and a pharmaceutically acceptable carrier and/or excipient.

In one embodiment, the composition is a pharmaceutical composition or vaccine or kit.

In a further aspect, the present disclosure provides the use of an RNA nucleic acid molecule as described in the present disclosure and/or obtained according to the in vitro transcription method of the present disclosure for the preparation of a medicament for the treatment or prevention of a disease or disorder selected from immune diseases, genetic diseases, cancer, infectious diseases, inflammatory diseases, allergies and/or for gene therapy and/or immunomodulation.

Drawings

The drawings are only for the purpose of illustrating the invention more clearly and are not to be taken as limiting the scope of the invention as regards its disclosure and protection in any way.

FIG. 1 shows the structure of a nucleic acid molecule according to an embodiment of the present disclosure: a DNA nucleic acid construct (fig. 1A); RNA nucleic acid molecules (fig. 1B) and preferably 5' -ARCA (fig. 1C).

FIG. 2 shows in vitro transcription of EGF and bFGF mRNA.

FIG. 3 shows the fluorescent signals of GFP mRNA transfected 293T cells.

FIG. 4 shows the results of quantification of intracellular EGF and bFGF mRNA in 293T cells by qPCR.

Figure 5 shows expression of EGF and FGF proteins in 293T cells. Molecular weights of EGF and bFGF mRNA were confirmed by gel electrophoresis (fig. 5A); after transfection of EGF and bFGF mRNA into 293T cells, whole cell lysates and cell culture media were analyzed by western blotting at the indicated time points, wherein the transfected 293T cells showed significant increases in extracellular hbFGF (fig. 5B), intracellular hbFGF (fig. 5C), extracellular hEGF (fig. 5D) and intracellular hEGF (fig. 5E).

Detailed Description

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the drawings and specific descriptions thereof. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended.

A detailed description of a cell-free and vector-free in vitro transcription method and constructs for use in such methods is provided below. These methods and constructs meet at least one need in the art.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

Unless explicitly defined otherwise, terms used herein should be construed according to their usual meaning in the art. Unless the context indicates otherwise or indicated, nouns without quantitative word modifications denote one or more than one.

Standard techniques and procedures are generally performed according to conventional methods and various general references in the art (see generally ,Sambrook et al.Molecular Cloning：ALaboratory Manual,2nd ed.(1989)Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.).

The term "about" as used herein, unless otherwise indicated, refers to +/-10%, more preferably +/-5%, such as +/-4%, +/-3%, +/-2% or +/-1% of the specified value.

It should be noted that, without conflict, the features of the embodiments and examples in the present disclosure may be combined with each other.

The present disclosure provides a novel in vitro transcribed RNA platform for the production of mRNA, e.g., an mRNA therapeutic agent. The platform can provide cost-effective mRNA production, has high safety and efficacy, and is easy to administer into cells. The present disclosure provides mrnas comprising a 5' -anti-reverse cap analogue (5 ' -ARCA), a human β globin 5' -untranslated region sequence, a signal sequence and a target or target gene coding sequence, a human α globin 3' -untranslated sequence, and a 3' -poly a tail. mRNA produced on this platform showed enhanced stability of gene expression and translation efficiency. Also, the novel platform shows maximum yields of mRNA of up to about 2.3mg/mL or even higher within 60 minutes of incubation. The present disclosure is based, at least in part, on the surprising discovery that: the combination of elements selected in the present disclosure not only enables maximum yields of mRNA of up to about 2.3mg/mL per hour or even higher to be obtained, but the resulting mRNA exhibits enhanced gene expression stability and translation efficiency.

As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to any DNA or RNA molecule, and is used interchangeably with polynucleotide. Where reference is made herein to a nucleic acid or nucleic acid sequence encoding a particular protein and/or peptide, the nucleic acid or nucleic acid sequence, respectively, preferably also comprises regulatory sequences, allowing expression, i.e. transcription and/or translation, of the nucleic acid sequence encoding the particular protein or peptide in a suitable host, e.g. a human.

RNA nucleic acid molecules

In one aspect, the present disclosure provides an RNA nucleic acid molecule comprising, from 5 'end to 3' end, a 5 '-cap structure, a human β -globin 5' untranslated region (5 '-UTR), at least one coding region, a human α -globin 3' untranslated region (3 '-UTR), and a3' -poly a tail, the at least one coding region operably linked to the 5'-UTR and the 3' -UTR.

Untranslated region (UTR)

As used herein, the term "untranslated region (UTR)" refers to an "untranslated region" located upstream (5 ') and/or downstream (3') of a coding region of a nucleic acid molecule described herein, and thus typically flanking the coding region. Thus, the term "UTR" generally includes a 5 '-untranslated region ("5' -UTR") and a 3 '-untranslated region ("3' -UTR"). UTRs may generally comprise or consist of nucleic acid sequences that are not translated into proteins. The UTR may comprise one or more regulatory elements.

As used herein, the term "regulatory element" refers to a nucleic acid sequence having gene regulatory activity that is capable of affecting transcription or translation of an operably linked (in cis or trans) transcribable nucleic acid sequence. The term may include promoters, enhancers, internal Ribosome Entry Sites (IRES), introns, leader sequences, transcriptional termination signals such as polyadenylation signals, and other expression control elements.

As used herein, the term "operably connected" refers to an arrangement of elements wherein the elements described in that term are assembled to perform their general function. For example, a given promoter operably linked to a nucleic acid sequence can affect the expression of that sequence in the presence of the appropriate enzyme. The promoter need not be contiguous with the sequence, so long as it directs expression of the sequence. Thus, for example, a spaced untranslated but transcribed sequence may be present between a promoter sequence and a nucleic acid sequence, and the promoter sequence is still considered to be "operably linked" to the coding sequence.

In some embodiments, UTRs are "operably linked", i.e., located in functional relationship upstream and downstream of the coding region, preferably upstream and downstream of the coding region in a manner that allows them to control (i.e., regulate or modulate, preferably enhance) expression of the coding sequence.

The inventors of the present application have unexpectedly found that a combination of 5' -and 3' -untranslated regions (UTRs), preferably in combination with the 5' -cap structure and poly a tail of the present disclosure, act synergistically to synergistically enhance expression of operably linked nucleic acid sequences. The nucleic acid molecules having the UTR combinations of the present application advantageously enable rapid and transient expression of large amounts of polypeptides or proteins delivered for gene therapy or immunotherapy purposes. Thus, the nucleic acid molecules provided herein are particularly useful for a variety of therapeutic applications in vivo, including, for example, gene therapy, cancer immunotherapy, or vaccination against infectious agents.

The synergy test of UTR combinations (preferably in combination with the 5' -cap structure and poly a tail of the present disclosure) is a routine procedure for a person skilled in the art, e.g. synergy tests can be performed by luciferase expression after mRNA transfection to demonstrate that there is synergy, i.e. not just additive.

As used herein, the term "5'-UTR" refers to a portion of a nucleic acid molecule that is located 5' (i.e., "upstream") of an open reading frame and is not translated into a protein. In the context of the present disclosure, the 5' -UTR starts at the transcription start site and ends one nucleotide before the start codon of the open reading frame.

As used herein, the term "3'-UTR" refers to a portion of a nucleic acid molecule that is located 3' (i.e., "downstream") of the open reading frame and is not translated into a protein. In the context of the present disclosure, the 3'-UTR corresponds to a sequence located between the 3' of the stop codon of the protein coding sequence (preferably immediately adjacent to the stop codon of the protein coding sequence) and the polyadenylation sequence.

In one embodiment, the UTR combinations of the present disclosure are the 5 'untranslated region (5' -UTR) of human β -globin and the 3 'untranslated region (3' -UTR) of human α -globin. In one embodiment, the UTR combinations of the present disclosure include (a) a human β -globin 5 'untranslated region or a homolog, variant, or fragment thereof and (b) a human α -globin 3' untranslated region or a homolog, variant, or fragment thereof. In some embodiments, the homologue or variant and the UTR have nucleotide differences, e.g., one or more nucleotide differences, such as 1-20, 2-15, 3-10, 4-9, 5-8, 6-7 nucleotide differences, only outside the regulatory element of the UTR. In some embodiments, a fragment of a UTR comprises all regulatory elements of the UTR.

In some embodiments, the human β -globin 5' -UTR comprises or consists of a sequence according to SEQ ID NO:1, or with an RNA sequence according to SEQ ID NO:1, has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the nucleic acid sequence according to SEQ ID NO:1 and the RNA sequence having said identity to the RNA sequence of SEQ ID NO:1, only outside the regulatory elements in UTR, there are nucleotide differences, e.g. one or more nucleotide differences, such as 1-20, 2-15, 3-10, 4-9, 5-8, 6-7 nucleotide differences.

SEQ ID NO:1 (RNA sequence of human beta-globin 5' -UTR)

5’-ACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACC-3’

In some embodiments, the human α -globin 3' -UTR comprises or consists of a sequence according to SEQ ID NO:2, or with an RNA sequence according to SEQ ID NO:2, has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the nucleic acid sequence according to SEQ ID NO:2 and the RNA sequence having said identity to the RNA sequence of SEQ ID NO:2, only outside the regulatory elements in UTR, there are nucleotide differences, e.g. one or more nucleotide differences, such as 1-20, 2-15, 3-10, 4-9, 5-8, 6-7 nucleotide differences.

SEQ ID NO:2 (RNA sequence of human alpha-globin 3' -UTR)

5’-GCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUG-3’

5' -Cap structure

According to a particularly preferred embodiment of the present invention, the RNA nucleic acid molecules of the present disclosure are modified by the addition of a "5' -cap structure" such that said RNA nucleic acid molecules can be further stabilized.

The "5' -cap structure" may be formed from modified nucleotides, in particular from derivatives of guanine nucleotides. Preferably, the 5 '-cap is attached to the 5' terminus by a 5'-5' -triphosphate bond. In some embodiments, the 5 'cap may be methylated, e.g., m7GpppN, where N is the 5' terminal nucleotide of the 5 '-capped nucleic acid, typically the 5' -end of the mRNA. m7 gppppn is a 5' -cap structure (cap 0 structure) that occurs naturally in mRNA transcribed by polymerase II.

Other examples of 5' -cap structures known in the art include glyceryl, inverted deoxyabasic residues (moieties), 4',5' -methylene nucleotides, 1- (. Beta. -D-erythro ribofuranosyl) nucleotides, 4' -thio nucleotides, carbocyclic nucleotides, 1, 5-anhydrohexitol nucleotides, L-nucleotides, alpha-nucleotides, modified base nucleotides, threo-pentofuranribonucleotides, acyclic 3',4' -secoisolaricic nucleotides, acyclic 3, 4-dihydroxybutyl nucleotides, acyclic 3, 5-dihydroxyamyl nucleotides, 3' -3' -inverted nucleotide moieties, 3' -3' -inverted abasic moieties, 3' -2' -inverted nucleotide moieties, 3' -2' -inverted abasic moieties, 1, 4-butanediol phosphate, 3' -phosphoramidates, hexyl phosphate, 3' -phosphates, 3' -phosphorothioates, phosphorodithioates or bridged or unbridged methylphosphonate moieties.

The 5' -cap structure may be cap 0, cap 1 (ribose of adjacent nucleotide of m7G is methylated), cap 2 (ribose of 2 nd nucleotide downstream of m7G is additionally methylated), cap 3 (ribose of 3 rd nucleotide downstream of m7G is additionally methylated), cap 4 (ribose of 4 th nucleotide downstream of m7G is methylated).

In some embodiments, a particularly preferred 5' -cap structure useful in the present disclosure is ARCA (anti-reverse cap analogue). In one embodiment, the ARCA is 7m G (3' -O-Me) pppG. The ARCA is obtained by substituting the 3' -OH group of the 7mG residue in 7mGpppG with OCH ₃ ("OMe"). Several types of ARCA analogs are known in the art (see, e.g., U.S. patent No. 7,074,596). However, the present application surprisingly found that the use of 7m G (3 '-O-Me) pppG compared to other ARCA analogs does not require a large molar excess of ARCA relative to pppG to ensure that most mRNA transcript molecules have a 5' -cap structure. In some embodiments, the molar ratio of 7m G (3' -O-Me) pppG to pppG (GTP) is no greater than 4:1, e.g., 3:1, 2:1, or 1:1.

The present application also finds that the use of ARCA, particularly 7m G (3' -O-Me) pppG, synergistically enhances expression of operably linked nucleic acid sequences in combination with UTRs described in the present disclosure. Nucleic acid molecules having the ARCA of the application, particularly 7m G (3' -O-Me) pppG, and UTR combinations advantageously enable rapid and transient expression of large amounts of polypeptides or proteins delivered for gene therapy or immunotherapy purposes.

3' -Poly (A) tail

According to further preferred embodiments, the RNA nucleic acid molecules of the invention may comprise a polyadenylation sequence.

As used herein, the term "polyadenylation sequence" is also referred to as "polyadenylation tail" or "3' -polyadenylation tail", which refers to a sequence of adenosine nucleotides, e.g. up to about 400 adenosine nucleotides, e.g. a sequence of about 20 to about 400, preferably about 50 to about 400, more preferably about 50 to about 300, even more preferably about 50 to about 250, most preferably about 60 to about 250 adenosine nucleotides. As used herein, a "polyadenylation sequence" may also comprise from about 10 to 200 adenosine nucleotides, preferably from about 10 to 100 adenosine nucleotides, more preferably from about 40 to 80 adenosine nucleotides or even more preferably from about 50 to 70 adenosine nucleotides. The polyadenylation sequence is usually located at the 3' end of the RNA, particularly mRNA.

The polyadenylation sequence in the RNA nucleic acid molecule may preferably be obtained from the DNA template by RNA in vitro transcription. Alternatively, the polyadenylation sequence may be obtained in vitro by conventional methods of chemical synthesis and may not be transcribed from the DNA template.

Furthermore, the polyadenylation sequence or polyadenylation tail can be produced by enzymatic polyadenylation of the RNA nucleic acid molecule using commercially available polyadenylation kits and corresponding protocols as are known in the art. Polyadenylation is generally understood to be the addition of a polyadenylation sequence to an RNA nucleic acid molecule, such as mature pre-mRNA. Polyadenylation may be induced by so-called polyadenylation signals. The signal is preferably located within a nucleotide fragment at the 3' end of the nucleic acid (RNA) sequence to be polyadenylation. The polyadenylation signal generally comprises the hexamer, preferably the hexamer sequence AAUAAA, consisting of adenine and uracil/thymine nucleotides.

The inventors of the present application further found that adding a polyadenylic acid tail at the 3' end of the RNA nucleic acid molecules of the present disclosure further increases the stability of the RNA nucleic acid molecules. The poly a tail synergistically enhances the stability of operably linked nucleic acid sequences and their expression yields in combination with the ARCA described in the present disclosure, particularly 7m G (3' -O-Me) pppG, and UTR described in the present disclosure.

Coding region

The nucleic acid molecules according to the invention comprise at least one coding region or coding sequence which is operably linked (typically flanked) to at least one 3'-UTR element and at least one 5' -UTR element as defined herein. The terms "coding sequence" or "cds" and "coding region" are used interchangeably herein to refer to a segment or portion of a nucleic acid encoding a (gene) product of interest. A gene product is a product of gene expression, including polypeptides and nucleic acids, and in general, at least one coding region of a nucleic acid molecule of the present disclosure may encode at least one polypeptide or protein, referred to as a "polypeptide or protein of interest. The coding region is typically composed of exons bordered at their 5 'end by a start codon (e.g., AUG) and at their 3' end by a stop codon (e.g., UAG, UAA or UGA). In the nucleic acid molecules of the present disclosure, the coding region is bordered by at least one 5'-UTR element and at least one 3' -UTR element as defined herein.

The polypeptide or protein of interest generally includes any polypeptide or protein that can be encoded by a nucleic acid sequence having at least one coding region, and can be expressed under appropriate conditions to produce a functional polypeptide or protein product. Herein, the term "functional" means "capable of performing a desired biological function" and/or "exhibiting a desired biological property". The polypeptide or protein of interest may have a variety of functions and include, for example, growth factors, antibodies, enzymes, signaling proteins, receptors, receptor ligands, peptide hormones, transport proteins, structural proteins, neurotransmitters, serum proteins, vectors, drugs, immunomodulators, oncogenes, cancer inhibitors, toxins, tumor antigens, and the like. In some embodiments, the polypeptide or protein of interest may be a therapeutic, antigenic, and allergenic polypeptide or protein.

Cell growth factor

In some embodiments, at least one coding region of a nucleic acid molecule of the disclosure may encode at least one growth factor. As used herein, the term "growth factor" refers to a class of polypeptides that regulate multiple effects of cell growth and other cellular functions by binding to specific, highly compatible cell membrane receptors. In some embodiments, the growth factor may be selected from the group consisting of platelet-based growth factors (platelet-derived growth factor, PDGF); osteosarcoma-derived growth factor (ODGF), epidermal growth factor type (epidermal growth factor EGF, transforming growth factors TGFα and TGFβ), fibroblast growth factor (αFGF, βFGF), insulin-like growth factor (IGF-I, IGF-II), nerve Growth Factor (NGF), interleukin-like growth factor (IL-1, IL-3, etc.), erythropoietin (EPO), colony Stimulating Factor (CSF), etc.

Therapeutic polypeptides or proteins

In some embodiments, at least one coding region of a nucleic acid molecule of the disclosure may encode at least one "therapeutic polypeptide or protein". The term "therapeutic polypeptide or protein" refers to a polypeptide or protein that is capable of mediating a desired diagnostic, prophylactic or therapeutic effect, preferably resulting in the detection, prevention, amelioration and/or cure of a disease.

Preferably, a nucleic acid molecule according to the present disclosure may comprise at least one coding region encoding a therapeutic protein that replaces a deleted, defective or mutated protein; therapeutic proteins useful for the treatment of genetic or acquired diseases, infectious diseases or tumors, such as cancers or neoplastic diseases; auxiliary or immunostimulatory therapeutic proteins; a therapeutic antibody or antibody fragment, variant or derivative; peptide hormones; a gene editing agent; immune checkpoint inhibitors; t cell receptors, or T cell receptor fragments, variants or derivatives; and/or enzymes.

Antigenic polypeptides or proteins

At least one coding region of a nucleic acid molecule of the present disclosure may encode at least one "antigenic polypeptide or protein". The term "antigenic polypeptide or protein" or simply "antigen" generally refers to any polypeptide or protein "antigenic peptide or protein" capable of interacting with/being recognized by a component of the immune system, e.g., an antibody or immune cell, through its antigen receptor, e.g., B Cell Receptor (BCR) or T Cell Receptor (TCR), under appropriate conditions, and preferably capable of eliciting an immune response, preferably through its "epitope" or "antigenic determinant" interacting with a component of the immune system.

The choice of a particular antigenic polypeptide or protein will generally depend on the disease to be treated or prevented. In general, a nucleic acid molecule may encode any antigenic polypeptide or protein associated with a disease (e.g., cancer, infectious disease) that may be treated by inducing an immune response against the antigenic infectious disease.

Preferably, an artificial nucleic acid molecule according to the invention may comprise at least one coding region which codes for a tumor antigen, a pathogenic antigen, a self-antigen, an alloantigen or an allergen antigen. Particularly preferred are the tumor antigens NY-ESO-1, 5T4, MAGE-C1, MAGE-C2, muc-1, PSA, PSMA, PSCA, STEAP and PAP.

Allergenic polypeptides or proteins

At least one coding region of a nucleic acid molecule of the present disclosure may encode at least one "allergenic polypeptide or protein". The term "allergenic polypeptide or protein" or "allergen" refers to a polypeptide or protein that is capable of inducing an allergic reaction, i.e. a pathological immune reaction characterized by an altered bodily responsiveness (e.g. hypersensitivity) when exposed to a subject. In general, "allergen" is associated with "atopy", i.e. an adverse immune response involving immunoglobin E (IgE). Thus, the term "allergen" generally refers to a substance (herein a polypeptide or protein) that is related to atopy and induces IgE antibodies. In some embodiments, the allergen may include insect-derived allergens, mammalian allergens, mollusc-derived allergens, plant allergens, fungal allergens, and the like.

Other domains, tags, linkers, sequences or elements

Preferably, in addition to encoding at least one polypeptide or protein of interest, at least one coding region of a nucleic acid molecule of the present disclosure may encode other polypeptide domains, tags, linkers, sequences or elements. The nucleic acid sequence encoding the other domain, tag, linker, sequence or element is operably linked in frame to a region encoding the polypeptide or protein of interest such that expression of the coding sequence preferably results in a fusion product of the polypeptide or protein of interest coupled to the other domain, tag, linker, sequence or element.

The at least one coding region of the nucleic acid molecules of the present disclosure may also encode at least one of: signal peptide, peptide or protein tag, localization signal or sequence, peptide linker, etc.

The term "signal peptide" (also sometimes referred to as a secretion signal peptide or signal sequence) refers to a typical short peptide (which may typically be 16 to 30 amino acids in length) that is usually present at the end of a protein to be secreted via the secretory pathway.

Preferably, a signal peptide may be introduced into the polypeptide or protein of interest to promote secretion of the polypeptide or protein. In particular, where a nucleic acid encoding an antigenic polypeptide or protein is fused to a signal peptide, proper secretion may assist in triggering an immune response against the antigen. The signal peptide may also be effectively combined with any of the other polypeptides or proteins disclosed herein. When encoded in combination with a polypeptide or protein of interest, such signal peptide may be located at the N-terminus, C-terminus and/or internally of the polypeptide or protein of interest, preferably at the N-terminus. At the nucleic acid level, the coding sequence for such a signal peptide is typically placed in frame (i.e., in the same reading frame).

Exemplary signal peptides that may be used in the present disclosure include, but are not limited to, signal sequences of classical or non-classical MHC molecules (e.g., signal sequences of MHC I molecules and MHC II molecules, e.g., signal sequences of MHC class I molecules HLA-A x 0201), signal sequences of cytokines or immunoglobulins, signal sequences of immunoglobulin or antibody constant chains, signal sequences of Lamp1, tapasin, erb 57, calretikulin, calnexin, PLAT, EPO, or albumin, and signal sequences of other membrane-associated proteins or proteins associated with the Endoplasmic Reticulum (ER) or endosomal-lysosomal compartments.

The term "peptide or protein tag" is a short amino acid sequence that is introduced into a polypeptide or protein of interest to confer a desired biological function or property. In general, a "peptide tag" can be used to detect, purify, isolate, or add certain desired biological properties or functions.

A "peptide linker" or "spacer" is a short amino acid sequence that connects domains, portions or parts of a polypeptide or protein of interest disclosed herein, e.g., domains, portions or parts of a multi-domain protein or fusion protein. The polypeptide or protein, or a domain, portion or part thereof, is preferably functional, i.e. performs a specific biological function.

DNA nucleic acid molecules

The RNA nucleic acid molecules of the present disclosure may preferably be obtained from DNA templates by RNA in vitro transcription. Alternatively, the polyadenylation sequence may be obtained in vitro by conventional methods of chemical synthesis and may not be transcribed from the DNA template.

In one embodiment, the RNA nucleic acid molecules of the present disclosure may preferably be obtained from a DNA template by RNA in vitro transcription.

Thus, in one aspect, the present disclosure provides a DNA nucleic acid molecule comprising, from the 5 'end to the 3' end, a promoter, a human β -globin 5 '-untranslated region (5' -UTR), at least one coding region, a human α -globin 3 '-untranslated region (3' -UTR), and a transcription terminator, said at least one coding region being operably linked to said 5'-UTR and said 3' -UTR.

In one embodiment, the DNA nucleic acid molecules of the present disclosure include a UTR combination of a human β -globin 5 'untranslated region (5' -UTR) and a human α -globin 3 'untranslated region (3' -UTR). In one embodiment, the UTR combinations of the present disclosure include (a) a human β -globin 5 'untranslated region or a homolog, variant, or fragment thereof and (b) a human α -globin 3' untranslated region or a homolog, variant, or fragment thereof. In some embodiments, the homologue or variant and the UTR have nucleotide differences, e.g., one or more nucleotide differences, such as 1-20, 2-15, 3-10, 4-9, 5-8, 6-7 nucleotide differences, only outside the regulatory element of the UTR. In some embodiments, a fragment of a UTR comprises all regulatory elements of the UTR.

In some embodiments, the human β -globin 5' -UTR comprises or consists of a sequence according to SEQ ID NO:3, or with an RNA sequence according to SEQ ID NO:3, has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the nucleic acid sequence according to SEQ ID NO:3 and the DNA sequence having said identity to the DNA sequence of SEQ ID NO:3, there is only a nucleotide difference, e.g. one or more nucleotide differences, such as 1-20, 2-15, 3-10, 4-9, 5-8, 6-7 nucleotide differences, outside the regulatory element in UTR.

SEQ ID NO:3 (DNA sequence of human beta-globin 5' -UTR)

5’-ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC-3’

In some embodiments, the human α -globin 3' -UTR comprises or consists of a sequence according to SEQ ID NO:4, or with a DNA sequence according to SEQ ID NO:4, has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the nucleic acid sequence according to SEQ ID NO:4 and the DNA sequence of SEQ ID NO:4, there is only a nucleotide difference, e.g. one or more nucleotide differences, such as 1-20, 2-15, 3-10, 4-9, 5-8, 6-7 nucleotide differences, outside the regulatory element in UTR.

SEQ ID NO:4 (DNA sequence of human alpha-globin 3' -UTR)

5’-GCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTG-3’

In some embodiments, the respective promoter and transcription terminator or termination element are selected according to the RNA polymerase employed. RNA polymerases useful in the present disclosure include, but are not limited to, T3, T7, sny, or SP6 RNA polymerase. Thus, promoters and transcription terminators useful in the present disclosure may be selected from the group consisting of promoters and transcription terminators or termination elements of T3, T7, sny, or SP6 RNA polymerase. Exemplary promoters are selected from the T3, T7, sny, or SP6 promoters, preferably the T7 promoter. An exemplary transcription terminator or termination element may be a T7 transcription terminator.

In some embodiments, DNA templates for transcription of RNA molecules of the present disclosure may include linear templates obtained by PCR methods or annealing chemically synthesized oligonucleotides, clonally constructed plasmids, and cDNA templates obtained by first and second strand synthesis (e.g., an acrna amplification) based on RNA precursors.

Plasmid vectors used as transcription templates need to be linearized by restriction enzyme digestion. Linearization ensures that RNA transcripts of defined length and sequence are obtained, since the transcription reaction will continue to the end of the DNA template.

In some embodiments, the DNA nucleic acid molecules of the present disclosure further comprise a first restriction site located upstream of the promoter and a second restriction site located downstream of the transcription terminator.

In some embodiments, the first restriction site and the second restriction site allow insertion of the DNA nucleic acid molecule into a vector, such as a plasmid. As used herein, the term "restriction endonuclease" or "restriction endonuclease" refers to a class of enzymes that can recognize and attach a particular sequence of deoxyribonucleotides and cleave a phosphodiester bond between two deoxyribonucleotides at a particular position in each strand. The cleavage method is to cleave the bond between the sugar molecule and the phosphate, thereby generating a nick on each of the two DNA strands without damaging the nucleotide and the base. There are two types of cleavage formats, each of which produces a sticky end with protruding single-stranded DNA and a smooth end with a flat end without protrusions. Since the broken DNA fragments can be joined by DNA ligase, different restriction fragments on the chromosome or DNA can be joined together by splicing. In some embodiments, the first restriction site and the second restriction site may be the same or different, and both the first restriction site and the second restriction site are EcoRI. Restriction enzymes useful in the present disclosure may include, but are not limited to: ecoRI, pstI, xbaI, bamHI, hindIII, taqI, notI, hinfI, sau3, A, povII, smaI, haeIII, aluI, salI, dra, etc. Insertion of the target polynucleotide is performed using standard molecular biology methods, e.g., as described in Sambrook et al.(Sambrook et al.Molecular Cloning:A Laboratory Manual,Cold Spring Harbour Laboratory Press,1989) and/or Ausubel et al.(Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience(1988). Methods of ligating nucleic acids are apparent to those skilled in the art and are described, for example, in Sambrook et al molecular Cloning: A Laboratory Manual, cold Spring Harbour Laboratory Press,1989 and/or Ausubel et al (editors), current Protocols in Molecular Biology, greene Pub.associates and Wiley-Interscience (1988). In one example, the nucleic acid is ligated using a ligase (e.g., T4DNA ligase).

In some embodiments, the DNA nucleic acid molecules of the present disclosure further comprise a third restriction site located between the promoter and the 5'-UTR and a fourth restriction site located between the 3' -UTR and the transcription terminator. In some embodiments, the third restriction site and the fourth restriction site allow for universal cloning. In some embodiments, the third restriction site and the fourth restriction site are independently selected from EcoRI, pstI, xbaI, bamHI, hindIII, taqI, notI, hinfI, sau, A, povII, smaI, haeIII, aluI, salI and Dra. In some embodiments, the third restriction site and the fourth restriction site are different, e.g., salI and NotI, respectively.

In vitro transcription method

In one aspect, the present disclosure provides an in vitro transcription method comprising the steps of: (a) Providing a DNA nucleic acid molecule according to the present disclosure as a transcription template; (b) optionally amplifying the DNA nucleic acid molecule; (c) Subjecting the DNA nucleic acid molecule to in vitro transcription in the presence of a 5 '-anti-reverse cap analogue (5' -ARCA), preferably 7m G (3 '-O-Me) pppG, to obtain a reaction mixture comprising a 5' -ARCA-capped mRNA; (d) Optionally, removing the transcription template from the reaction mixture comprising 5' -ARCA-capped mRNA by adding dnase; and (e) adding a polyadenylic acid polymerase reaction mixture to the reaction mixture comprising 5 '-ARCA-capped mRNA for 3' -polyadenylic acid tail addition to obtain a 5 '-ARCA-capped mRNA having a 3' -polyadenylic acid tail.

The term "in vitro transcription" of RNA relates to a method of synthesizing RNA from a DNA template in a cell-free system (in vitro). DNA, preferably linear DNA (e.g., linearized plasmid DNA, linearized dbDNA) is used as a template for the generation of RNA transcripts. DNA templates for the in vitro transcription of RNA can be obtained by cloning nucleic acids, in particular cDNA corresponding to the corresponding RNA to be transcribed in vitro, and introducing it into a suitable vector for the in vitro transcription of RNA, for example into plasmid DNA.

Templates for in vitro transcription may also be prepared by PCR amplification using the DNA nucleic acid molecules of the present disclosure as templates. After purification of the PCR product, RNA synthesis can be performed by standard in vitro transcription methods. The PCR product obtained is mixed with an in vitro RNA synthesis premix comprising a 5' -anti-reverse cap analogue (5 ' -ARCA) and incubated (e.g. for 30 minutes at 37 ℃) to generate 5' -ARCA-capped RNA. In some embodiments, the in vitro RNA synthesis premix comprises: buffers suitable for in vitro transcription (e.g., tris-HCl, pH 7.9), cap analogues (e.g., 5' -anti-reverse cap analogues), ribonucleoside triphosphates (ATP, UTP, CTP and GTP), ribonuclease inhibitors and RNA polymerase (e.g., T7 RNA polymerase). In some embodiments, the in vitro RNA synthesis premix may further comprise at least one or all of MgCl ₂, antioxidants, and polyamines (e.g., spermidine).

In order to obtain high quality RNA suitable for use in RNA-based therapies, DNA templates can be efficiently and reliably removed from the final RNA product to ensure efficacy and safety of the RNA-based therapeutics. Removal of the DNA template from the RNA in vitro transcription reaction may be achieved, for example, by enzymatic (e.g. DNase I) digestion of the DNA and purification of the RNA.

The 3' -polyadenylation tail is carried out by means of the additional polyadenylation polymerase reaction mixture. In some embodiments, the polyadenylation polymerase reaction mixture comprises buffer (e.g., tris-HCl, pH 8.1) and polyadenosine polymerase and salts (e.g., one or both of NaCl and MgCl ₂) and incubated (e.g., 30 minutes at 37 ℃) to obtain 5 '-ARCA-capped mRNA with a 3' -polyadenylation tail.

Compositions and vaccines

In another aspect, the present disclosure provides a composition comprising an RNA nucleic acid molecule of the present disclosure, and at least one pharmaceutically acceptable carrier and/or excipient. In some preferred embodiments, the composition is provided as a pharmaceutical composition. According to a further preferred embodiment, the composition may be provided as a vaccine. "vaccine" is generally understood to mean a prophylactic or therapeutic material that provides at least one antigen, preferably an antigenic peptide or protein. By "providing at least one antigen" is meant, for example, that the vaccine comprises an antigen or that the vaccine comprises, for example, a molecule encoding an antigen. Thus, a vaccine of the present disclosure may comprise at least one RNA nucleic acid molecule encoding at least one antigenic polypeptide or protein as defined herein, which antigenic polypeptide or protein may for example be derived from a tumor antigen, a bacterial antigen, a viral antigen, a fungal antigen or a protozoan antigen, an autoantigen, an allergen or an alloantigen, and preferably induces an immune response against the respective antigen when expressed and presented to the immune system.

The compositions or vaccines of the present disclosure preferably comprise at least one RNA nucleic acid molecule described herein. Each RNA nucleic acid molecule in the compositions or vaccines of the present disclosure can encode at least one or at least two (the same or different) multiple polypeptides or proteins of interest. The RNA nucleic acid molecules may be provided in the composition or vaccine in "complexed" or "free" form or a mixture thereof. The composition or vaccine may further comprise at least one additional active agent for the treatment of a disease or disorder treated with the RNA nucleic acid molecule or the composition or vaccine comprising the same.

Preferably, the composition or vaccine according to the invention comprises at least one pharmaceutically acceptable carrier and/or excipient. The term "pharmaceutically acceptable" refers to a compound or agent that is compatible with one or more active agents and does not interfere with and/or significantly reduce the pharmaceutical effect thereof. The pharmaceutically acceptable carriers and excipients are preferably of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to a subject to be treated.

Pharmaceutically acceptable excipients may perform various functional roles and include, but are not limited to, diluents, fillers, bulking agents, carriers, disintegrants, binders, lubricants, glidants, coatings, solvents and co-solvents, buffers, preservatives, adjuvants, antioxidants, wetting agents, antifoaming agents, thickening agents, sweeteners, flavoring agents and humectants.

For compositions in liquid form, useful pharmaceutically acceptable carriers and excipients include solvents, diluents or vehicles such as (pyrogen-free) water, (isotonic) saline solutions such as phosphate or citrate buffered saline, fixed oils, vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like); lecithin; a surfactant; preservatives such as benzyl alcohol, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like; isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride; aluminum monostearate or gelatin; antioxidants such as ascorbic acid or sodium bisulphite; chelating agents such as ethylenediamine tetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate; and agents for modulating tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The buffer may be hypertonic, isotonic or hypotonic with respect to the specific reference medium, i.e. the buffer may have a higher, the same or a lower salt content with respect to the specific reference medium, wherein preferably such a concentration of the above mentioned salts may be used which does not cause cell damage due to osmosis or other concentration effects. The reference medium is, for example, a liquid produced in an "in vivo" method, such as blood, lymph, cytosolic liquid, or other body fluids, or a liquid that can be used, for example, as a reference medium in an "in vitro" method, such as a commonly used buffer or liquid. Such usual buffers or liquids are known to the skilled person.

For compositions in (semi) solid form, useful pharmaceutically acceptable carriers and excipients include binders, such as microcrystalline cellulose, gum tragacanth or gelatin; starch or lactose; sugars such as lactose, glucose, and sucrose; starches, such as corn starch or potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate; disintegrants, such as alginic acid; lubricants, such as magnesium stearate; glidants, such as stearic acid, magnesium stearate; calcium sulfate, colloidal silica, and the like; sweeteners, such as sucrose or saccharin; and/or flavoring agents, such as peppermint, methyl salicylate, or orange flavoring.

Suitable pharmaceutically acceptable carriers and excipients can generally be selected based on the desired formulation of the composition.

Liquid compositions for administration by injection, particularly intravenous injection, should be sterile and stable under the conditions of manufacture and storage. Such compositions are typically formulated as parenterally acceptable aqueous solutions which are pyrogen free, have a suitable ph, are isotonic and maintain the stability of the active ingredient. Particularly useful pharmaceutically acceptable carriers and excipients for liquid compositions according to the invention include water, typically pyrogen-free water; isotonic saline or buffer solutions, for example phosphate, citrate and the like. In particular for injection of the compositions of the present disclosure, water or a preferred buffer, more preferably an aqueous buffer, comprising a sodium salt, preferably at least 50mM sodium salt, may be used; a calcium salt, preferably at least 0.01mM calcium salt; and optionally a potassium salt, preferably at least 3mM potassium salt.

According to a preferred embodiment, the sodium, calcium and optionally potassium salts may be present in the form of their halides, for example chloride, iodide or bromide, in the form of their hydroxides, carbonates, bicarbonates or sulphates etc. In addition, the buffer may contain an organic anion of the cation described above.

According to a preferred embodiment, the buffer suitable for injection purposes as defined above may comprise a salt selected from sodium chloride (NaCl), calcium chloride (CaCl ₂) and optionally potassium chloride (KCl), wherein other anions may be present in addition to chloride ions. CaCl ₂ may also be replaced with another salt, such as KCl. Typically, the concentration of salt in the injection buffer is at least 50mM sodium chloride (NaCl), at least 3mM potassium chloride (KCl), and at least 0.01mM calcium chloride (CaCl ₂).

Compositions for topical application may be formulated as emulsions, ointments, gels, pastes or powders using suitable liquid and/or (semi) solid excipients or carriers as described herein and herein. Compositions for oral administration may be formulated as tablets, capsules, liquids, powders or sustained release forms using suitable liquid and/or (semi) solid excipients or carriers as described elsewhere herein.

According to some preferred embodiments, the compositions or vaccines of the present disclosure are administered parenterally, in particular by intradermal or intramuscular injection, orally, intranasally, pulmonary, inhaled, topically, rectally, buccally, vaginally or by an implanted reservoir, and are provided in liquid or lyophilized formulations for parenteral administration as discussed elsewhere herein. Parenteral formulations are typically stored in vials, iv bags, ampoules, cartridges or pre-filled syringes and may be administered in the form of injections, inhalants or aerosols, preferably in the form of injections.

According to a preferred embodiment, the composition or vaccine of the present disclosure may comprise the RNA nucleic acid molecule of the present disclosure complexed with a lipid, which may be in the form of a lipid nanoparticle, a liposome, a lipid complex or an emulsion, for example.

According to other preferred embodiments, the composition or vaccine of the present disclosure is provided in lyophilized form. Preferably, the lyophilized composition or vaccine is reconstituted in a suitable buffer prior to administration, which buffer is advantageously based on an aqueous carrier, such as a lactated ringer's solution, a phosphate buffer, preferably a lactated ringer's solution.

According to a preferred embodiment, the composition or vaccine of the present disclosure may further comprise at least one adjuvant. An "adjuvant" or "adjunct component" in a broad sense is typically a pharmacological and/or immunological agent that can alter, for example, the effect of other active agents, such as therapeutic agents or vaccines. In this context, "adjuvant" may be understood as any compound suitable to support the administration and delivery of the compositions of the present disclosure. In particular, adjuvants may be preferred to enhance the immunostimulatory properties of the composition or vaccine to which they are added. In addition, such adjuvants may, but are not limited to, elicit or enhance an immune response of the innate immune system, i.e., a non-specific immune response.

Kit for detecting a substance in a sample

In another aspect, the present disclosure relates to a kit or kit of parts comprising an RNA nucleic acid molecule and/or a composition or vaccine of the present disclosure.

In the kits or kits of the present disclosure, at least one RNA nucleic acid molecule in lyophilized or liquid form, optionally together with one or more pharmaceutically acceptable carriers and/or excipients.

Optionally, the kit or kit of the present disclosure may further comprise other reagents, such as antimicrobial agents, rnase inhibitors, solubilizing agents, and the like.

The kit of parts may be a two or more part kit and typically contains the components thereof in a suitable container. For example, each container may be in the form of a vial, bottle, squeeze bottle, jar, sealed pouch, or the like, or any other suitable form, provided that the container is configured to prevent premature mixing of the components. Each of the different components may be provided separately or may be provided with some of the different components (i.e., in the same container). The kit may also contain instructions for any administration and dosage information regarding its components.

Medical use and treatment

The RNA nucleic acid molecules or compositions or vaccines or kits of the present disclosure may be used in humans, as well as for veterinary purposes, preferably for human medical purposes.

According to another aspect, the invention thus relates to an RNA nucleic acid molecule, composition, vaccine, or kit of the present disclosure for use as a medicament.

The RNA nucleic acid molecules, compositions, or vaccines or kits of the present disclosure can be used to treat genetic diseases, cancers, autoimmune diseases, inflammatory diseases, and infectious diseases or other diseases or conditions.

According to another aspect, the present invention thus relates to an RNA nucleic acid molecule, composition or vaccine or kit of the present disclosure for use in the treatment of genetic diseases, cancer, autoimmune diseases, inflammatory diseases and infectious diseases or other diseases or conditions.

"Gene therapy" preferably involves modulating gene expression in a subject to achieve a therapeutic effect. To this end, gene therapy generally involves introducing nucleic acids into cells. Gene therapy may involve in vivo or in vitro transformation of host cells.

The term "treating" a disease includes preventing the disease (i.e., causing no development of clinical symptoms); inhibiting the disease (i.e., preventing or inhibiting the progression of clinical symptoms); and/or to alleviate the disease (i.e., cause regression of clinical symptoms). It will be appreciated that it is not always possible to distinguish between "preventing" and "inhibiting" a disease or condition, as one or more of the final evoked events may be unknown or potential. Thus, the term "prevention" will be understood to constitute a type of "treatment" that encompasses both "prevention" and "inhibition". Thus, the term "treatment" includes "prophylaxis".

As used herein, the term "subject," "patient," or "individual" generally includes humans and non-human animals, and preferably includes mammals (e.g., non-human primates including marmosets, silk monkeys, spider monkeys, owl monkeys, long tail monkeys, squirrels monkeys, and baboons, macaque, chimpanzees, gorillas, cattle, horses, sheep, pigs, chickens, cats, dogs, mice, rats, rabbits, guinea pigs, etc.), including chimeric and transgenic animals and disease models. In this context, the term "subject" preferably refers to a non-human primate or human, most preferably a human.

Thus, the present disclosure also provides a method of treating a disease disclosed herein by administering to a subject in need thereof a pharmaceutically effective amount of an RNA nucleic acid molecule, composition or vaccine or kit, comprising administering to a patient/subject in need thereof a pharmaceutically effective amount of the RNA nucleic acid molecule, composition or vaccine or kit.

The RNA nucleic acid molecules or compositions or vaccines or kits of the present disclosure can be administered, for example, systemically or locally. Systemic routes of administration generally include, for example, transdermal, oral, parenteral routes including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injection and/or intranasal routes of administration. Topical routes of administration generally include, for example, topical routes, but also include intradermal, transdermal, subcutaneous or intramuscular injection or intralesional, intratumoral, intracranial, intrapulmonary, intracardiac, and sublingual injection.

According to a preferred embodiment, the RNA nucleic acid molecule, composition or vaccine or kit is administered by a parenteral route, preferably by an intradermal, subcutaneous or intramuscular route. Preferably, the RNA nucleic acid molecule composition or vaccine or kit may be administered by injection, e.g. subcutaneous, intramuscular, or intradermal injection, which may be needle-free injection and/or needle injection. Thus, in a preferred embodiment, the medical use and/or method of treatment according to the invention comprises administering the RNA nucleic acid molecule, composition or vaccine or kit by subcutaneous, intramuscular or intradermal injection, preferably by intramuscular or intradermal injection, more preferably by intradermal injection. Such injection may be performed by using conventional needle injection or (needle-free) jet injection, preferably by using (needle-free) jet injection.

The RNA nucleic acid molecules, (pharmaceutical) compositions or vaccines or kits of the present disclosure may be administered to a subject in need thereof several times daily, every other day, weekly or monthly; and may be administered sequentially or simultaneously.

If different RNA nucleic acid molecules, or compositions or vaccines or kits comprising several components, e.g., different RNA nucleic acid molecules and optionally other agents described herein alone, are administered, each component may be administered simultaneously (by the same or different routes of administration) or separately (by the same or different routes of administration at different times).

The RNA nucleic acid molecules, compositions or vaccines or kits of the present disclosure can preferably be administered in a therapeutically effective amount. As used herein, "therapeutically effective amount" refers to an amount of an active agent sufficient to elicit the desired biological or pharmaceutical response in the tissue, system, animal or human being sought. The therapeutically effective amount is preferably sufficient to induce a positive change in the disease to be treated, i.e. to alleviate symptoms of the disease to be treated, to reduce disease progression or to prevent symptoms of the disease to be prevented. At the same time, however, the "therapeutically effective amount" is preferably small enough to avoid serious side effects, that is to say to allow a reasonable relationship between advantage and risk, i.e. a safe and therapeutically effective amount.

The "therapeutically effective amount" will also vary with the particular condition being treated, as well as the age, physical condition, weight, sex and diet of the patient being treated, the severity of the condition, the duration of the treatment, the nature of the concomitant treatment, the particular pharmaceutically acceptable carrier or excipient used, the treatment regimen, and the like.

The "therapeutically effective amount" of an RNA nucleic acid molecule may also be selected according to the type of RNA nucleic acid molecule, e.g. monocistronic, bicistronic or even polycistronic RNA, since in case the amounts of monocistronic RNA are equal, the bicistronic or even polycistronic RNA may result in significantly higher expression of the encoded polypeptide or protein of interest.

Therapeutic efficacy and toxicity of the RNA nucleic acid molecules, compositions or vaccines or kits of the present disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective for 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD50/ED50 ratio. RNA nucleic acid molecules, compositions or kits that exhibit large therapeutic indices are generally preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.

For example, a therapeutically effective dose of an RNA nucleic acid molecule, composition or vaccine or kit described herein may be about 0.001mg to 10mg per dosage unit, preferably about 0.01mg to 5mg per dosage unit, more preferably about 0.1mg to 2mg per dosage unit or about 0.01nmol to 1mmol per dosage unit, in particular 1nmol to 1mmol per dosage unit, preferably 1 μmol to 1mmol per dosage unit. In some embodiments, a therapeutically effective dose of an RNA nucleic acid molecule, composition or vaccine or kit of the present disclosure may be about 0.01g/kg to 10g/kg, preferably about 0.05mg/kg to 5g/kg, more preferably about 0.1mg/kg to 2.5g/kg (per kg body weight).

Genetic disease

In a preferred embodiment, the RNA nucleic acid molecule, (pharmaceutical) composition or vaccine or kit is used for the treatment or prevention of a genetic disorder.

As used herein, the term "genetic disease" includes any disease, disorder or condition that is caused by, characterized by, or associated with genomic abnormalities (i.e., deviations from wild-type, healthy, and asymptomatic states). Such abnormalities may include changes in chromosome copy number (e.g., aneuploidy) or partial changes thereof (e.g., deletions, duplications, amplifications); or changes in chromosome structure (e.g., translocation, point mutation). Genomic abnormalities may be genetic (recessive or dominant) or non-genetic. Genomic abnormalities may be present in some cells of an organism or in all cells of the organism, and include autosomal abnormalities, X-linked abnormalities, Y-linked abnormalities, and mitochondrial abnormalities.

Cancer of the human body

In a preferred embodiment, the RNA nucleic acid molecule, composition or vaccine or kit is for use in the treatment or prevention of cancer.

As used herein, the term "cancer" refers to neoplasms characterized by uncontrolled and often rapid proliferation of cells, tending to invade surrounding tissue and metastasize to distant body sites. The term includes benign tumors and malignant tumors. Malignant tumors in cancer are often characterized by meta-changes, invasion and metastasis; benign tumors, however, often do not possess these properties. The term includes neoplasms that grow as tumors, as well as cancers of the blood and lymphatic systems.

In some embodiments, RNA nucleic acid molecules, compositions or vaccines or kits according to the present disclosure may be used as a medicament, in particular for the treatment of a tumor or cancer disease. In this case, the treatment preferably involves intratumoral administration, in particular by intratumoral injection. Thus, the RNA nucleic acid molecules, compositions or vaccines or kits according to the present disclosure may be used for the preparation of a medicament for the treatment of a tumor or cancer disease, which medicament is particularly suitable for intratumoral application (administration) for the treatment of a tumor or cancer disease.

Preferably, the tumor and cancer diseases mentioned herein are selected from tumor or cancer diseases preferably comprising: such as acute lymphoblastic leukemia, acute myelogenous leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal carcinoma, appendicular carcinoma, astrocytoma, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, osteosarcoma/malignant fibrous histiocytoma, brain stem glioma, brain tumor, cerebellar astrocytoma, brain astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuro-ectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma/carcinoid, Burkitt's lymphoma, childhood carcinoid tumor, gastrointestinal carcinoid tumor, unknown primary central nervous system lymphoma, childhood cerebellar astrocytoma, childhood brain astrocytoma/glioblastoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disease, colon cancer, cutaneous T-cell lymphoma, connective tissue proliferative microcylindrical tumor, endometrial cancer, ependymoma, esophageal cancer, ewing's sarcoma in a ewing's family of tumors, childhood extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, intraocular melanoma, retinoblastoma, cholecystocarcinoma, Gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, extracranial, extragonadal or ovarian germ cell tumors, gestational trophoblastoma, brain stem glioma, childhood brain astrocytoma, childhood visual pathway and hypothalamic glioma, gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, hodgkin's lymphoma, hypopharyngeal cancer, childhood hypothalamic and visual pathway glioma, intraocular melanoma, islet cell carcinoma, kaposi's sarcoma, renal cancer, laryngeal carcinoma, leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphoblastic leukemia, chronic granulocytic leukemia, hairy cell leukemia, lip and oral cancer, Liposarcoma, liver cancer, non-small cell lung cancer, lymphoma, AIDS-related lymphoma, burkitt's lymphoma, cutaneous T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, primary central nervous system lymphoma, megalobulinemia, malignant fibrous histiocytoma of bone/osteosarcoma, childhood medulloblastoma, melanoma, intraocular (ocular) melanoma, mercker's cell carcinoma, adult malignant mesothelioma, childhood mesothelioma, metastatic squamous neck carcinoma with occult primary, oral carcinoma, childhood multiple endocrine tumor syndrome, multiple myeloma/plasmacytoma, mycosis fungoides, myelodysplastic syndrome, Myelodysplasia/myeloproliferative disease, chronic myelogenous leukemia, adult acute myelogenous leukemia, pediatric acute myelogenous leukemia, multiple myeloma, chronic myelodysplastic, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal carcinoma, osteosarcoma/bone malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic islet cell pancreatic cancer, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, throat cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pediatric pineal tumor and on-screen primitive neuroectodermal tumor, Pituitary adenoma, plasmacytoma/multiple myeloma, pleural pneumoblastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, childhood rhabdomyosarcoma, salivary gland carcinoma, ewing family sarcoma, kaposi' S sarcoma, soft tissue sarcoma, uterine sarcoma, se zary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), merck cell skin cancer, small intestine cancer, squamous cell carcinoma, metastatic squamous neck cancer with occult primary, primary neuroectodermal tumor on childhood, testicular cancer, pharyngeal and laryngeal cancer, childhood thymoma, thymoma and thymus cancer, Thyroid cancer, childhood thyroid cancer, renal pelvis and ureter transitional cell carcinoma, gestational trophoblastic tumor, urinary tract cancer, endometrial carcinoma, uterine sarcoma, vaginal cancer, childhood visual pathway and hypothalamic glioma, vulvar cancer, megaloblastic and childhood wilms' tumor (renal cancer).

Infectious diseases

In a preferred embodiment, the RNA nucleic acid molecule, composition or vaccine or kit is used for the treatment or prevention of an infectious disease.

The term "infection" or "infectious disease" refers to the invasion and proliferation of microorganisms such as bacteria, viruses and parasites that are not normally present in the body. Infection may not cause any symptoms and is subclinical, or may cause symptoms and be clinically significant. The infection may remain localized or may spread through the blood or lymphatic system to become a systemic infection. In this case, the infectious disease preferably includes a viral, bacterial, fungal or protozoal infectious disease.

Autoimmune diseases

In a preferred embodiment, the RNA nucleic acid molecule, composition or vaccine or kit is used for the treatment or prevention of an autoimmune disease.

The term "autoimmune disease" refers to any disease, disorder or condition in a subject characterized by damage to cells, tissues and/or organs caused by the subject's immune response to its own cells, tissues and/or organs. In general, an "autoimmune disease" is caused or exacerbated by antibodies that are reactive with autoantigens (i.e., antigens expressed by healthy human cells).

Autoimmune diseases can be categorized as systemic symptoms including, but not limited to, systemic Lupus Erythematosus (SLE), sjogren's syndrome, scleroderma, rheumatoid arthritis, and polymyositis; or local syndrome, which may be endocrine (type I diabetes, hashimoto thyroiditis, addison's disease, etc.), dermatological (pemphigus vulgaris), hematological (autoimmune hemolytic anemia), nervous system (multiple sclerosis) or may involve almost any defined body tissue. In this context, the autoimmune disease may be selected from a type I or a type II or a type III or a type IV autoimmune disease, such as Multiple Sclerosis (MS), rheumatoid arthritis, diabetes, type I diabetes (type 1 diabetes), chronic multiple arthritis, sudden ocular goiter, autoimmune forms of chronic hepatitis, ulcerative colitis, type I allergic disease, type II allergic disease, type III allergic disease, type IV allergic disease, fibromyalgia, alopecia, bie Hejie sev, crohn's disease, myasthenia gravis, neurodermatitis, polymyalgia rheumatica, progressive Systemic Sclerosis (PSS), rette syndrome, rheumatoid arthritis, psoriasis, vasculitis and type II diabetes.

Inflammatory diseases

In a preferred embodiment, the RNA nucleic acid molecule, composition or vaccine or kit is for use in the treatment or prevention of an inflammatory disease.

The term "inflammatory disease" refers to any disease, disorder or condition in a subject characterized by, caused by, or associated with inflammation, preferably chronic inflammation. Autoimmune diseases may or may not be associated with inflammation. Furthermore, inflammation may or may not be caused by autoimmune diseases. Thus, certain diseases may be characterized as both autoimmune and inflammatory diseases.

Herein, exemplary inflammatory diseases include, but are not limited to, rheumatoid arthritis, crohn's disease, diabetic retinopathy, psoriasis, endometriosis, alzheimer's disease, ankylosing spondylitis, arthritis (e.g., osteoarthritis, rheumatoid Arthritis (RA), psoriatic arthritis), asthma, atherosclerosis, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable Bowel Syndrome (IBS), systemic Lupus Erythematosus (SLE), nephritis, parkinson's disease, and ulcerative colitis.

Allergy reaction

In a preferred embodiment, the RNA nucleic acid molecule, composition or vaccine or kit is for use in the treatment or prevention of allergy.

The term "allergy" or "allergic hypersensitivity" refers to any disease, disorder or condition that is usually caused by or characterized by an immune mechanism against allergy-induced hypersensitivity in genetically susceptible individuals (atopy). Allergies may be antibody-mediated or cell-mediated. In most patients, antibodies that normally elicit allergic reactions belong to the IgE isotype (IgE-mediated allergic reactions, type I allergic reactions). In non-IgE-mediated allergies, antibodies may belong to the IgG isotype. Allergies may be classified according to the source of the antigen that causes the allergy. In this context, the allergy may be selected from (a) food allergy, (b) drug allergy, (c) house dust allergy, (d) insect venom or biting allergy, and (e) pollen allergy. Or the allergies may be classified according to their main symptoms. In this context, the allergy may be selected from (a) asthma, (b) rhinitis, (c) conjunctivitis, (d) rhinoconjunctivitis (rhinoconjuctivitis), (e) dermatitis, (f) urticaria and (g) allergy.

Combination therapy

The RNA nucleic acid molecules, compositions or vaccines or kits of the present disclosure may also be used in combination therapies. Any other therapies useful for treating or preventing the diseases and conditions defined herein may be combined with the uses and methods disclosed herein.

For example, a subject receiving an RNA nucleic acid molecule, composition, or vaccine or kit of the present disclosure may be a patient suffering from cancer or a related disorder that has received chemotherapy (e.g., first-line or second-line chemotherapy), radiation therapy, chemoradiation therapy (a combination of chemotherapy and radiation therapy), tyrosine kinase inhibitors (e.g., EGFR tyrosine kinase inhibitors), antibody therapy, and/or inhibitory and/or stimulatory checkpoint molecules (e.g., CTLA4 inhibitors), or a patient who has achieved partial remission or disease stabilization after receiving one or more of the above. Or a subject receiving an RNA nucleic acid molecule, composition or vaccine or kit of the present disclosure may be a patient suffering from an infectious disease, preferably as defined herein, receiving antibiotic, antifungal or antiviral treatment.

Administration of the RNA nucleic acid molecules, compositions, or vaccines or kits of the present disclosure can be performed prior to, concurrently with, and/or subsequent to administration of another therapeutic agent or subjecting the patient to another treatment useful for treating a particular disease or disorder.

The present disclosure describes a novel cell-free in vitro transcribed RNA platform or system for producing therapeutic mRNA, taking as an example mRNA encoding human basic fibroblast growth factor (hbFGF), human epidermal growth factor (hEGF), and Green Fluorescent Protein (GFP). In this system there are one or more of the following advantages: the mRNA produced has high stability; can be easily applied to cells; the translation efficiency is better; has rapid production time (not longer than 1 hour); the yield can reach 2mg/ml/h, even about 2.3mg/ml/h; easy to manage; and low production cost.

Examples

The disclosure is described herein by the following examples, which are intended to be illustrative only and are not limiting on the scope of the disclosure.

Materials and methods:

Design of DNA oligonucleotides for RNA in vitro transcription

As shown in FIG. 1A, the DNA oligonucleotide construct was designed starting from EcoRI before the T7 promoter. The signal sequence and coding sequence (hEGF, hbFGF, GFP) of the target gene are added between the 5 'untranslated sequence of human beta globin and the 3' untranslated sequence of human alpha globin, respectively. To terminate transcription, a T7 terminator was added at the 5' end of the construct. For general cloning, salI and NotI were added to the spacer sequence. The DNA oligonucleotide was synthesized from ThermoFisher Scientific. The sequences of the DNA oligonucleotide constructs are shown in SEQ ID NO. 5-7 respectively.

SEQ ID NO:5(hEGF)

5'-GGGAGAGTCGACAAATAAGAGAGAAAAGAAGAGTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGCTGCTCACTCTTATCATTCTGTTGCCAGTAGTTTCAAAAAATAGTGACTCTGAATGTCCCCTGTCCCACGATGGGTACTGCCTCCATGATGGTGTGTGCATGTATATTGAAGCATTGGACAAGTATGCATGCAACTGTGTTGTTGGCTACATCGGGGAGCGATGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGCTGAGCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGGCGGCCGCCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATGAATTCGC-3'

SEQ ID NO:6(hbFGF)

5'-GGGAGAGTCGACAAATAAGAGAGAAAAGAAGAGTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGCAGCCGGGAGCATCACCACGCTGCCCGCCTTGCCCGAGGATGGCGGCAGCGGCGCCTTCCCGCCCGGCCACTTCAAGGACCCCAAGCGGCTGTACTGCAAAAACGGGGGCTTCTTCCTGCGCATCCACCCCGACGGCCGAGTTGACGGGGTCCGGGAGAAGAGCGACCCTCACATCAAGCTACAACTTCAAGCAGAAGAGAGAGGAGTTGTGTCTATCAAAGGAGTGTGTGCTAACCGTTACCTGGCTATGAAGGAAGATGGAAGATTACTGGCTTCTAAATGTGTTACGGATGAGTGTTTCTTTTTTGAACGATTGGAATCTAATAACTACAATACTTACCGGTCAAGGAAATACACCAGTTGGTATGTGGCACTGAAACGAACTGGGCAGTATAAACTTGGATCCAAAACAGGACCTGGGCAGAAAGCTATACTTTTTCTTCCAATGTCTGCTAAGAGCTGAGCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGGCGGCCGCCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATGAATTCGC-3'

SEQ ID NO:7(GFP)

5'-GGGAGAGTCGACAAATAAGAGAGAAAAGAAGAGTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGGCGGCCGCCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATGAATTCGC-3'

In vitro transcription of RNA

PCR templates for in vitro transcription were prepared by PCR extension using the above designed DNA oligonucleotides as templates with the oligonucleotides P1 (5'-AATTCATCCGGATATAGTTC-3' (SEQ ID NO: 8)) and P2 (5'-TGTACATAATACGACTCACTAT-3' (SEQ ID NO: 9)) as primers. PCR products were obtained by NEW ENGLAND Biolabs (Isplasivelqi, mass.)The PCR & DNA Cleanup kit was purified and then subjected to standard mRNA synthesis, which was performed as described by NEW ENGLAND Biolabs. Mu.g of purified PCR product was mixed with [1.5mM ATP,1.25mM UTP,1.25mM CTP,1mM GTP,4mM 7m G (3 '-O-Me) pppG (ARCA), 1U/. Mu.l RNase inhibitor, 0.4U/. Mu. l T7 RNA polymerase, 40mM Tris-HCl (pH 7.9), 6mM MgCl ₂, 2mM spermidine ] and incubated at 37℃for 30 minutes to produce 5' -ARCA-capped mRNA. Then 0.2U/. Mu.l DNase I was added to the reaction mixture and incubated at 37℃for 15 minutes to remove the PCR template. Poly (A) tailing was performed by an additional Poly (A) polymerase reaction mixture [50mM Tris-HCl (pH 8.1), 250mM NaCl,10mM MgCl ₂, 0.05U/. Mu.l Poly (A) polymerase ] and incubated at 37℃for 30 min. The final mRNA product was obtained by NEW ENGLAND Biolabs (Isplasivisqi, mass., U.S.A.)The RNA clearup kit was purified and saved for analysis. The structure of the mRNA obtained is shown in FIG. 1B, and the sequences of the transcribed mRNAs are shown in SEQ ID NOs: 10-12.

SEQ ID NO:10(hEGF)

5'-GGGAGAGUCGACAAAUAAGAGAGAAAAGAAGAGUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCAUGCUGCUCACUCUUAUCAUUCUGUUGCCAGUAGUUUCAAAAAAUAGUGACUCUGAAUGUCCCCUGUCCCACGAUGGGUACUGCCUCCAUGAUGGUGUGUGCAUGUAUAUUGAAGCAUUGGACAAGUAUGCAUGCAACUGUGUUGUUGGCUACAUCGGGGAGCGAUGUCAGUACCGAGACCUGAAGUGGUGGGAACUGCGCUGAGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGGCGGCCGC-3'

SEQ ID NO:11(hbFGF)

5'-GGGAGAGUCGACAAAUAAGAGAGAAAAGAAGAGUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCAUGGCAGCCGGGAGCAUCACCACGCUGCCCGCCUUGCCCGAGGAUGGCGGCAGCGGCGCCUUCCCGCCCGGCCACUUCAAGGACCCCAAGCGGCUGUACUGCAAAAACGGGGGCUUCUUCCUGCGCAUCCACCCCGACGGCCGAGUUGACGGGGUCCGGGAGAAGAGCGACCCUCACAUCAAGCUACAACUUCAAGCAGAAGAGAGAGGAGUUGUGUCUAUCAAAGGAGUGUGUGCUAACCGUUACCUGGCUAUGAAGGAAGAUGGAAGAUUACUGGCUUCUAAAUGUGUUACGGAUGAGUGUUUCUUUUUUGAACGAUUGGAAUCUAAUAACUACAAUACUUACCGGUCAAGGAAAUACACCAGUUGGUAUGUGGCACUGAAACGAACUGGGCAGUAUAAACUUGGAUCCAAAACAGGACCUGGGCAGAAAGCUAUACUUUUUCUUCCAAUGUCUGCUAAGAGCUGAGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGGCGGCCGC-3'

SEQ ID NO:12(GFP)

5'-GGGAGAGUCGACAAAUAAGAGAGAAAAGAAGAGUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCAUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAAGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGGCGGCCGC-3'

MRNA transfection

Transfection of mRNA was accomplished with lipid nanoparticles. Briefly, DC-cholesterol (3β - [ N- (N ', N' -dimethylaminoethane) -carbamoyl ] cholesterol hydrochloride) and DOPE (dioleoyl phosphatidylethanolamine) were dissolved at 25mg/mL in chloroform. 40. Mu.L of DC-cholesterol was mixed with 80. Mu.L of DOPE and dried in a vacuum concentrator for 15 minutes to evaporate chloroform. When the lipids were resuspended in 1mL nuclease free water and emulsified in a 35kHz ultrasonic bath for 1 hour, then extruded in a small extruder.

10. Mu.L of lipid nanoparticles were diluted in 100. Mu.L of serum-free DMEM medium, then mixed with 1. Mu.g of the obtained mRNA and incubated at room temperature for 15 minutes. The mRNA-lipid nanoparticle complex was then added to 293T cells for incubation for the indicated time.

Protein detection

24 Hours after transfection, 293T cells transfected with GFP mRNA were observed under PlanApo 20x/0.75DIC lens under a Nikon ECLIPSE TI inverted microscope equipped with an Andor EM-CCD camera. The bright field and GFP images were taken with the corresponding filter settings as shown in fig. 3. 293T cells transfected with hEGF and hbFGF mRNA were incubated for 24 hours. Cells and media were collected 0, 2,4, 6, 8, 24 hours post-transfection. Cells were lysed in lysis buffer (pbs+1% triton and complete protease inhibitor). Cell lysate samples were analyzed for hbFGF and hEGF expression by Western blot. The results are shown in fig. 5.

Real-time PCR

Transfected 293T cells were lysed and their total RNA was extracted with RNAzol reagent (molecular research center). The yield of RNA was quantified using Qubit. 100ug of RNA was reverse transcribed with GoScript reverse transcriptase (Promega) using Oligodt primers. cDNA samples were analyzed by pre-designed real-time PCR primers/probes and hEGF and hbFGF were detected with QuantStudio and SYBR Green I premix. All samples were run in triplicate. Average gene expression was calculated in 3 independent experiments. As shown in fig. 2, the in vitro transcription yields of EGF and bFGF mRNA collected at different time points were quantified with Qubit. When in vitro transcription was performed for one hour, the maximum yield reached 2250. Mu.g/mL.

Results:

engineering and in vitro transcription of mRNA

The DNA oligonucleotide consists of a T7 promoter, a human beta globin 5 '-untranslated sequence, a signal sequence and a coding sequence (hEGF, hbFGF and GFP) of a target gene, a human alpha globin 3' -untranslated sequence and a T7 terminator. The oligonucleotides act as stable in vitro transcribed backbones for hEGF, hbFGF and GFP. mRNA was transcribed by T7 polymerase and anti-reverse cap analogs and polyA tails were added at the 5 'and 3' ends, respectively, to enhance mRNA stability and translation efficiency. Various time points during in vitro transcription were used for analysis. When the reaction time was 1 hour, the maximum yield per ml of mRNA was about 2.3mg.

FIG. 4 shows quantification of intracellular EGF and bFGF mRNA in 293T cells by qPCR. 0.5. Mu.g EGF and bFGF mRNA encapsulated with lipid nanoparticles were transfected into 293T cells. EGF and bFGF mRNA were quantified in cells collected at different time points after transfection using qPCR. As can be seen from fig. 4, the concentrations of EGF and bFGF mRNA remained stable for 8 hours under standard 293T cell culture conditions and eventually decreased only after transfection to 24 hours.

GFP-expressing mRNA transfection 293T cells

293T cells were transfected with lipid nanoparticles encapsulating 0.5ug of GFP-expressing mRNA and incubated for 24 hours. Under fluorescence microscopy, more than 85% of transfected cells showed fluorescent signals under GFP filter, as shown in figure 3. mRNA encoding GFP encapsulated by lipid nanoparticles has been successfully transfected into 293T cells. The fluorescent signal of GFP mRNA transfected 293T cells increased over time.

QPCR detection of hEGF and hbFGF mRNA

293T cells were transfected with lipid nanoparticles encapsulating 0.5ug hEGF mRNA and 0.5ug hbFGF mRNA, respectively. RNA probes with high specificity for hEGF and hbFGF were used to quantify mRNA levels. The levels of hEGF and hbFGF mRNA remained stable for the first 8 hours of the entire culture, and only slightly decreased at 24 hours, demonstrating that mRNA was very stable, as shown in fig. 4.

Western blotting method for detecting hEGF and hbFGF

293T cells transfected with hEGF and hbFGF mRNA were collected at 0, 2, 4, 6, 8, 24 hours post-transfection. Cell lysates and cell culture media were prepared and analyzed by western blotting for hEGF and hbFGF antibodies. As shown in fig. 5, expression of both hEGF and hbFGF in cell lysates increased significantly after 4 hours post-transfection; on the other hand, hEGF and hbFGF levels in the cell culture medium were observed and significantly increased after 6 hours post-transfection.

Although various embodiments of methods and constructs for RNA in vitro transcription have been described in great detail herein, these embodiments are provided merely as non-limiting examples of the disclosure described herein. Accordingly, those skilled in the art will appreciate that various changes and modifications can be made to the arrangements described in the present disclosure without departing from the spirit of the invention. Indeed, the present disclosure is not intended to be exhaustive or to limit the scope of the invention.

Further, in the description of representative embodiments, the present disclosure has presented the methods and/or processes of the present invention in a particular sequence of steps. However, the method or process should not be limited to the particular sequence of steps described. Other sequences of steps are possible. Therefore, the particular order of the steps disclosed herein should not be construed as limitations on the invention. Furthermore, the disclosure directed to methods and/or processes should not be limited to performing their steps in the order described. Such order may be altered and still be within the scope of the invention.

Sequence listing

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<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

gggagagucg acaaauaaga gagaaaagaa gaguacauuu gcuucugaca caacuguguu 60

cacuagcaac cucaaacaga caccauggug agcaagggcg aggagcuguu caccggggug 120

gugcccaucc uggucgagcu ggacggcgac guaaacggcc acaaguucag cguguccggc 180

gagggcgagg gcgaugccac cuacggcaag cugacccuga aguucaucug caccaccggc 240

aagcugcccg ugcccuggcc cacccucgug accacccuga ccuacggcgu gcagugcuuc 300

agccgcuacc ccgaccacau gaagcagcac gacuucuuca aguccgccau gcccgaaggc 360

uacguccagg agcgcaccau cuucuucaag gacgacggca acuacaagac ccgcgccgag 420

gugaaguucg agggcgacac ccuggugaac cgcaucgagc ugaagggcau cgacuucaag 480

gaggacggca acauccuggg gcacaagcug gaguacaacu acaacagcca caacgucuau 540

aucauggccg acaagcagaa gaacggcauc aaggugaacu ucaagauccg ccacaacauc 600

gaggacggca gcgugcagcu cgccgaccac uaccagcaga acacccccau cggcgacggc 660

cccgugcugc ugcccgacaa ccacuaccug agcacccagu ccgcccugag caaagacccc 720

aacgagaagc gcgaucacau gguccugcug gaguucguga ccgccgccgg gaucacucuc 780

ggcauggacg agcuguacaa guaagcugga gccucgguag ccguuccucc ugcccgcugg 840

gccucccaac gggcccuccu ccccuccuug caccggcccu uccuggucuu uggcggccgc 900

Claims

1. An RNA nucleic acid molecule comprising from 5' end to 3' end a 5' -cap structure, a human β -globin 5' -UTR, at least one coding region, a human α -globin 3' -UTR, and a 3' -poly a tail, said at least one coding region operably linked to said 5' -UTR and said 3' -UTR, said 5' -UTR comprising or consisting of a sequence according to SEQ ID NO:1, and a sequence of RNA; and wherein the 3' -UTR comprises or consists of a sequence according to SEQ ID NO:2, said coding region is the RNA sequence of hEGF,

The sequence of the RNA nucleic acid molecule comprises or consists of a sequence according to SEQ ID NO:10, an RNA sequence;

The in vitro transcription of the RNA nucleic acid molecule has a production time of not more than 1 hour and the yield can reach 2mg/ml/h.

2. The RNA nucleic acid molecule of claim 1, wherein the 5 '-cap structure is a 5' -anti-reverse cap analogue.

3. The RNA nucleic acid molecule of claim 2, wherein the 5 '-anti-reverse cap analogue is 7m G (3' -O-Me) pppG.

4. The RNA nucleic acid molecule of any one of claims 1-3, wherein the 3' -poly a tail comprises 10 to 200 adenine nucleotides.

5. A DNA nucleic acid molecule comprising, from the 5' end to the 3' end, a promoter, a human β -globin 5' -UTR, at least one coding region, a human α -globin 3' -UTR, and a transcription terminator, said at least one coding region being operably linked to said 5' -UTR and said 3' -UTR, said 5' -UTR comprising or consisting of a sequence according to SEQ ID NO:3, a DNA sequence of 3; and wherein the 3' -UTR comprises or consists of a sequence according to SEQ ID NO:4, said coding region is the DNA sequence of hEGF,

The sequence of the DNA nucleic acid molecule comprises or consists of a sequence according to SEQ ID NO:5, a DNA sequence;

in vitro transcription of RNA nucleic acid molecules obtained by using the DNA nucleic acid molecules as transcription templates has a production time of not more than 1 hour, and the yield can reach 2mg/ml/h.

6. The DNA nucleic acid molecule of claim 5, wherein the promoter is selected from the group consisting of a T3, T7, sny, or SP6 promoter, and the transcription terminator is a T7 transcription terminator.

7. An in vitro transcription method comprising the steps of:

(a) Providing a DNA nucleic acid molecule according to any one of claims 5-6 as a transcription template;

(b) Amplifying the DNA nucleic acid molecule;

(c) Subjecting the DNA nucleic acid molecule to in vitro transcription in the presence of a 5 '-anti-reverse cap analogue to obtain a reaction mixture comprising a 5' -ARCA-terminated mRNA;

(d) Removing the transcription template from the reaction mixture comprising 5' -ARCA-terminated mRNA by adding dnase; and

8. A composition comprising an RNA nucleic acid molecule according to any one of claims 1-4 and/or obtained according to the method of claim 7, and a pharmaceutically acceptable carrier and/or excipient.