Alphavirus-based replicons for administration of biotherapeutics

Abstract

To provide RNA replicons useful for administering a heterologous protein or peptide into a mammal and eliciting a reduced immune response or no immune response from the mammal.SOLUTION: The RNA replicon comprises RNA sequences encoding a heterologous protein or peptide, New World alphavirus nonstructural proteins nsP1, nsP2, and nsP4, and an alphavirus nsP3 protein macro domain, central domain, and hypervariable domain. The encoded hypervariable domain may have an amino acid sequence derived from an Old World alphavirus nsP3 hypervariable domain, or may have an amino acid sequence derived from a portion of a New World alphavirus nsP3 hypervariable domain and another portion derived from an Old World alphavirus nsP3 hypervariable domain.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed on October 1, 2018, the disclosure of which is incorporated herein by reference in its entirety. This application claims priority to U.S. Provisional Patent Application No. 62 / 742,868, filed on August 8, 2008. be.

[0002] Reference to an electronically submitted sequence listing The application has a file name " 689405.107WO_SL" as an ASCII format sequence table in EFS - Submitted electronically via the Web, including a sequence listing. The sequence listing provided is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Alphavirus-based self-amplifying RNA (replicon) is a versatile platform for vaccines. Replicons have been used for decades as a platform for The two properties that make it a successful candidate are 1) high and long-lasting protein expression and 2) robust cell proliferation. Platform autoadjuvants that boost cytotoxic, humoral, and mucosal immunity These properties make replicons excellent vaccine platforms. However, in other areas, platforms are becoming increasingly popular, particularly for the in vivo expression of biologics. This may interfere with the replicon's performance as a system.

[0004] The use of biological agents in medicine is increasing. However, recombinantly produced These proteins are often recognized as foreign and trigger immune responses and anti-drug antibodies (AD). A) and consequently reduce the therapeutic efficacy of the protein. A) The factors responsible for the occurrence of a response are diverse, but the biological agent or biologic is delivered The resulting inflammatory environment may promote and / or enhance the ADA response. Therefore, for the delivery of replicon-derived biologics that are naturally self-adjuvanting and proinflammatory. This may enhance the ADA response and reduce the clinical efficacy of the encoded biologic. downregulating the immune response to the heterologous protein expressed from the replicon. This ability to inhibit ADA production reduces the risk of developing biologics in vivo. This will likely increase the usefulness of replicons in the current situation.

[0005] Therefore, it is possible to produce a human or animal model with reduced or eliminated risk of eliciting an unwanted immune response. It is useful to have compositions and methods that allow for the administration of biologic molecules to a subject or animal. It is thought to be useful. Summary of the Invention

[0006] The present invention provides a method for administering heterologous molecules to humans or animals, and detecting the immunity of the humans or animals to the heterologous molecules. The RNA replicons of the present invention are useful for reducing or eliminating an immune response. A replicon is an RNA sequence (e.g., For example, the gene of interest (GOI) is contained in the RNA sequence, and the RNA sequence is the nonstructural template of the New World alphavirus. proteins nsP1, nsP2, and nsP4; and nsP3 proteins of alphaviruses It encodes the protein macrodomain, central domain, and hypervariable domain (HVD). The encoded hypervariable domain is similar to the Old World alphavirus nsP3 hypervariable domain. or may have an amino acid sequence derived from a New World alphavirus nsP3 superfamily. Amino acid sequences derived from parts of the variable domain and the Old World alphavirus nsP3 superfamily The chimeric nsP3 hypervariable domain may also have another portion derived from the chimeric nsP3 hypervariable domain. When New World alphavirus-based replicons are modified as described herein, Reduced immune response triggered by the encoded foreign protein or peptide or was found to be eliminated.

[0007] RNA replicons are useful for the administration of biopharmaceutical molecules such as proteins and peptides. The replicon of the present invention, together with the biological agent encoded by the replicon, administered to humans or animals and encoding biological products (e.g., heterologous proteins or The polypeptide (or peptide) is expressed in a human or animal.

[0008] In a first aspect, the present invention provides a method for the preparation of a heterologous protein or peptide encoding an RNA sequence. 5' and 3' alphavirus untranslated regions and nonstructural regions of New World alphaviruses RNA encoding amino acid sequences derived from proteins nsP1, nsP2, and nsP4 A sequence encoding an amino acid sequence derived from the alphavirus nsP3 macrodomain. and an amino acid sequence derived from the central domain of alphavirus nsP3. RNA sequences encoding amino acids derived from the Old World alphavirus nsP3 hypervariable domain RNA sequences encoding hypervariable domains with nucleotide sequences, or New World alphaviruses Portions derived from the nsP3 hypervariable domain and the Old World alphavirus nsP3 hypervariable domain RNA replicas having RNA sequences encoding amino acid sequences containing portions derived from the Provide a recon.

[0009] In some embodiments, the alphavirus nsP3 macrodomain and the alphavirus The nsP3 central domain is derived from a New World alphavirus, although in other embodiments, The lufavirus nsP3 macrodomain and alphavirus nsP3 central domain Derived from an Old World alphavirus. In various embodiments, the Old World alphavirus is Selected from the group consisting of CHIKV, SINV, and SFV. New World Alphavirus The virus is Venezuelan equine encephalitis virus (VEEV) or Western equine encephalitis virus (WEEV). ), or Eastern Equine Encephalitis Virus (EEEV). In various embodiments, Old World alphaviruses include Sindbis virus (SINV), Chikungunya virus (Chikugunya), and others. ngunya virus (CHIKV), Semliki Forest virus (SFV), Ross River virus RSV, Sagiyama virus (SAGV), Getah virus (GETV), Middle bar MIDV, Bebale virus (BEBV), Onyongnyong virus (ON NV), Nudum (NDUV), and Barmah Forest virus (BFV) stomach.

[0010] In some embodiments, a portion derived from an Old World alphavirus nsP3 hypervariable domain is selected from the group consisting of FGDF (SEQ ID NO: 18) and FGSF (SEQ ID NO: 19). The portion derived from the Old World alphavirus nsP3 hypervariable domain contains the motif FGDF / FGDF (SEQ ID NO: 20) repeats, FGSF / FGSF (SEQ ID NO: 21) repeats, FGDF / FGSF (SEQ ID NO: 22) repeats, and FGSF / FGDF (SEQ ID NO: 23) repeats; and the repeat sequence can have repeats selected from the group consisting of: The amino acids may be separated by at least 10 and no more than 25 amino acids. In the example, the repeat sequence is NEGEIESLSSELLT (SEQ ID NO: 6), SDGEIDELS RRVTTESEPVL (SEQ ID NO: 7), and DEHEVDALASGIT (SEQ ID NO: 8).

[0011] In any of the embodiments of the RNA replicon, an Old World alphavirus hypervariable domain The portion derived from amino acids 479 to 482 or 483 of CHIKV nsP3 HVD 97-500 or 479-500 or 335-517; or SFV Amino acids 451 to 454 or 468 to 471 or 451 to 454 of nsP3 HVD or amino acids 490-493 of the SINV nsP3 HVD; can have 513-516 or 490-516 or 335-538 In any of these embodiments (or any embodiment described herein) , New World alphaviruses can be VEEV, New World alphavirus hypervariable The region derived from the domain is amino acids 478-51 of the VEEV nsP3 hypervariable domain. 8; or amino acids 478 to 545 of the VEEV nsP3 hypervariable domain or does not contain amino acids 335 to 518 of the VEEV nsP3 hypervariable domain In other embodiments, the New World alphavirus can be EEEV, and the New World The portion derived from the alphavirus hypervariable domain is the amino acid sequence of the EEEV hypervariable domain. 531-547 are not included. Alternatively, New World Alphaviruses may be referred to as WEEV. The portion derived from the New World alphavirus hypervariable domain is the WEEV hypervariable domain. It does not contain amino acids 504 to 520 of the amino acid.

[0012] In any of the embodiments, the RNA replicon comprises an RNA sequence encoding a heterologous protein. a subgenomic promoter operably linked to the sequence and controlling translation of the RNA sequence The RNA replicon may also have a 5' cap and a 3' polyA tail. The RNA replicon can have a positive-sense single-stranded RNA. In various embodiments, the RNA replicon can have an RNA of 10-12 kb. and / or have a diameter of 30 to 50 nm.

[0013] In various embodiments, the heterologous protein is a biologic protein or peptide. These include, for example, antibodies or engineered chimeric antibodies or antibody fragments, antigenic polypeptides, or any other therapeutic or immunogenic polypeptide or peptide It can be done.

[0014] In some particular embodiments of the replicon, the New World alphavirus is VEEV; The portion derived from the New World alphavirus nsP3 hypervariable domain is VEEV nsP3 It does not contain the hypervariable domain amino acids 335-518 and is different from Old World alphavirus nsP3. The portion derived from the variable domain is amino acids 490–516 of SINV nsP3 HVD. or the Old World alphavirus is SINV, including Old World alphaviruses ns The portion derived from the P3 hypervariable domain is amino acid 335–336 of the SINV nsP3 HVD. Including 538.

[0015] In any of the embodiments, the RNA sequence encoding the heterologous protein or peptide is operably linked to RNA sequences encoding nsP1, nsP2, and nsP4 can be done.

[0016] In another aspect, the present invention provides a method for administering a heterologous protein or peptide to a mammal. The method of the present invention provides a method for producing a heterologous protein or peptide, as described herein, Administering the RNA replicon described above to a mammal, wherein the heterologous protein or The peptide is expressed in a mammal. Any of the listed ones is acceptable.

[0017] In another aspect, the present invention provides a method for producing a heterologous protein or peptide comprising: Derived from the nonstructural proteins nsP1, nsP2, and nsP4 of New World alphaviruses RNA sequences encoding the amino acid sequences corresponding to the nsP3 proteins of Old World alphaviruses and an RNA sequence encoding an amino acid sequence derived from the protein. and the first 1 to 6 amino acids at the N-terminus and / or C-terminus of the nsP3 protein. provides an RNA replicon derived from a New World alphavirus sequence. 1 to 6 amino acids may be present at the junction between nsP2 and nsP3; or 1 to Six amino acids may be present at the junction between nsP3 and nsP4. The Old World alphavirus may be any of those described herein. If the alphavirus is VEEV, the nsP2 / nsP3 junction sequence is LHEAGC / APSY (SEQ ID NO: 12); if the junction is an nsP3 / nsP4 junction, In this case, the sequence can be RFDAGA / YIFS (SEQ ID NO: 13). In either of the following, the penultimate glycine (also referred to as its single-letter code "G") can be conserved, and the remaining amino acids of nsP3 can be varied as described herein. The junction sequence can be optionally preceded by a stop codon (TGA), This can be a read-through stop codon. New World alphaviruses are known as EEEV In other embodiments, the nsP2 / nsP3 junction sequence is QHEAGR / APAY (sequence The penultimate G is conserved. If the virus is EEEV, the sequence at the nsP3 / nsP4 junction is RYEAGA / YI FS (SEQ ID NO: 15), with the penultimate glycine optionally being conserved. The remaining amino acids of nsP3 can be varied as described herein. These sequences can also be preceded by a read-through stop codon (TGA). In another embodiment, the New World alphavirus is WEEV and has an nsP2 / nsP3 junction. The combination sequence can be RYEAGR / APAY (SEQ ID NO: 16), with the penultimate The G is conserved, but the remaining amino acids at the nsP2 / nsP3 junction are as described herein. In the case of the nsP3 / nsP4 junction of WEEV, the sequence is RYEAGA / Y IFS (SEQ ID NO: 17), where the penultimate glycine is conserved and the remaining The amino acids of nsP3 in the The sequence may be preceded by a read-through stop codon (TGA). The sequences of columns 12 to 17 may also contain one or two or more amino acids on the N-terminal and / or C-terminal sides. may contain three substitutions.

[0018] The above summary of the invention is not limiting, and other features and advantages of the invention may be found in the following detailed description of the invention. This will become apparent from the following detailed description and from the claims. Section headings or subheadings are provided solely as a convenience to the reader and are not intended to be limiting. It does not represent a departure from the current discussion or necessarily an entirely new subject area. may be discussed or disclosed under any section heading or subheading.

[0019] The foregoing summary, as well as the following detailed description of the present invention, is best understood in conjunction with the accompanying drawings. It will be understood that the invention is not limited to the precise embodiments shown in the drawings. Please understand that this is not the case. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 provides a pictorial illustration of a wild-type alphavirus. The viral particle is shown as an enveloped, spherical, icosahedral capsid with a diameter of 65-70 nm and T=4 icosahedral symmetry composed of 240 monomers. The envelope contains 80 spikes, each a trimer of E1 / E2 proteins. [Figure 2]Figure A2 is a diagrammatic illustration of a VEEV-based alphavirus replicon encoding red firefly luciferase (rFF). Three embodiments are shown: one with the wild-type VEEV HVD (WT) in nsP3; one VEEV / SINV hybrid (VEEV / SINV) with a portion of the SINV HVD (by substituting amino acid residues 335-538 of the VEEV HVD for amino acids 335-538 of the SINV HVD); and another hybrid (VEEV / CHIKV) with a portion of the CHIKV HVD (by substituting amino acid residues 335-518 of the VEEV HVD for amino acids 335-517 of the CHIKV HVD). Figures 2B and 2C are diagrammatic illustrations showing that replicons containing mutant nsP3 proteins replicate to the same levels (Figure 2B) and express the same levels of rFF (Figure 2C) as replicons containing wild-type nsP3. [Figure 3A] Figure 3A shows the results of monitoring in vivo luciferase activity, reported as total flux. Ten micrograms of each of the three VEEV-based alphavirus replicon RNAs described in Figure 2A in saline was delivered intramuscularly to the quadriceps muscle of BALB / c mice. α.SGI.rFF encodes VEEV WT, α.SGI.SINV.rFF encodes VEEV / SINV, and α.SGI.CHIKV.rFF encodes VEEV / CHIKV. [Figure 3B] Figure 3B shows the same results as in Figure 3A, but monitored using 1 μg of replicon RNA. Replicons expressing mutant forms of nsP3 exhibited similar levels of luciferase activity as replicons with wild-type nsP3. [Figure 4] 1 is a plot and bar graph showing the results of an in vivo study of VEEV-based replicons expressing HA from H5N1 influenza virus. The data show that replicons encoding VEEV / CHIKV HVD chimeras did not elicit HA-specific IgG titers compared to replicons expressing wild-type HVD. [Figure 5A] Figure 5A provides plots in a diagrammatic format demonstrating the frequency of HA-specific short-lived effector CD8+ T cells (SLECs) in BALB / c mice immunized with the indicated VEEV-based replicons expressing H5N1 HA. WT refers to the unmodified replicon backbone derived from the TC-83 strain of VEEV; SGI refers to the replicon backbone modified to be interferon-resistant. [Figure 5B] Figure 5B provides plots in a diagrammatic format demonstrating the frequency of HA-specific memory precursor effector CD8+ T cells (MPEC) in BALB / c mice immunized with the indicated VEEV-based replicons expressing H5N1 HA. WT refers to the unmodified replicon backbone derived from the TC-83 strain of VEEV; SGI refers to the replicon backbone modified to be interferon-resistant. [Figure 6]FIG. 1 provides a partial domain structure and sequence alignment of nsP3 proteins from representative members of New and Old World alphaviruses. The schematic diagram of the nsP3 protein shows three predicted structural domains: the macrodomain, the alpha domain, and the HVD. Sequence alignment of the nsP3 proteins from various alphaviruses was performed using Clustal Omega. Domain sequences are underlined in the same color as used in the schematic diagram. Exemplary nsP3 protein sequences were derived from the following viruses: SFV (GenBank accession number NP_740667.1) (protein shown is SEQ ID NO: 24), SINV (GenBank accession number P03317.1) (protein shown is SEQ ID NO: 25), CHIKV (GenBank accession number NP_690588.1) (protein shown is SEQ ID NO: 26), VEEV (GenBank accession number P27282.2) (protein shown is SEQ ID NO: 27), and EEEV (GenBank accession number Q4QXJ8.2) (protein shown is SEQ ID NO: 28). Image from Foy et al., Journal of Virology, Vol. 87, No. 4, pp. 1997-2010 (2013). [Figure 7]Figure 1 provides an illustration showing various regions in the HVD of the encoded nsP3 proteins of various New and Old World viruses (copied from Figure 2 in Gotte et al., Viruses, 2018, 10, 105). The Uniprot entries for the nsP3 sequences used in the figure are: MAYV (Q8QZ73), RRV (P13887), SFV (P08411), CHIKV (Q8JUX6), ONNV (Q8QZ73), BFV (P87515), SINV (P03317), VEEV (P36328), EEEV (Q4QXJ8), WEEV (P13896). G3BP binding sites exist for the following Old World virus nsP3 proteins: amino acids 470–473 for MAYV; amino acids 512–515 and 523–526 for RRV; amino acids 451–454 and 468–471 for SFV; amino acids 479–482 and 497–500 for CHIKV; amino acids 519–522 and 537–540 for ONNV; amino acids 429–432 and 447–450 for BFV; and amino acids 490–493 and 513–516 for SINV. In the case of New World P1234, the binding sites for the viral protein G3BP (and FXR) are as follows: in VEEV, amino acids 478–545 contain the FXR binding site; in EEEV, amino acids 471–483 contain the G3BP binding site, and amino acids 531–547 encode the FXR binding site; in WEEV, amino acids 504–520 contain the FXR binding site. DETAILED DESCRIPTION OF THE INVENTION

[0021] Unless otherwise specified, all technical and scientific terms used herein are those of the present invention. The terms "term" and "expression" have the same meaning as commonly understood by one of ordinary skill in the art to which they pertain. Certain terms used herein have the meanings set forth herein. All patents, published patent applications, and publications cited herein are hereby incorporated by reference. The present specification and the accompanying patent claims are incorporated by reference as if fully set forth. The singular forms "a," "an," and "the" used in the scope of the claim are used where the context is clear. It should be noted that plural references are included unless stated otherwise.

[0022] Unless otherwise specified, the percent sequence identity or percent sequence identity ranges described herein are not intended to be limiting. Any numerical value is modified in all instances by the term "about." Therefore, numerical values ​​are typically ±10% of the stated value. For example, a dosage of 10 mg includes 9 mg to 11 mg. In this case, the use of a numerical range includes all possible Subranges, all individual numbers within that range, including integers and fractions of values ​​within that range Explicitly include the value.

[0023] Throughout this specification and the claims that follow, unless the context requires otherwise, Unless otherwise specified, the word "comprise" and variations thereof, such as "comprise" "prises" and "comprising" mean any integer or state step or group of integers or steps, but may include any other integer or step or It will be understood that no exclusion of groups of integers or steps is implied. When used in this document, the term "comprising" shall mean the term "including / containing" "containing" or "including" or, as the case may be, may be substituted for the term "having" when used herein. can.

[0024] As used herein, "consisting of" means that the Any element, step, or ingredient not specified in the elements of is excluded. If "consisting essentially of" f) excludes substances or steps that do not materially affect the basic and novel characteristics of the claim. The foregoing terms "comprising", "containing" and "contain" "ining," "including," and "having" are Whenever used herein in the context of any aspect or embodiment of the present invention, However, to broaden the scope of this disclosure, the term "consisting of" may be used. " or "consisting essentially of " can be replaced with

[0025] In one general aspect, the invention provides an RNA sequence encoding a heterologous protein or peptide. and methods for using RNA replicons and administering them to humans or animals. The RNA replicon of the present invention is an RNA encoding a heterologous protein or peptide. A sequence and the New World alphavirus nsP1, nsP2, and nsP4 proteins The replicon also contains an RNA sequence that encodes an amino acid sequence derived from the protein. RNA sequence encoding amino acid sequence derived from the rufa virus nsP3 macrodomain , and R, which encodes an amino acid sequence derived from the alphavirus nsP3 central domain. The RNA replicon of the present invention has an Old World alphavirus nsP3 superpolymer sequence. Amino acid sequences derived entirely from the variable domain, or from the New World alphavirus nsP3 superfamily A portion derived from the variable domain and a portion derived from the Old World alphavirus nsP3 hypervariable domain and an RNA sequence encoding an amino acid sequence having the HV portion. D can be a hybrid or chimeric New World / Old World sequence. As used herein, "polypeptide," "peptide," or "protein" refers to a peptide. A molecule containing at least two amino acid residues linked by bond to form a polypeptide. It means child.

[0026] As used herein, alphavirus nsP3 or alphavirus nsP For the three hypervariable domains (HVD), the amino acid residues are the same as those of wild-type alphavirus nsPs. The amino acid sequences are numbered relative to the amino acid sequence of wild-type alphavirus nsP3. The amino acid sequences are described herein or otherwise available from GenBank data. For example, for SFV nsP3, Acid residues are numbered relative to wild-type SFV nsP3 of SEQ ID NO: 24; SI For NV nsP3, the amino acid residues are the same as those of wild-type SINV nsP3 of SEQ ID NO: 25. For CHIKV nsP3, the amino acid residues are numbered relative to the sequence number. No. 26 are numbered relative to wild-type CHIKV nsP3; VEEV nsP For nsP3, the amino acid residues are numbered as compared to wild-type VEEV nsP3 of SEQ ID NO: 27. for EEEV nsP3, the amino acid residues are the wild-type of SEQ ID NO: 28 Numbered relative to EEEV nsP3; relative to WEEV nsP3, The amino acid residues are numbered relative to wild-type WEEV nsP3 in SEQ ID NO:29.

[0027] nsP1, nsP2, nsP3, and nsP4 encoded by the replicon Each of the proteins is a functional or biologically active protein. The plicon may also encode a 3' untranslated region (UTR) and a 5' UTR, These can be the 3'UTR and 5'UTR of the alphavirus. The pluricons contain regulatory elements (e.g., one or more subgenomic promoters) and The promoter, 5'UTR and / or 3'UTR may also encode a polyA tail. The UTRs, as well as the RNA sequence encoding the heterologous protein or peptide, are When introduced into an organism, the RNA self-amplifies and the foreign protein or peptide is delivered to the organism. The vector can be operably linked to the host so that it is expressed in the host.

[0028] The inventors of the present application have unexpectedly discovered a novel alphavirus derived from the New World alphavirus (NW) genome. In the RNA replicon, at least a portion of the RNA encoding the nsP3 protein is , encoding at least a portion of nsP3 from Old World alphavirus (OW) R Substitution with NA allows for targeting of the heterologous protein or peptide encoded in the replicon. The present inventors have discovered that immunogenicity in mammals containing the IgG is significantly reduced or eliminated. In some embodiments of the replicon, the macrodomain and central domain of nsP3 can be derived from New World alphavirus sequences, while HVD can be derived from a) Old World alphavirus sequences a) derived from an Old World alphavirus HVD sequence; or b) derived from an Old World alphavirus HVD sequence. It has a portion derived from and a portion derived from a New World alphavirus HVD sequence.

[0029] In another embodiment, the macrodomain and central domain are from an Old World alphavirus. The HVD is derived from the sequences of the clonal and central domains of a) Old World alphaviruses b) a portion derived from an Old World alphavirus HVD sequence; or It contains a portion derived from a New World alphavirus HVD sequence.

[0030] In another embodiment, the macrodomain is derived from a New World alphavirus macrodomain sequence. The central domain is derived from an Old World alphavirus central domain sequence, and the HVD is a a) derived from an Old World alphavirus HVD sequence, or b) an Old World alphavirus It has a portion derived from the HVD sequence and a portion derived from the New World alphavirus HVD sequence. do.

[0031] In another embodiment, the macrodomain is derived from an Old World alphavirus macrodomain sequence. The central domain is derived from a New World alphavirus central domain sequence, and the HVD is a a) derived from an Old World alphavirus HVD sequence, or b) an Old World alphavirus It has a portion derived from the HVD sequence and a portion derived from the New World alphavirus HVD sequence. do.

[0032] In some embodiments, the replicon comprises a portion derived from a New World alphavirus HVD sequence. Hybrid or chimeric New World HVD sequences with portions derived from both the Old World and New World HVD sequences. In various embodiments, the Old World portion encodes at least At least 5 or at least 10 or at least 15 or at least 20 or less At least 25 or at least 30 or at least 52 or at least 53 or at least 75 or at least 100 or at least 125 or less at least 150, or at least 175, or at least 200 amino acids Parts of both worlds can be combined to form wild-type Old World or New World alpha The HVD sequence may be the same length as the wild-type HVD sequence. up to 10 or even 20 more sequences than the Genus or New World alphavirus HVD sequences. or up to 30 amino acids shorter; or up to 10 or up to 20 or up to 30 or up to 40 or up to 50 or up to 60 or is up to 70, up to 80, up to 90, or up to 100 amino acids long It can be made easier.

[0033] In some embodiments, the N-terminal portion of the HVD is derived from a New World nsP3 HVD sequence. and the C-terminal amino acids of the HVD are the same as those of the wild-type OW alphavirus HVD. The sequences may be derived from, for example, at least five or at least one of the HVDs. 0 or at least 15 or at least 20 or at least 25 or less At least 30 or at least 31 or at least 32 or at least 33 or at least 34 or at least 35 or 35-55 or 35-65 or at least 40 or at least 45 or at least 50 or at least 52 or at least 53 or at least 60 or at least 70 or fewer At least 80 or at least 100 or at least 125 or at least 1 50 or at least 175 C-terminal amino acids are derived from amino acids of OW HVD. The amino acid sequence of these embodiments may be the amino acid sequence of (and optionally the corresponding amino acid sequence of) In either case, the HVD must also be less than 200 or less than 175 or less than 150 or less than 125, or less than 100, or less than 80 amino acids. In further embodiments, the C-terminal amino acids are, for example, 1 to 5 or 5 or 5-10 or 10-12 or 10-13 or 10-15 or 15-2 0 amino acids can be retained from the NW alphavirus C-terminal HVD sequence. On the other hand, the remaining C-terminal amino acids are derived from the OW alphavirus HVD as described. can be done.

[0034] In any of the embodiments described herein, the New World alphavirus is VEEV or may be EEEV or WEEV or any of the above described or known in the art. The New World alphaviruses can be classified as Old World alphaviruses, such as CHIKV. , SINV, or SFV or any of the above-described or known in the art. Any Old World alphavirus may be used. New World and Old World alphaviruses can be used in the present invention in any combination, and all possible combinations and subcombinations are disclosed as if fully set forth herein. is shown.

[0035] Alphavirus replicons Alphaviruses are classified as viruses in group 4, the family Togaviridae These viruses typically contain positive-sense oligonucleotides in the 11-12 kb range. The alphavirus replicon of the present invention is 11 kb in length and carries a single-stranded RNA genome. ~12kb, or 10-13kb in length, or 7-20kb, or 7-25kb and can have a 5' cap and a 3' polyA tail, which can be The 5' cap and 3' polyA tail of the lufa virus may be used. The caps may be any of those known to those skilled in the art, such as a 7-methylguanylic acid cap, or an amplicon. Chile reverse cap analog 3'-O-Me-m7G(5')ppp(5')G or Other analogous cap structures are possible. Alphaviruses are generally enveloped. They are viruses, spherical in shape and have a diameter of about 70 nm. They are also isometric The replicon may encode a single piece of RNA. Alphavirus genomes and replicons can be non-structural and structural. It has two open reading frames (ORFs). The unstructured part of the genome is These proteins encode proteins nsP1 to nsP4, which are involved in the transcription and replication of viral RNA. It plays a role in the synthesis of ribosomal proteins, is produced as a polyprotein, and is the viral replication mechanism. However, a replicon may contain one, two, or more open reading frames. Any of the alphavirus replicons of the present invention can have a capsid, Lacking or not containing a nucleocapsid, coat protein, or nucleoprotein or not contained in or associated with them. The alphavirus replicon of the present invention can be an RNA molecule.

[0036] The structural portion of the genome consists of core nucleocapsid protein C, and a heterodimer and The RNA fragment of the present invention encodes the envelope proteins P62 and E1, which associate with each other. The plicon has any one or more of the described properties of an alphavirus. In some embodiments, the RNA replicon of the present invention can be an alphavirus construct. lacking sequences encoding the recombinant protein; or alphavirus (or optionally any In some embodiments, the RNA replica of the present invention does not encode any structural proteins (other than the structural proteins). The compounds are intended to be used in all combinations and combinations as if fully set forth herein. Protein C, P62, 6K, and E1, any one of which may be used in combination In some embodiments, the RNA replicon of the present invention does not encode one or more It does not encode any one of tein C, P62, 6K, and E1.

[0037] The geographic isolation of the alphavirus family has led to the distribution of these viruses to their unique environments. Circulating alphavirus serocomplexes may be a factor in the evolution and adaptation of viruses. can be further classified as either Old World or New World alphaviruses Both Old World and New World alphaviruses may be utilized in the present invention as described herein. As a New World alphavirus, any New World alphavirus Equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEE) V), Western Equine Encephalitis Virus (WEEV), Fort Morgan (FMV), Highland J virus (HJV), Buggy Creek virus (BCRV), Mucamba virus (MUC V), and Pixnavirus (PIXV). Old World alphaviruses and and any Old World alphavirus, e.g., Sindbis virus (SINV), Semi Riki Forest virus (SFV), Chikungunya virus (CHIKV), Bebaru virus ( BEBV), Onyong-nyong virus (ONNV), Ross River virus (RRV), Giyama virus (SAGV), Getah virus (GETV), Middleburg virus (MI DV), Nudum virus (NDUV), Barmah Forest virus (BFV), Mayaro virus MAYV, Aura virus, Una virus, Wataroa virus, Baba virus New World and Old World viruses include the genus avian flu, flu virus, and flu virus. and these sequences may be used in any combination or sub-combination in the RNA replicon of the present invention. may be used in any combination, whether or not they are fully described herein. Thus, all possible combinations and subcombinations are disclosed.

[0038] The RNA replicon of the present invention can be derived from an alphavirus genome, This means that the replicon has some of the structural characteristics of the alphavirus genome or The RNA replicon of the present invention is a modified alphavirus. In some embodiments of the replicons disclosed herein, the genome may be a gene. One or more sequences of a precon can be provided in "trans," i.e., The sequence of the replicon is provided on two or more RNA molecules. All of the sequences of the con are present on a single RNA molecule, which can also be used as described herein. The compound can be administered to a mammal to be treated.

[0039] Origin The RNA replicon of the present invention can be a wild-type New World or Old World alphavirus genome ( or the amino acid sequence encoded thereby) Any of the inventive RNA replicons disclosed herein can be used to express wild-type alphaviruses. may contain RNA sequences "derived from" or "based on" genome sequences, This means that these RNA sequences may be derived from either New World or Old World alphavirus genomes. RNA sequences from wild-type RNA alphavirus genomes that can be used as genomes (equivalent and at least 60%, or at least 65%, or At least 68% or at least 70% or at least 80% or at least 85% % or at least 90% or at least 95% or at least 97% or less At least 98% or at least 99% or 100% or 80-99% or 90-10 0% or 95~99% or 95~100% or 97~99% or 98~99%, Any of the nucleic acid or amino acid sequences disclosed herein has sequence identity of can also be functional or biologically active, and can be an alphavirus or replicon The molecule may be operably linked to additional sequences necessary for its self-replication. A gene is considered functional or wild-type if it performs at least 50% of the same activity as the corresponding molecule (either nucleotide or wild-type). Although biologically active, a functional molecule is one that is identical to its natural (or wild-type) counterpart. at least 60%, or at least 70%, or at least 90%, or less of the same activity In some cases, the RNA replicon is at least 95% or even 100%. An amino acid sequence derived from or based on the amino acid sequence of a virus This means that these RNA sequences can encode either New World or Old World genes. The wild-type RNA alphavirus genome can be considered as the global alphavirus genome. The amino acid sequence encoded by the or at least 60% or at least 65% or at least 68% or at least 70% or At least 80% or at least 90% or at least 95% or at least 97% % or at least 98% or at least 99% or 100% or 80-99% or 90-100% or 95-99% or 95-100% or 97-99% or The term "sequence identity" refers to a sequence that has 98-99% sequence identity. Sequences derived from other sequences are not identical to the original sequence. Up to 5%, up to 10%, up to 20%, or up to 30% longer or shorter than the column In any of the embodiments, the sequence identity is determined by the binding site (or sequences) of G3BP or FXR. For any nucleotide sequence that encodes the amino acid sequence At least 95% or at least 97% or at least 98% or at least 99 % or 100%. These sequences can also be up to 5% smaller than the original sequence. Or it may be up to 10%, or up to 20%, or up to 30% longer or shorter.

[0040] For example, in some embodiments, nsP1, nsP2, nsP3 macrodomains, nsP 3 central domain, the nsP3 hypervariable domain, and / or the nsP4 protein The RNA sequence encoding one or more of the alphaviruses is derived from the corresponding wild-type alphavirus sequence. A "corresponding" sequence may be a similar sequence from another type of alphavirus. The corresponding sequences are disclosed herein and are known to those skilled in the art. It can also be determined by alignment tools (e.g., Clustal Omega). Figure 6 shows the Old and New World alignments obtained using Clustal Omega. Illustrates the corresponding sequences of nsP3 proteins from representative members of the lufavirus family. A sequence alignment is shown; however, other sequence alignment tools recognized by those skilled in the art may be used. A useful program for performing sequence alignments is Molecular Systems Bio ology (2011) 7, 539. Therefore, nsPs from New World alphaviruses The sequences 1, nsP2, nsP3, and nsP4 are derived from Old World alphaviruses, respectively. The sequences "correspond" to the nsP1, nsP2, nsP3, and nsP4 sequences of The corresponding amino acid sequence may be at least five or at least At least 10 or at least 15 or at least 20 or at least 25 or At least 30 or at least 52 or at least 53 or at least 75 or at least 100 or at least 125 or 150 or at least It can be 175 or at least 200 amino acids, and can be up to 175 amino acids longer than the native sequence. May be up to 5% or up to 10% or up to 20% or up to 30% longer or shorter; equivalent The nucleic acid sequence may be at least 15, or at least 30, or at least 45, or At least 60 or at least 75 or at least 90 or at least 1 56 or at least 159 or at least 225 or at least 300 or at least 375 or at least 450 or at least 525 or less Such a sequence may be at most 600 nucleotides long. It may be up to 5% or up to 10% or up to 20% or up to 30% longer or shorter.

[0041] In some embodiments of the replicon, the sequences of nsP1, nsP2, and nsP4 Each can be derived from or based on a New World alphavirus genome. In some embodiments, an RNA replicon derived from a wild-type New World alphavirus genome is The virus or RNA replicon based on it is derived from a wild-type New World alphavirus genome. at least one RNA sequence (including at least one heterologous protein or peptide sequence) that is not The RNA sequence may contain the sequence of nsP3 or the sequence of nsP3. or at least the sequence of the central domain and / or macrodomain of the HVD In some embodiments, the sequence may be a portion of a New World alphavirus genome. The RNA replicons derived from the corresponding sequences from wild-type Old World alphavirus genomes. nsP3, or a domain of nsP3, or a part of a domain of nsP3, substituted with a When referring to the entire replicon, "derived from" can have an RNA sequence that encodes a portion of the replicon. "Based on" or "based on" refers to a gene encoding at least one heterologous protein or peptide. Optionally, any combination or subcombination of the RNA sequences, without counting the RNA sequences that are In this case, the nsP3 protein, or the macrodomain, central domain, and Not counting sequences encoding any one or more of the IL-1 and / or HVD domains. It's okay.

[0042] The term "RNA replicon" refers to a nucleic acid molecule that may be human, mammalian, or animal cell. Contains all of the genetic information necessary to induce its own amplification or self-replication within a host cell RNA replicon refers to the RNA that 1) encodes an RNA-dependent RNA polymerase; This may be a protein, nucleic acid, or ribonucleoprotein derived from a virus or a host cell. The nonstructural proteins can interact with n to catalyze the RNA amplification process. 2) 3'UTR and 5'UTR (non-structural) Genomes and subunits such as alphavirus nucleotide sequences for protein-mediated amplification cis-acting RNA sequences required for replication and transcription of genomic RNA, and / or subgenomic RNA sequences These sequences can be used to direct the expression of self-encoded proteins or non-self-encoded proteins. Self-encoded cell-derived proteins, nucleic acids or ribonucleoproteins, or components thereof Some of the components can be combined during the replication process to form complexes. In embodiments, the modified RNA replicon molecule typically contains the following order of elements: 5' viral RNA sequences required in cis for replication (e.g., 5' UTR and 5' CSE), sequences encoding biologically active nonstructural proteins (e.g., nsP123 4) a promoter for transcribing the subgenomic RNA, 3' required in cis for replication Viral sequences (e.g., 3'UTR), and polyadenylation tract, as well as optional Alternatively, a heterologous protein or peptide may be expressed behind or under the control of a subgenomic promoter. A sequence (or more than one sequence) that encodes a specific peptide. The term can refer to positive-sense (or message-sense) molecules, and The A replicon was of a length different from that of any known naturally occurring RNA virus. In any of the embodiments of the present disclosure, the RNA replicon may be a structural viral protein. Proteins (e.g., nucleocapsid protein C, as well as envelope protein P6 lacking (or not containing) at least one (or all) of the sequences of In these embodiments, the gene encoding one or more structural genes may be The sequence may be linked, for example, to at least one heterologous protein or peptide (or other gene of interest). can be replaced with one or more heterologous sequences, such as the coding sequence of a gene of intermediary interest (GOI) .

[0043] In various embodiments, the RNA replicon disclosed herein is an engineered RNA replicon. Whether it is a recombinant RNA replicon, a synthetic RNA replicon, or a recombinant RNA replicon, As used herein, the term recombinant refers to a humanized version of a polynucleotide. any molecule that is or indirectly results from the treatment of As a non-limiting example, cDNA refers to a recombinant DNA molecule. Similarly, the phospholipids produced by in vitro polymerase reactions or Carriers are attached to or incorporated into vectors such as cloning vectors or expression vectors. A recombinant RNA replicon is any nucleic acid molecule that contains one of the following: 1) For example, the synthesis of nucleic acid molecules using chemical or enzymatic techniques. by using chemical nucleic acid synthesis or by replication, polymerization, exonucleation, ase digestion, endonuclease digestion, ligation, reverse transcription, transcription, base modification (e.g. enzymes involved in recombination (including homologous and site-specific recombination), including methylation, 2) naturally occurring (using a compound), synthesized or modified in vitro; 3) a bound nucleotide sequence that is not bound in the native nucleotide sequence or engineered using molecular cloning techniques to have multiple nucleotide deletions and 4) one or more sequence changes or modifications relative to the native nucleotide sequence. have been treated using molecular cloning techniques to have a rearrangement.

[0044] As used herein, "percent identity" with respect to a nucleic acid or polypeptide sequence "Sex" or "Homology" or "Shared Sequence Identity" or "Percent (%) Sequence Identity" The term "aligns" refers to aligning sequences for maximum percent identity and, if necessary, aligning sequences for maximum percent identity. Identical to a known polypeptide after introducing caps to obtain the maximum percent homology; It is defined as the percentage of nucleotides or amino acid residues in the candidate sequence. Terminal or C-terminal insertions or deletions shall not be construed as affecting homology and may be within approximately 3 polypeptides of less than 0, less than about 20, or less than about 10 or less than 5 amino acid residues Internal deletions and / or insertions into the peptide sequence should not be construed as affecting homology. Homology or identity at the nucleotide sequence level or amino acid sequence level is determined by the sequence classification. The programs blastp, blastn, blastx, and others are tailored for similarity searches. tblastn and tblastx (Altschul (1997), Nucleic Acids Res. 25, 3389 -3402 and Karlin (1990), Proc. Natl. Acad. Sci. USA 87, 2264-2268). Using the algorithm, BLAST (Basic Local Alignment This can be determined by BLAST (Block Search Tool) analysis. The more commonly used approach is to first compare the query sequence with the data, with or without gaps. The similarity segments between the database sequences are considered, and then statistics of all the matches identified are compiled. Finally, we evaluate the significance of those matches by a preselected significance threshold. In a similarity search of a sequence database, For a discussion of the fundamental issues involved, see Altschul (1994), Nature Genetics 6, 119-129. See histogram, description, alignment, expectation (i.e., database alignment) Statistical significance threshold for reporting matches for columns, cutoff, matrix The search parameters of the search and filter (low complexity) can be set to the defaults. Definitions used by astp, blastx, tblastn and tblastx The default scoring matrix is ​​the BLOSUM62 matrix (Henikoff (1999) 92), Proc. Natl. Acad. Sci. USA 89, 10915-10919), and lengths greater than 85 (nucleobases). Recommended for query sequences (nucleotides or amino acids).

[0045] In the case of blastn, which is designed to compare nucleotide sequences, the scoring matrix The formula is a function of M (i.e., the reward score for a pair of matched residues) and N (i.e., the number of mismatches). The default values ​​for M and N are set by the ratio of the penalty score for the residue to the penalty score for the You can set the four blastn parameters as follows: Allowed: Q=10 (gap creation penalty); R=10 (gap extension penalty) wink=1 (generates word hits at every wink along the query) and gapw=16 (sets the window width in which gapped alignments occur) The equivalent Blastp parameter settings for comparing amino acid sequences are Q=9, R=2, wink=1, and gapw=32. BESTFIT® comparisons between sequences available in version 10.0 are based on DNA parameters. GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) penalty) can be used, and the equivalent settings for protein comparison are GAP=8 and and LEN=2.

[0046] In disclosing nucleic acid or polypeptide sequences herein, for example, the disclosed nsPs 1, nsP2, nsP3, nsP3 macrodomain, nsP3 central domain, nsP3 superdomain The sequences of the variable domains, nsP4, RdRp, and P1234 may also be based on the native sequence. or sequences believed to be derived therefrom. The full-length polypeptide sequence of any of the polypeptide sequences described herein, for example, SEQ ID NOs: 1 to 5, 29 (and a nucleotide sequence encoding any of SEQ ID NOs: 1 to 29), and and fragments thereof, and at least 40%, at least 45%, at least 50%, at least 55% %, at least 60%, at least 65%, at least 70%, at least 75%, at least at least 80%, or at least 85%, for example at least 86%, at least 87%, At least 88%, at least 89%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% or 85 ~99% or 85~95% or 90~99% or 95~99% or 97~99% or a polypeptide sequence having 98-99% sequence identity. Also disclosed are fragments or portions of any of the sequences disclosed herein. The total sequence contains at least 5, at least 7, or at least 1 0 or at least 20 or at least 30, at least 50, at least at least 75, at least 100, at least 125, 150 or more, or 5-10 or 10-12 or 10-15 or 15-20 or 2 0-40 or 20-50 or 30-50 or 30-75 or 3 a sequence having 0 to 100 amino acid residues (or a nucleic acid encoding such a fragment), Or at least 100 or at least 200 or at least 300 or at least 400 or at least 500 or at least 600 or At least 700 or at least 800 or at least 900 or At least 1000 or 100-200 or 100-500 or 10 A sequence having 0 to 1000 or 500 to 1000 amino acid residues (or a nucleic acid encoding a fragment thereof, or any of these amounts, but or less than 500 or less than 700 of any of the fragments disclosed herein or less than 1000 or less than 2000 consecutive amino acids, or such fragments It may also include, for example, at least one, two, or three nucleic acids encoding the or four or five amino acid residues are inserted at the N-terminus of the disclosed sequences containing insertions and substitutions. at the terminal and / or C-terminal end and / or inserted within the sequence Variants of the sequences are also disclosed, as are nucleic acid sequences encoding such variants. , may additionally or alternately include: for example, homologous recombination or site-specific or containing predetermined mutations by PCR mutagenesis, and are limited to these. corresponding polypeptides or nucleic acids of other species, including those described herein, Alleles or variants of a family of polypeptides or nucleic acids containing insertions and substitutions and / or other variants of the polypeptides, which contain insertions and substitutions. Substitution, chemical means, or other means with moieties other than natural amino acids (e.g., detectable moieties such as enzymes) Derivatives that are covalently modified by enzymatic or other suitable means. The nucleic acid sequence described in can be an RNA sequence.

[0047] Any of the components or sequences of the RNA replicon may be The components or sequences of the RNA replicon can be operably linked to any of the host cells. At least one heterologous protein or peptide in the host cell or treated organism. for expression of a gene (or biological agent) and / or the ability of the replicon to self-replicate. The term "operably connected" means that refers to an operative linkage between two or more sequences that are configured to perform their usual function. Therefore, a promoter or UTR operably linked to a coding sequence may be used. When the correct enzyme is present, it can affect the transcription and expression of the coding sequence. The promoter need not be adjacent to the coding sequence as long as it functions to direct its expression. Therefore, the RNA sequence encoding the heterologous protein or peptide and the regulatory sequence An operable linkage between a polynucleotide of interest (e.g., a promoter or a UTR) The term "operably linked" refers to a functional linkage that allows expression of the RdRp. a sequence encoding nsP1 to nsP4 (e.g., nsP4), nsP1 to nsP4, a UTR, a promoter, and It can also refer to sequences such as other sequences encoded within an RNA replicon, and these sequences The sequence allows transcription and translation of the biologic molecule and / or replication of the replicon The UTRs are the building blocks for ribosomal recognition of other encoded sequences. and translation of the sequence and spacing necessary for the sequence and translation of the sequence can be provided to operably link the two.

[0048] G3BP and FXR G3BP (Ras-GTPase-activating protein (Src-homology 3 (SH3)) Both FXR (fragile X family protein) and FXR (fragile X family protein) It is an RNA-binding protein that self-assembles to form ribonucleoprotein complexes (RNPs). Both bind to the HVD domain. The RNP complex formed by G3BP and FXR The body performs distinct functions within cells. For example, G3BP acts as a stress conjugate in the immune response. Stress granules are crucial for the nucleation and formation of stress granules. and shutting down, as well as type I interferons and other cytokines. These activities together regulate the induction and secretion of cytokines. It reinforces the antiviral state in the body and promotes adaptive immune responses. Proteins are not thought to play a role in innate immunity, but polyribosomal These RNA-binding proteins associate with the nucleosomes to form RNA transport granules in neurons. Proteins bind to alphavirus sequences on various alphavirus nsP3 HVDs. indicates the area.

[0049] Nonstructural proteins The alphavirus genome contains the nonstructural proteins nsP1, nsP2, nsP3, and These encode nsP4, and these are P1234 (or nsP1-4 or nsP1234 It is produced as a single polyprotein precursor, sometimes referred to as a polyprotein complex, and is broken down into proteins. Through proteolytic processing, nsP1 is cleaved into the mature protein, approximately 60 kD in size. a, which has methyltransferase activity and is involved in the viral capping reaction nsP2 is approximately 90 kDa in size and is a helicase and protease. On the other hand, nsP3 is approximately 60 kDa and consists of three domains: clo domain, central (or alphavirus-specific) domain, and hypervariable domain (HVD) (see Figure 6). nsP4 is approximately 70 kDa in size. , which contains the core RNA-dependent RNA polymerase (RdRp) catalytic domain. After infection, The alphavirus genomic RNA is translated to produce the P1234 polyprotein, which is a It is cleaved into various proteins.

[0050] nsP3 The nsP3 protein of alphaviruses has three domains: a) macrodomain, b) It contains a central (or alpha) domain, and c) a hypervariable domain (HVD). Three domains of some representative members of the Old and New World alphaviruses The corresponding amino acid sequence is shown in Figure 6. In various embodiments, the replicon of the present invention is The nsP3 macrodomain from live alphavirus nsP3 and wild-type alphavirus The RNA sequence encodes the nsP3 central domain derived from the nsP3 of the species. In some embodiments, the macrodomain and the central domain are both derived from a New World wild-type antigen. may be derived from the rufavirus nsP3, both of which are Old World wild-type alphaviruses. In some embodiments, the macrodomain may be derived from the nsP3 protein of the New World The central domain may be derived from a wild-type alphavirus macrodomain of an Old World wild type alphavirus. It may be derived from a live alphavirus central domain, or vice versa. The various domains can be any of the sequences described herein.

[0051] Hypervariable domain (HVD) In some embodiments, the replicon is C-terminal to the amino acid at which the FXR binding site begins. The flanking sequences were deleted and a replacement sequence of the Old World wild-type alphavirus HVD sequence or part thereof was obtained. It can have a New World Alphavirus HVD, which can be replaced by a sequence. The World Alphavirus replacement sequences are described herein. If the virus is VEEV, then the amino acid sequence C-terminal to amino acid 478 of nsP3 If the New World alphavirus is EEEV, the amino acid in nsP3 can be deleted. The amino acids C-terminal to 531 can be deleted; New World alphaviruses If it is EEV, then the amino acids C-terminal to amino acid 504 of nsP3 are deleted. In any of these embodiments, the replacement sequence can be As otherwise described herein, New World Arginine may be substituted as described in the specification. It still retains some of the C-terminal amino acids of the avian virus HVD at the C-terminal end of the Old World sequence. It is possible.

[0052] In some embodiments, a small amount of a sequence encoding the FXR binding site of a New World alphavirus is At least a portion of the sequence may be deleted and replaced with a replacement sequence as described herein. Therefore, if the New World alphavirus is VEEV, amino acids 478-5 of nsP3 Deletion of 17 or 478-545 and replacement with a replacement sequence from an Old World alphavirus Alternatively, if the New World alphavirus is VEEV, the amino acid sequence of nsP3 can be At least one repeat present between amino acids 478 and 545 was deleted to obtain the Old World Alpha The New World alphaviruses can optionally be substituted with E. coli replacement sequences. If it is EEV, amino acids 531 to 547 of nsP3 are deleted and replaced with the Old World replacement sequence. If the New World alphavirus is WEEV, the amino acid sequence of nsP3 can be replaced. Acids 504-520 can be deleted and replaced with an Old World substitution sequence. In some embodiments, the entire sequence encoding the FXR binding site can be deleted, or the FXR binding site can be removed. At least 50%, or at least 70%, or at least 80%, or At least 90% can be deleted and optionally replaced with a replacement sequence In any of the embodiments, the sequence shown can be deleted and a replacement sequence inserted. That's fine.

[0053] Old World alphavirus replacement sequences contain one or more G3BP binding sites, or G3 The BP-binding site can include an amino acid fragment having at least a portion of the BP-binding site. The replacement sequence may be FGDF (SEQ ID NO: 18) or FGSF (SEQ ID NO: 19). The replacement sequence should be at least as long as the wild-type nsP3 hypervariable domain of the Old World alphavirus. Further examples of Old World alphavirus substitution sequences are listed below. The Old World alphavirus replacement sequences are described in the New World alphaviruses described herein. The present invention can be applied to replicons having any of the HVD sequences. In either case, the New World alphavirus is referred to as VEEV, EEEV, WEEV, or the alphaviruses described herein. The virus may be any New World alphavirus described in the literature.

[0054] If the Old World alphavirus is CHIKV, the replacement sequence is CHIKV nsP3 The amino acids may be 479 to 582, 479 to 500, or 479 to 500.

[0055] If the Old World alphavirus is SINV, the replacement sequence is the SINV nsP3 The sequence may include amino acids 490-493, 513-516, or 490-516. .

[0056] If the Old World alphavirus is SFV, the replacement sequence is The sequence may include acids 451-471, or 451-454, or 468-471. .

[0057] If the Old World alphavirus is MAYV, the replacement sequence is the nucleotide sequence of MAYV nsP3. The sequence may include amino acids 470 to 473.

[0058] If the Old World alphavirus is an RRV, the replacement sequence is the amino acid sequence of RRV nsP3. The sequence may include acids 412-426, or 512-515, or 523-526. .

[0059] If the Old World alphavirus is ONNV, the replacement sequence is the ONNV nsP3 a sequence containing amino acids 519 to 540, or 519 to 522, or 537 to 540; obtain.

[0060] If the Old World alphavirus is BFV, the replacement sequence is The sequence may include acids 429-450, or 429-432, or 447-450. .

[0061] The New World and Old World alphaviruses may be any of those described herein, and any of the possible can be combined in any combination or subcombination, all of which , are disclosed as if fully set forth herein.

[0062] Alphavirus genomes encode the core RNA-dependent RNA polymerase nsP4 Polyprotein cleavage can occur at the nsP2 / 3 junction, resulting in the genomic It affects the RNA template used in the replication process. After cleavage, nsP3 is converted to nsP The two proteins can form a ring structure surrounding the 2, and these two proteins are essentially inter- Therefore, the nsP2 / 3 and / or nsP3 / 4 junctions Conservation of surrounding sequences can be useful.

[0063] Thus, in some embodiments, the macro and / or central and Each domain of the HVD and / or HVD is an amino acid sequence derived from a New World alphavirus. The nucleotide sequence may have a C-terminal portion and / or an N-terminal portion (as described herein). whereas the remainder of the domain(s) is derived from Old World alphavirus sequences. For example, each domain of the Macro and / or Central and / or HVD may have a corresponding Paleozoic The sequences may be derived from the alphavirus domain (nsP1, nsP2, nsP3, nsP4, nsP5, nsP6, nsP7, nsP8, nsP9, nsP10, nsP11, nsP12, nsP13, nsP14, nsP15, nsP16, nsP17, nsP18, nsP19, nsP19, nsP19 New World alphaviruses from which nsP2 and nsP4 are derived The first four or four N- and / or C-terminal residues of nsP3 derived from the lufavirus sequence It may have 5, 6, 4-6, 6-8, or 6-10 amino acids. Thus, the replicon can be amplified by the Old World alphavirus nsP3 macro and and / or R encoding amino acid sequences derived from each of the central and / or HVD domains. and / or a polypeptide having an NA sequence as described herein, and or the first 1-3 or 1-4 or 1-5 or 1-6 or 1 ~7 or 1-8 amino acids are derived from or related to the New World alphavirus domain may have one, two or three substitutions on top of. When used in this context The terms "C-terminus" and "N-terminus" do not refer to true termini, but rather to the specific end of the polyprotein. The polypeptides (e.g., P1234) are separated into individual polypeptides (e.g., nsP1, nsP2, nsP The nsP coding sequence is cut into nsP3 and nsP4. Normally, transcription would stop at that point. When treated as a read-through stop codon, the terminus is as shown in SEQ ID NOs: 12-17. It can be " / ", which can represent the N-terminus and / or C-terminus of the nsP. The junction sequence can be one to six such amino acids on either side of the terminus, e.g., on the nsP3 side. Such an embodiment is advantageous in that the nsP3 sequence is derived from an Old World sequence. This allows the nsP2 / nsP3 and nsP3 / nsP4 junctions to be maintained. The conservation of these junctions allows for the identification of P123 using New World alphavirus enzymes. In some embodiments, cleavage of the penultimate group may be possible. The lysine is conserved at the junction. Old World alphaviruses are any of the alphaviruses described herein. For example, if the New World alphavirus is VEEV, nsP2 / nsP3 The sequence can be LHEAGC / APSY (SEQ ID NO: 12), and can be followed by a slash (" / " ) represents the boundary between nsP2 and nsP3, and the penultimate G is conserved, while n The remaining amino acids at the sP2 / nsP3 junction are varied as described herein. For the nsP3 / nsP4 junction of EV, the sequence is RFDAGA / YIFS (SEQ ID NO: 13 ), where the penultimate glycine is again conserved, and the remaining nsP3 The amino acids can be changed as described herein. These sequences are preceded by a stop codon (TGA), which acts as a read-through stop codon, as described above. If the New World alphavirus is EEEV, it may be possible to treat it with nsP2 / The nsP3 sequence can be QHEAGR / APAY (SEQ ID NO: 14), (" / ") represents the boundary between nsP2 and nsP3, and the penultimate G is a conserved site. Meanwhile, the remaining amino acids at the nsP2 / nsP3 junction are altered as described herein. In the case of the nsP3 / nsP4 junction of EEEV, the sequence is RYEAGA / YIFS (sequence number 15), where the penultimate glycine is again conserved and the remaining n The amino acids of sP3 can be varied as described herein. It can also be preceded by a read-through stop codon (TGA) as shown above. If the virus is WEEV, the nsP2 / nsP3 sequence is RYEAGR / APAY (sequence column number 16), and a slash (" / ") can be used at the end or terminal of nsP2 (and and the junction between nsP2 and nsP3), the penultimate G is conserved, while The remaining amino acids at the nsP2 / nsP3 junction are varied as described herein. For the nsP3 / nsP4 junction of EEV, the sequence is RYEAGA / YIFS (SEQ ID NO: 1 7), where the penultimate glycine is again conserved, and the remaining nsP3 The amino acids in these sequences can be varied as described herein. These can also be preceded by a read-through stop codon (TGA) as described in the literature. Each of the sequences (SEQ ID NOs: 12 to 17) has one or more amino acids at the N-terminus and / or C-terminus. It may also contain two or three substitutions.

[0064] Repeated motifs Alphaviruses have similar or distinct nucleic acid sequences or polypeptides across species. Conserved sequence elements (CSEs) can be included that are identical sequences. It may also occur in the HVD of World or Old World alphavirus nsP3, and is well known in the art. It is known.

[0065] Old World alphaviruses also contain FGDF (SEQ ID NO: 18) or FGSF (SEQ ID NO: 19). 19) It can contain amino acid motifs, which can be repeated within the sequence to form repetitive or or repeat motifs. In this study, the HVD of Old World alphaviruses was determined to be FGDF / FGDF (SEQ ID NO: 20) reaction. repeats, or FGSF / FGSF (SEQ ID NO: 21) repeats, or FGDF / FGSF (SEQ ID NO: 22) repeats, or FGSF / FGDF (SEQ ID NO: 23) repeats. In all embodiments where repeats are present, the two repeat motifs can be separated by one or more In various embodiments, the two repeat motifs can be separated by an amino acid residue. , 5 or 6 or 7 or 8 or 9 or 10 or less At most 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 amino acid residues They may be separated by 25 or more amino acid residues, and in one embodiment, can be random amino acids. In one embodiment, a motif or repeating motif are separated by at least 10 and not more than 25 amino acids, In various embodiments, the two repeat motifs can be: NEGEIESLSSELLT (SEQ ID NO: 6), SDGEIDELSRRVTTESEP VL (SEQ ID NO: 7), or DEHEVDALASGIT (SEQ ID NO: 8), or The sequences may be separated by sequences derived from either Thus, repeat motifs separated by SEQ ID NO: 6, 7, or 8 can be used As disclosed, these motifs include: 1) an FGDF (SEQ ID NO: 18) motif at both ends; 3) an FGSF (SEQ ID NO: 19) motif at either the 3' or 5' end; 18) motif and at the opposite end a FGSF (SEQ ID NO: 19) motif. In various embodiments, the amino acid sequence can also be followed by a second motif. the amino acid sequence DDVLRLGRAGA (SEQ ID NO: 11) or EPGEVN SIISSRSAVSFPLRKQRRRRRSRRTEY (SEQ ID NO: 10) or L PGEVDDLTDSDWSTCSDTDDELRLDRAGG (SEQ ID NO: 9) The present invention also includes sequences derived from any of these, any of which may be used in conjunction with the motifs disclosed herein. It can follow a repeating motif.

[0066] Untranslated region Any of the replicons of the present invention may contain 5' and 3' untranslated regions (UTRs). The UTRs can be wild-type New World or Old World alphavirus UTR sequences, or The sequence may be derived from any of these. The length of the fragment may be approximately 60 nt, 50-70 nt, or 40-80 nt. In some embodiments, the 5'UTR may also comprise a conserved primary or Alphaviruses that have secondary structure (e.g., one or more stem loops) In some embodiments, the 3' U may be involved in the replication of the 3' U or replicon RNA. A TR can be up to several hundred nucleotides, e.g., 50 to 900 or 100 to 1000 nucleotides. 900 or 50-800 or 100-700 or 200nt-700nt The 3'UTR may also have a secondary structure, such as a step loop, This may be followed by an adenylation tract or a poly A tail. In either case, the 5' and 3' untranslated regions are encoded by the replicon. The UTR can be operably linked to any of the sequences of other coding regions. by providing the sequences and spacing necessary for recognition and transcription of heterologous proteins. or operably linked to a promoter and / or sequence encoding the peptide. can be done.

[0067] In one embodiment, the RNA replicon of the present invention can be used to encode a heterologous protein or peptide (e.g. a protein or peptide encoding a monoclonal antibody or biologic RNA sequences and nsP1, nsP2, and nsP4 of wild-type New World alphaviruses The RNA sequence encoding the amino acid sequence derived from each protein sequence and the 5'UTR sequence and 3'UTR sequences (for non-structural protein-mediated amplification). The NA replicon also has a 5' cap and a polyadenylated (or polyA) tail. RNA replicons can also be used in New World alphavirus macrodomains. and amino acid sequences derived from the central domain of New World alphaviruses. and an amino acid sequence derived from an Old World alphavirus hypervariable domain. In an alternative embodiment, the RNA replicon can be a genomic vector, as described herein. Some have amino acid sequences derived from Old World alphavirus hypervariable domains, while others have amino acid sequences derived from Old World alphavirus hypervariable domains. and another portion having an amino acid sequence derived from the variant domain.

[0068] The immunogenicity of a heterologous protein or peptide can be determined by several methods known to those skilled in the art. Assays, e.g., for short-lived effector and memory precursor effector CD8+ T cells Intracellular or secreted cytokines by epitope-specific T cell populations, such as or by immunostaining to quantify the frequency and total number of epitope-specific T cells and their differentiation and activation state. Epidemiology measures antibody-mediated immune responses, e.g., by measuring serum IgA or IgG titers. It can also be determined by measuring antibody production by the antibody.

[0069] Heterologous Proteins and Peptides The RNA replicon of the present invention is an RNA sequence that is heterologous to the alphavirus. and also to humans, mammals, or animals that express the above RNA sequence in their bodies. At least one protein or peptide that can be (but is not necessarily) a seed In any embodiment, the replicon comprises two or more RNA sequences encoding the peptides. has RNA sequences encoding three or more heterologous proteins or peptides In various embodiments, the heterologous protein or peptide can be any of the proteins or peptides described herein. Administration of the biologic molecule to a human, mammal, or animal However, according to the present invention, it is possible to prevent the development of an anti-drug antibody immune response in humans, mammals, and administration of the replicon to the human, mammal, or animal body and While the expression of biologics is possible, the replicon is administered and the biologic molecule is expressed. The immune response from human, mammalian, or animal cells expressing the In any of the embodiments, the sequence encoding the heterologous protein or peptide is The columns may be modified to include one or more other sequences of the replicon (e.g., promoter or 5' UTR). sequence or 3'UTR sequence) and can be operably linked to a subgenomic The heterologous protein or peptide can be under the control of a promoter, such that the heterologous protein or peptide is expressed in a human The gene is expressed in a mammal or animal.

[0070] The heterologous protein or peptide can be any protein or peptide, for example cytokines, growth factors, immunoglobulins, monoclonal antibodies (Fab fragments), fragments, including Fc fusion proteins), hormones, interferons, interleukins, These include regulatory peptides and proteins. Monoclonal antibodies that may be heterologous proteins Specific examples of antibodies include raxibacumab, tocilizumab, brentuzimab vedotin, and Phase IX Factor Fc fusion protein, rilonacept, ofatumumab, bevacizumab, belimumab, Certolizumab pegol, ramucirumab, factor VIII Fc fusion protein, etanerce pt, vedolizumab, cetuximab, aflibercept, obinutuzumab, trastuzumab adalimumab, canakinumab, infliximab, ado-trastuzumab emtansine , pembrolizumab, alemtuzumab, ranibizumab, romiplostim, belatacept, Abatacept, pertuzumab, denosumab, infliximab, catumaxomab, infliximab Riximab, abciximab, rituximab, golimumab, basiliximab, eculizama ustekinumab, siltuximab, palivizumab, natalizumab, panitumumab, Nosumab, omalizumab, ipilimumab, divaflibercept, and ibritumomab In other embodiments, the heterologous protein or peptide is an endothelial Growth factors (e.g., vascular EGF), hormones (e.g., insulin, relaxin), exocytosis skipping oligonucleotides, morpholino oligomers, morpholino antisense oligomers The nucleic acid sequence may be a ligomer or an RNA encoding a tumor-specific antigen. In some embodiments, the heterologous protein or peptide may be up to 5 kb, up to 6 kb, or up to 7 kb. b or up to 8kb, or up to 9kb, or up to 10kb, or up to 11kb, or up to The heterologous protein can also be encoded by a single-chain antibody molecule. A child is also fine.

[0071] The alphavirus replicons of the present invention also can be used for the expression of heterologous proteins or peptides. As used herein, a "subgenomic promoter" may have a subgenomic promoter. The term "promoter" refers to the promoter of the subgenomic mRNA of the viral nucleic acid. As used herein, an "alphavirus subgenomic promoter" refers to an alphavirus induces transcription of subgenomic messenger RNA as part of the viral replication process , the promoter originally defined in the wild-type alphavirus genome.

[0072] Used in reference to a polynucleotide, gene, nucleic acid, polypeptide, protein, or enzyme When used in a heterologous context, the term "heterologous" refers to polynucleotides, genes, or nucleic acids that are not native to the host species. For example, as used herein, "heterologous" refers to a heterologous acid, polypeptide, protein, or enzyme. A "gene" or "heterologous nucleic acid sequence" is a gene or nucleic acid sequence derived from a species other than the species of the host organism into which it is introduced. Heterologous sequences may also be synthetic and may be derived from an organism. The gene regulatory sequence may be one that is not present in the genome or that is not found in nature. When referring to a gene sequence (e.g., 5' untranslated region, 3' untranslated region, polyA fragment, etc.), Additional sequences, intron sequences, splice sites, ribosome binding sites, internal ribosome entry sequences auxiliary nucleic acid sequences used to direct expression of the target gene (e.g., sequences, genomic homology regions, recombination sites, etc.) or encoding a protein domain or protein localization sequence. When referring to a nucleic acid sequence, "heterologous" refers to a regulatory or auxiliary sequence or protein. The sequence encoding the domain or localization sequence may be a regulatory or auxiliary nucleic acid sequence or Nucleic acid sequences encoding protein domains or localization sequences are located in genomes, chromosomes, or epitopes. This means that the genes juxtaposed in the genome are from different sources. Therefore, in its natural state (e.g., in the genome of a non-genetically engineered organism), A promoter operably linked to a gene that is not operably linked to a gene other than the promoter described herein .... The document states that a promoter must be from the same species (or in some cases, the same species) as the gene to which it is linked. Even if the promoter can be derived from the same organism, it is still referred to as a "heterologous promoter." , referring to the protein localization sequence or protein domain of the engineered protein. In this case, "heterologous" means that the localization sequence or protein domain is derived from a gene that has been genetically engineered. This means that it is derived from a protein other than the one into which it is incorporated.

[0073] As used herein, the term "recombinant" or "engineered" nucleic acid molecule refers to a nucleic acid molecule that is As a non-limiting example, cDNA refers to a nucleic acid molecule that has been modified by the intervention of a recombinant DNA sequence. NA molecules, as well as those generated by in vitro polymerase reactions, are linked to a linker, or are cloning vectors, expression vectors, replicons, etc. Non-limiting examples of recombinant nucleic acid molecules include: 1) For example, by using chemical or enzymatic techniques (e.g., chemical nuclease) of a nucleic acid molecule. By using acid synthesis or by replication, polymerization, exonuclease digestion, endonuclease cleavage, ligation, reverse transcription, transcription, base modification (e.g., including methylation) ), or by using enzymes for recombination (including homologous and site-specific recombination) 2) essentially unbound, synthesized or modified in vitro; 3) those containing one or more nucleotides relative to the naturally occurring nucleic acid molecule sequence; engineered using molecular cloning techniques to lack a specific peptide; and / or 4) have one or more sequence changes or rearrangements relative to the native nucleic acid sequence. As such, these have been treated using molecular cloning techniques. cDNA is a recombinant DNA molecule, similar to the DNA produced by in vitro polymerase reactions. produced by a reaction, or to which a linker is attached, or which is Any vector, such as the present vector, or an RNA replicon. It is a nucleic acid molecule of interest.

[0074] method The invention also provides methods of administering nucleic acids to a human, mammal, or animal patient. The nucleic acid may be a protein or peptide (heterologous protein or peptide). The method can be an RNA sequence encoding the RNA replicase described herein. administering to the patient a replicon, wherein the replicon encodes a tag. The protein or peptide is expressed (or transcribed) in the patient, which is This can be done using cellular components. The mammal may be a human, a livestock animal, a food animal, or a companion animal. any bird, fish (e.g., of the Salmonidae family), poultry, or large fowl; For example, chickens, ducks, geese, turkeys, ostriches, emus, and swans. It can be a cormorant, peacock, pheasant, partridge, or guinea fowl. The solution may be dissolved in a pharmaceutically acceptable carrier, such as saline, water, or another acceptable carrier. It may be administered.

[0075] As used herein, a "pharmaceutically acceptable carrier" refers to a compound that is suitable for administration to an individual. For example, a pharmaceutically acceptable carrier may be phosphate buffered saline. It may be a sterile aqueous solution such as physiological saline (PBS) or water for injection. Pharmaceutically acceptable carriers include buffers, preservatives, isotonicity agents, stabilizers, surfactants, wetting agents, milk The additives may be additives, antioxidants, bulking agents, or chelating agents. The other ingredients are required not to adversely affect the overall stability of the pharmaceutical formulation of the present invention. do.

[0076] The replicon may also be administered as a pharmaceutically acceptable salt. When used herein, the term "pharmaceutically acceptable salt" refers to a chemical compound such as a nucleic acid compound or a polynucleotide. physiologically and pharmaceutically acceptable salts of the compounds, i.e., salts that possess the desired biological activity of the parent compound. and does not impart any undesirable toxicological effects thereto. Pharmaceutically acceptable acidic / anionic salts for the preparation of medicaments include, but are not limited to, Acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, edetate Calcium carbonate, camsilate, carbonate, chloride, citrate, dihydrochloride, edetate, edetate Disilate, estolate, esilate, fumarate, gluceptate, Gluconate, glutamate, glycolyl arsanilate, hexylresorcinol, Hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethione acid salts, lactate, lactobionate, malate, maleate, mandelate, mesylate Salt, methyl bromide, methyl nitrate, methyl sulfate, mucoate, napsylate, nitrate, paprika Molate, Pantothenate, Phosphate / Diphosphate, Polygalacturonate, Salicylate , stearates, basic acetates, succinates, sulfates, tannates, tartrates, thiamin These include chlorate, tosylate, and triethiodide. Examples include, but are not limited to, hydroiodic acid, perchloric acid, sulfuric acid, phosphoric acid, propionic acid, acid, glycolic acid, methanesulfonic acid, hydroxyethanesulfonic acid, oxalic acid, 2- Naphthalenesulfonic acid, p-toluenesulfonic acid, cyclohexanesulfamic acid, carboxylic acid, or trifluoroacetic acid. Pharmaceutically acceptable bases / cations The reactive salts include, but are not limited to, aluminum, 2-amino-2-hydroxymethyl methyl-propane-1,3-diol (tris(hydroxymethyl)aminomethane, thromboxane) (also known as "TRIS"), ammonia, benzathine, t-butyl acetone amine, calcium, chloroprocaine, choline, cyclohexylamine, diethanolamine amine, ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl D-glucamine, piperidine, potassium, procaine, quinine, sodium, triglyceride ethanolamine, or zinc.

[0077] The present invention also provides a method for administering a heterologous protein or peptide to a mammal, comprising the steps of: The RNA replicon described herein encoding the heterologous protein or peptide is introduced into a mammal. administering to an animal, wherein the heterologous protein or peptide is expressed in the mammal. According to the method of the present invention, a naked heterologous protein and and administration of the peptide, resulting in a lower or eliminated immune response from the mammal. This allows for the administration of heterologous proteins or peptides to mammals.

[0078] Embodiment Embodiment 1 is an RNA sequence encoding a heterologous protein or peptide; with the 5' and 3' alphavirus untranslated regions; Derived from the nonstructural proteins nsP1, nsP2, and nsP4 of New World alphaviruses an RNA sequence that encodes the resulting amino acid sequence; RNA encoding amino acid sequences derived from the alphavirus nsP3 macrodomain Array and; RNA sequences encoding amino acid sequences derived from the alphavirus nsP3 central domain. columns and; a. an amino acid sequence derived from an Old World alphavirus nsP3 hypervariable domain; and teeth b. Portions derived from the New World alphavirus nsP3 hypervariable domain and Old World alphavirus nsP3 hypervariable domain Amino acid sequence containing a portion derived from the lufa virus nsP3 hypervariable domain R encoding an amino acid sequence derived from the alphavirus nsP3 hypervariable domain containing NA array and It is an RNA replicon comprising:

[0079] Embodiment 2 is directed to an alphavirus nsP3 macrodomain and an alphavirus nsP4 macrodomain. 2. The R according to embodiment 1, wherein the P3 central domain is from a New World alphavirus. It is an NA replicon.

[0080] Embodiment 3 is directed to an alphavirus nsP3 macrodomain and an alphavirus nsP4 macrodomain. 2. The R according to embodiment 1, wherein the P3 central domain is from an Old World alphavirus. It is an NA replicon.

[0081] Embodiment 4 is a method for determining whether the alphavirus nsP3 hypervariable domain is an alphavirus nsP3 hypervariable domain derived from an Old World alphavirus. Any one of embodiments 1 to 3, comprising an amino acid sequence derived from the nsP3 hypervariable domain. An RNA replicon according to the embodiment.

[0082] Embodiment 5 is a method for preparing a virulent virus, comprising administering to a mammalian animal the virulent virus of the present invention, the virulent virus comprising: The RNA replicon according to any one of embodiments 1 to 4, selected from the group consisting of: is.

[0083] Embodiment 6 is a method for detecting a New World alphavirus, comprising administering to a mammalian animal the method ... The RNA replicon according to any one of embodiments 1 to 5, wherein

[0084] Embodiment 7 is a method for detecting a New World alphavirus, the method comprising: The RNA replicon according to any one of embodiments 1 to 5, wherein

[0085] Embodiment 8 is a method for preparing a novel alphavirus, the New World alphavirus being a Venezuelan equine encephalitis virus (VEEV). , Western equine encephalitis virus (WEEV), and Eastern equine encephalitis virus (EEEV), The RNA replicon according to any one of embodiments 1 to 5, selected from the group consisting of: is.

[0086] Embodiment 9 is a method for treating an alphavirus, the method comprising administering to a subject ... Old World alphavirus, including Sindbis virus (SINV), chikungunya virus (CHV), or the like. Chikunya virus (CHIKV), Semliki Forest virus (SFV), Ross River virus (RRV), Sagiyama virus (SAGV), Getah virus (GETV), Middleburg Virus (MIDV), Bebar virus (BEBV), Onyongnyong virus (ONN V), Nudum virus (NDUV), and Barmah Forest virus (BFV). The RNA replicon according to any one of embodiments 1 to 8 is selected from the group consisting of:

[0087] Embodiment 10 is a method for producing a nucleotide sequence comprising the steps of: A moiety selected from the group consisting of FGDF (SEQ ID NO: 18) and FGSF (SEQ ID NO: 19). The RNA replicon according to any one of embodiments 1 to 9, comprising a chief.

[0088] Embodiment 11 is a method for producing a nucleotide sequence comprising the steps of: FGDF / FGDF (SEQ ID NO: 20) repeats, FGSF / FGSF (SEQ ID NO: 21) repeats, FGDF / FGSF (SEQ ID NO: 22) repeats, and FGSF / FGDF (SEQ ID NO: 23) repeats, and further, the repeat sequence comprises at least 10 repeats selected from the group consisting of and separated by no more than 25 amino acids, The RNA replicon according to any one of the first to tenth embodiments.

[0089] Embodiment 12 is a method for producing a nucleotide sequence comprising the steps of: NEGEIESLSSELLT (SEQ ID NO: 6), SDG EIDELSRRVTTESEPVL (SEQ ID NO: 7), and DEHEVDALASGI T (SEQ ID NO: 8). 11.

[0090] Embodiment 13 is a method for producing a nucleotide sequence comprising the steps of: CHIKV nsP3 HVD amino acids 479-482 or 497-500 or 479-500 or 335-517; or SFV nsP3 HVD amino acids 451-454 or 468-471 or 451-471; or SINV nsP3 HVD amino acids 490-493 or 513-516 490~516 or 335~538 The RNA replicon according to any one of embodiments 1 to 12, comprising:

[0091] Embodiment 14 is a method for producing a nucleotide sequence comprising the steps of: amino acids 479–500 or 335–517 of CHIKV nsP3 HVD; or amino acids 451–471 of SFV nsP3 HVD; or Amino acids 490–516 of SINV nsP3 HVD The RNA replicon according to any one of embodiments 1 to 12, comprising:

[0092] Embodiment 15 is a method for treating a New World alphavirus, wherein the New World alphavirus is VEEV. The hypervariable domain-derived portion is located at amino acid 478 of the VEEV nsP3 hypervariable domain. The RNA replicon of embodiment 13, which does not comprise -518.

[0093] Embodiment 16 is a method for treating a New World alphavirus, wherein the New World alphavirus is VEEV. The hypervariable domain-derived portion is located at amino acid 478 of the VEEV nsP3 hypervariable domain. The RNA replicon of embodiment 13, which does not comprise -545.

[0094] Embodiment 17 is a method for treating a New World alphavirus, wherein the New World alphavirus is VEEV. The hypervariable domain-derived portion is located at amino acid 335 of the VEEV nsP3 hypervariable domain. The RNA replicon of embodiment 13, which does not comprise -518.

[0095] Embodiment 18 is a method for treating an inflammatory bowel disease in which the Old World alphavirus is CHIKV and the Old World alphavirus is CHIKV. The portion derived from the hypervariable domain of CHIKV nsP3 corresponds to amino acids 335 to 517 of nsP3. 15. The RNA replicon of embodiment 14, comprising:

[0096] Embodiment 19 is a method for treating a bacterial infection in a mammal, the method comprising administering to a mammal the method of claim 1, wherein the Old World alphavirus is SINV. The portion derived from the hypervariable domain contains amino acids 335 to 538 of SINV nsP3. , an RNA replicon as described in embodiment 14.

[0097] Embodiment 20 is a method for treating a New World alphavirus, wherein the New World alphavirus is EEEV. The hypervariable domain-derived portion is located at amino acid 531 of the EEEV nsP3 hypervariable domain. The RNA replicon of embodiment 13, which does not comprise -547.

[0098] Embodiment 21 is a method for treating a New World alphavirus, wherein the New World alphavirus is EEEV. The hypervariable domain-derived portion is located at amino acid 531 of the EEEV nsP3 hypervariable domain. The portion derived from an Old World alphavirus hypervariable domain, excluding ~547, amino acids 479–500 of CHIKV nsP3 HVD; amino acids 451–471 of SFV nsP3 HVD; or Amino acids 490–516 of SINV nsP3 HVD 21. The RNA replicon of embodiment 20, comprising:

[0099] Embodiment 22 is a method for treating a New World alphavirus, wherein the New World alphavirus is WEEV. The hypervariable domain-derived portion is located at amino acid 504 of the WEEV nsP3 hypervariable domain. 14. The RNA replicon of embodiment 13, which does not comprise .beta.-520.

[0100] Embodiment 23 is a method for treating a New World alphavirus, wherein the New World alphavirus is WEEV. The hypervariable domain-derived portion is located at amino acid 504 of the WEEV nsP3 hypervariable domain. ~520 and derived from an Old World alphavirus hypervariable domain amino acids 479–500 of CHIKV nsP3 HVD; or amino acids 451–471 of SFV nsP3 HVD; or Amino acids 490–516 of SINV nsP3 HVD 23. The RNA replicon of embodiment 22, comprising:

[0101] Embodiment 24 is a method for preparing a nucleic acid sequence comprising: 24. The method of any one of embodiments 1 to 23, further comprising a subgenomic promoter regulating the translation of the A sequence. One of the RNA replicons is described in one embodiment.

[0102] Embodiment 25 is a method according to any one of embodiments 1 to 2, further comprising a 5' cap and a 3' polyA tail. 4. An RNA replicon according to any one of embodiments.

[0103] Embodiment 26 is any one of embodiments 1 to 25, comprising a positive-sense single-stranded RNA. An RNA replicon according to one embodiment.

[0104] Embodiment 27 is a method for producing a 30-50 nm diameter RNA containing 10-12 kb of RNA. An RNA replicon according to any one of embodiments 1 to 26.

[0105] Embodiment 28 is a method for treating a cancer, wherein the heterologous protein is a biologic protein or peptide. The RNA replicon according to any one of embodiments 1 to 27.

[0106] Embodiment 29 is any one of embodiments 1 to 28, wherein the heterologous protein is an antibody. The RNA replicon is the RNA replicon described in the embodiment.

[0107] Embodiment 30 is a method for preparing a nucleic acid sequence encoding an alphavirus, the nucleic acid sequence being a VEEV or alphavirus nsP. The 3 hypervariable domain contains amino acids 335 to 518 of the VEEV nsP3 hypervariable domain. The portion derived from the New World alphavirus nsP3 hypervariable domain, which is not included in the SINV n sP3 HVD amino acids 490-493 or 513-516 or 490-516 or a portion derived from the Old World alphavirus nsP3 hypervariable domain containing 335-538 and

[0108] Embodiment 31 is directed to a method for preparing a nucleotide sequence comprising the steps of: 31. The R according to embodiment 30, comprising amino acids 490 to 516 of SINV nsP3 HVD. It is an NA replicon.

[0109] Embodiment 32 is a method for treating a bacterial infection in a mammal, the method comprising administering to a mammal the method of claim 1, wherein the Old World alphavirus is SINV. The portion derived from the nsP3 hypervariable domain is located at amino acid 33 of the SINV nsP3 HVD. 5 to 538 are RNA replicons according to embodiment 30.

[0110] Embodiment 33 is a method for preparing a heterologous protein or peptide comprising the steps of: operably linked to RNA sequences encoding nsP1, nsP2, and nsP4, The RNA replicon according to any one of embodiments 1 to 32.

[0111] Embodiment 34 is a method of administering a heterologous protein or peptide to a mammal, comprising: 34. The nucleic acid according to any one of embodiments 1 to 33, encoding a heterologous protein or peptide. The method comprises administering the RNA replicon to a mammal, and administering the heterologous protein or peptide. The method is characterized in that the peptide is expressed in a mammal.

[0112] Embodiment 35 is a method for producing a VEEV-derived alphavirus, comprising the steps of: 35. The method of embodiment 34, wherein the replicon is a replicon of embodiment 14.

[0113] Embodiment 36 is an embodiment in which the RNA replicon is the replicon of embodiment 19. This is a method according to embodiment 34.

[0114] Embodiment 37 is an embodiment in which the RNA replicon is the replicon of embodiment 22. This is a method according to embodiment 34.

[0115] Embodiment 38 is an embodiment in which the RNA replicon is the replicon of embodiment 25. This is a method according to embodiment 34.

[0116] Embodiment 39 is an RNA sequence encoding a heterologous protein or peptide; Derived from the nonstructural proteins nsP1, nsP2, and nsP4 of New World alphaviruses an RNA sequence that encodes the resulting amino acid sequence; R encodes an amino acid sequence derived from the nsP3 protein of an Old World alphavirus and an RNA replicon comprising an NA sequence and an N-terminal and / or N-terminal sequence of the nsP3 protein. or the first 1-6 amino acids at the C-terminus are derived from New World alphavirus sequences, It is an NA replicon.

[0117] Embodiment 40 comprises, in the following order from the 5' end to the 3' end: (1) An alphavirus 5' non-translated vector to induce replication of the alphavirus replicon. Translation sequence and; (2) Alphavirus nonstructural proteins nsP1, nsP2, nsP3, and n an RNA sequence encoding sP4; (3) alphavirus subgenomic promoter sequences and; (4) an RNA sequence encoding one or more heterologous proteins or peptides; (5) Alphavirus 3' untranslated sequences and An alphavirus replicon RNA comprising: nsP1, nsP2, and nsP4 are members of one or more New World alphaviruses It is from nsP3 consists of, from the amino terminus to the carboxyl terminus, a macrodomain, a central domain, and It contains a main and hypervariable domain (HVD), The macrodomain and central domain are composed of one or more New World alphaviruses. are from the genus and / or Old World alphaviruses; HVD is from an Old World alphavirus or HVD is from a New World alphavirus Contains part of the HVD from a favirus and part of the HVD from an Old World alphavirus. , It is an alphavirus replicon RNA.

[0118] Embodiment 41 is a method for detecting nsP1, nsP2, and nsP4 from Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus (WEEV), and Eastern equine encephalitis virus (EE EV) from one or more New World alphaviruses 41. The alphavirus replicon RNA of embodiment 40, wherein:

[0119] Embodiment 42 is a method for detecting nsP1, nsP2, and nsP4 from Venezuelan equine encephalitis virus. 41. The alphavirus replicon RNA of embodiment 40, which is derived from (VEEV). It's A.

[0120] Embodiment 43 is a method for treating an alphavirus, comprising administering to a subject the method comprising administering to a subject the alphavirus, the Old World alphavirus, or a strain of the alphavirus, including, but not limited to, Sindbis virus (SINV), Chikungunya virus (CHV), or the like. Chikunya virus (CHIKV), Semliki Forest virus (SFV), Ross River virus RSV, Sagiyama virus (SAGV), Getah virus (GETV), Middle bar MIDV, Bebale virus (BEBV), Onyongnyong virus (ON NV), Nudum (NDUV), and Barmah Forest virus (BFV). 43. The alphavirus replica of any one of embodiments 40 to 42, It is conRNA.

[0121] Embodiment 44 is a method for treating an alphavirus, the method comprising administering to a subject the alphavirus ... The virus is either the Chikano virus (CHIKV) or the Semliki Forest virus (SFV). The alphavirus replicon RNA of embodiment 43.

[0122] Embodiment 45 is a method for preparing a macrodomain and a central domain comprising the steps of: 45. The method of claim 40, wherein the antibody is derived from a human avian influenza virus. It is a viral replicon RNA.

[0123] Embodiment 46 is a method for preparing a medicament for use in a medicament comprising administering to a subject the medicament, the ... macrodomain and the central domain, one or more New World algae. 45. The method of claim 40, wherein the antibody is derived from a human avian influenza virus. It is a viral replicon RNA.

[0124] Embodiment 47 is directed to nsP1, nsP2 and nsP4, the macrodomain and the central domain. The virus is from Venezuelan equine encephalitis virus (VEEV), and the HVD is from VEE Some of the HVDs from V, Sindbis virus (SINV), Chikungunya virus (C Old World albicans selected from the group consisting of HIKV and Semliki Forest virus (SFV) and a portion of the HVD from an alphavirus. It is a plicon RNA.

[0125] Embodiment 48 is a method for identifying a VEEV nsP3 polypeptide comprising: a polypeptide comprising VEEV nsP3 polypeptides having amino acid residues 335-538 of VEEV nsP3; Except for the substitution of amino acid residues 335–538 of sP3, HVD is identical to VEE 48. The alphavirus replicon RNA of embodiment 47, comprising a HVD from V .

[0126] Embodiment 49 is a method for preparing a VEEV nsP3 polypeptide comprising: Except for the substitution of amino acid residues 335–517 of nsP3, HVD is identical to VE 48. The alphavirus replicon RNA of embodiment 47, comprising a HVD from an EV. do. [Example]

[0127] [Example 1] Immunogenicity of VEEV-based replicons A VEEV-based alphavirus replicon encoding a mutant nsP3 was used as a vector for the V The nucleotide sequence encoding amino acids 335 to 518 of EEV nsP3 was used to identify the chikungunya virus. The nucleotide sequence encoding amino acids 335 to 517 of CHIKV nsP3 In place of the VEEV / CHIKV nsP3 chimera (SEQ ID NO: 30), This replacement allowed the VEEV-based replicon to be constructed. The first motif of the repeat sequence was deleted from the (in which the motif is replaced with the FGDF / FGDF (SEQ ID NO: 20) repeat from the CHIKV genome In a parallel experiment, amino acid 335 of VEEV nsP3 (HVD region) was replaced with ~538 of the amino acids (HVD region) of Sindbis virus (SINV) nsP3 The amino acids 335 to 538 of the VEEV / SINV nsP3 chimera (SEQ ID NO: A replicon encoding the nucleotide sequence of ... The repeat sequence was deleted from VEEV and replaced with FGSF from SINV by substitution. / FGSF (SEQ ID NO: 21) repeat sequence was replaced with WT, VEEV / CHIKV, or Contains VEEV / SINV chimeric nsP3 and is derived from subgenomic RNA (SGIα-rFF). Replicons expressing the red firefly luciferase (rFF) reporter from After electroporation, a portion of the cells was transferred to 6 wells of BHK-21 cells. Plated in one well of a 100-well plate and one well of a 96-well plate and incubated for 20 hours. Electroporated cells were stained for the presence of dsRNA and analyzed by flow cytometry. The frequency of dsRNA-positive cells was determined as a measure of replicon amplification. Replicons containing mutant nsP3 expressed the same level of nucleotides as replicons containing WT nsP3. The luciferase activity was analyzed and the cells were found to replicate up to 100 μg / ml (Figure 2B). However, no difference was observed between the WT-containing replicon and the indicated mutant forms of nsP3. (Figure 2C).

[0128] [Example 2] Expression of heterologous proteins from replicons In this example, a replicon encoding the mutant nsP3 shown in FIG. 2A (see Example 1) We investigated the in vivo expression of recombinant firefly luciferase (rFF) from 1) 1 or 10 micrograms of replicon RNA in saline was transfected into BALB / c mice. The luciferase activity was measured intramuscularly (IM) into the quadriceps of the mice at the indicated time points. In vivo monitoring was performed using a commercially available in vivo imaging system. The flux was reported as total flux (Figures 3A and 3B). The replicon expressing the mutant form was compared with the replicon containing the wild-type nsP3 from VEEV. showed similar levels of luciferase activity in vivo compared with control. be.

[0129] [Example 3] immunogenicity In this example, the immunogenicity and phenotype of a replicon carrying wild-type (WT) VEEV nsP3 were investigated. In comparison, a VEEV-based recombinant gene encoding a VEEV / CHIKV chimeric form of nsP3 was The immunogenicity of the replicons (from Example 1) was investigated. Each replicon contained a heterologous protein The hemagglutinin (HA) from the H5N1 strain of influenza was encoded and expressed as a vector. On day 0, 2.0 μg or 0.2 μg of RNA in saline was injected into the thigh of a BALB / c mouse. The mice were delivered intramuscularly to the quadriceps muscle and boosted on day 28 with the same replicon RNA and dose. Two weeks after the injection (day 42 after priming), spleens and serum were collected. Serum was analyzed by ELISA. The data show that VEEV / CHI In the replicon encoding the KV nsP3 chimera, the replicon with wild-type nsP3 The HA-specific IgG titer was significantly reduced compared to the control group.

[0130] In contrast, analysis of short-lived effector and memory precursor effector CD8+ T cells showed that there was no difference in the frequency of HA-specific cells among the different replicons tested. Figure 5A and 5B show that the wild-type, VEEV / SINV nsP3, and HA-specific short-lived effectors among the individual RNA replicons of VEEV / CHIKV nsP3 The frequency of memory effector CD8+ T cells is similar in Figure 5B. Similar results are shown for CD8+ T cells.

[0131] Those skilled in the art will appreciate that the present invention may be practiced in accordance with the teachings disclosed herein without departing from the scope and spirit of the present invention. It will be readily apparent that various alternatives and modifications can be made to the invention.

[0132] All patents and publications mentioned in this specification are indicative of the invention and are not intended to be limiting unless otherwise specified. It indicates the level of the person.

[0133] The inventions illustratively described herein include any inventions not specifically disclosed herein. The invention may be properly practiced without the element(s) or limitation(s). Thus, for example, in each example herein, the terms "comprising," "consisting essentially of," and " Either of the terms "comprising" and "consisting of" may be replaced with either of the other two terms. The terms and expressions used are intended to be words of description and not of limitation. In using such terms and expressions, the features illustrated and described, or portions thereof, It is not intended to exclude any equivalents of the claimed invention, and various modifications may be made to the claimed invention. It is understood that it is possible within the scope of the present invention to provide a method for manufacturing a marker. When described in terms of a brush group, one skilled in the art will understand that the invention also With respect to any individual member or subgroup of members of the Markush Group For example, X may be bromine, chlorine, or iodine. and The claims in which X is bromine and chlorine are also fully set forth. are within the scope of the following claims.

Claims

[Claim 1] The invention described in the present specification.

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