MODIFIED 5'UTRs

Abstract

The present disclosure relates to a modified 5'UTR derived from an alphavirus, such as the Venezuelan equine encephalitis virus (VEEV) wild-type 5'UTR sequence of SEQ ID NO 1 modified at least by a first nucleotide insertion, such as ribonucleotide insertion, between position 3 and position 4 of the corresponding wild-type 5'UTR sequence of SEQ ID NO 1, wherein the first nucleotide insertion is preferably guanine, adenine or uracil.

Description

[0001] MODIFIED 5'UTRs

[0002] TECHNICAL FIELD

[0003] The disclosure relates to modified 5'UTRs, as well as replicon RNA comprising the same, in particular for heterologous gene expression. The modified 5'UTRs are designed or engineered to produce desired levels of the heterologous gene product and / or with a desired induction of immune response. Specifically, alphavirus replicons comprising the modified 5'UTRs are disclosed.

[0004] BACKGROUND

[0005] The non-coding regions at the 5' and 3' ends of alphaviral genomes (5'UTR and 3'UTR respectively) play important roles in the regulation of viral gene expression, replication, translation and virus-host interactions. These functions are mediated by a combination of linear sequence and structural elements. (See Hyde J.L. et al. 2015). The stem-loop structures in the 5’-UTR (Untranslated Region) of alphaviruses play crucial roles in the viral replication and translation processes (Hyde et al. 2014). These structures are formed by base pairing between complementary nucleotides within the viral RNA sequence, resulting in a double-stranded stem region connected by a single-stranded loop.

[0006] WO2014170493 is concerned with alphavirus vectors engineered to produce altered levels of protein expression and / or interferon induction and describes 5'UTR modified vectors comprising G3A, G3A + A30G and G3A+A30G substitutions. US2018104359 describes 5'UTR modified replicon RNA having one or more nucleotide substitutions at position 1, 2, 4 or combinations thereof. W02020255055 also describes 5'UTR modified replicon RNA modified by substitutions at positions 1, 2 and / or 4.

[0007] BRIEF SUMMARY OF EXEMPLARY ASPECTS

[0008] At least several self-replicating RNA vectors are currently known. However, there remains a need in the art to further develop and differentiate the repertoire of selfamplifying RNA vectors featuring, for example, increased stability, decreased reactogenicity, altered immunological profile, increased and / or sustained expression levels of the gene of interest, etc. The present disclosure provides modified forms of 5'UTR sequences, in particular alphavirus 5'UTR sequences, which confer enhanced functionality of the overall RNA construct, such as in an alphavirus replicon RNA.

[0009] In some aspects, alphavirus 5'UTR sequences are provided such as alphavirus 5'UTR sequences which are derived from the Venezuelan equine encephalitis virus (VEEV) wild-type 5'UTR sequence having SEQ ID NO 1, modified by a first nucleotide insertion between position 3 and 4 of the wild-type sequence. Further alphavirus 5'UTR sequences are provided comprising one or more of the further modifications described herein.

[0010] Thus, according to a first aspect, the present disclosure provides a modified 5'UTR derived from the VEEV wild-type 5'UTR sequence of SEQ ID NO 1 modified at least by a first nucleotide insertion between position 3 and position 4 of the corresponding wild-type 5'UTR sequence of SEQ ID NO 1.

[0011] In some embodiments, the first nucleotide insertion is a ribonucleotide insertion. In some embodiments, the first nucleotide insertion is guanine, adenine or uracil. In certain embodiments, the first nucleotide insertion is adenine. In certain embodiments, the first nucleotide insertion is guanine.

[0012] In some embodiments, the modified 5'UTR further comprises a second nucleotide insertion between position 10 and 11 of the corresponding wild-type 5'UTR sequence of SEQ ID NO 1.

[0013] In some embodiments, the second nucleotide insertion is complementary to the first nucleotide insertion. In some embodiments, the second nucleotide insertion is an adenine or uracil.

[0014] In some embodiments, the modified 5'UTR further comprises one or more of the following modifications relative to the wild-type 5'UTR having the sequence of SEQ ID NO 1 : G4U in combination with A25U; G18A; AGIO; and insertion of 2 nucleotides between position 17 and 18 (17_18insXX) or between position 18 and 19 (18_19insXX), optionally insertion of 2 uridine nucleotides (17_18insUU or 18_19insUU). In some embodiments, the modified 5'UTR comprises the nucleotide substitutions G4U and A25U relative to the wild-type 5'UTR sequence of SEQ ID NO 1. Alternatively or additionally, the modified 5'UTR comprises the nucleotide substitution G18A relative to the wild-type 5'UTR sequence of SEQ ID NO 1. Alternatively or additionally, the modified 5'UTR comprises the nucleotide deletion AGIO relative to the wild-type 5'UTR sequence of SEQ ID NO 1. Alternatively or additionally, the modified 5'UTR comprises an insertion of 2 nucleotides between position 17 and 18 (17_18insXX) relative to the wild-type 5'UTR sequence of SEQ ID NO 1. Alternatively or additionally, the modified 5'UTR comprises an insertion of 2 nucleotides between position 18 and 19 (18_19insXX) relative to the wild-type 5'UTR sequence of SEQ ID NO 1. Optionally, the insertion of 2 nucleotides comprises insertion of 2 uridine nucleotides (17_18insUU or 18_19insUU). In some embodiments, the modified 5'UTR is selected from any one of SEQ ID NOs 2 to 41.

[0015] Further provided are recombinant replicon RNAs (e.g., an alphaviral replicon RNA) wherein the wild-type 5'UTR sequence is replaced by one of the engineered 5'UTR sequences described herein. In particular, the replicon RNAs further comprise one or more heterologous sequences each of which is operably linked to one or more sub- genomic promoter and / or internal ribosome entry site (IRES) elements, and a 3’ UTR.

[0016] It follows that, according to a second aspect, the present disclosure provides a replicon RNA comprising: a modified 5'UTR as described herein; one or more heterologous sequence each of which is operably linked to one or more sub-genomic promoter and / or IRES, and a 3’ UTR. The one or more heterologous sequences typically encodes one or more heterologous gene products (e.g. proteins and / or RNAs).

[0017] One or more of the heterologous sequences may encode a heterologous protein. In some embodiments, the heterologous protein is an antigenic protein. In some embodiments, the heterologous protein is a therapeutic protein. Alternatively, or additionally, one or more of the heterologous sequences may encode a heterologous RNA (e.g. siRNA, gRNA).

[0018] In some embodiments, the replicon RNA is an alphavirus replicon RNA. The replicon RNA may comprise one or more sequence encoding a biologically active non- structural protein. For example, the replicon RNA may comprise sequences encoding nsPl, nsP2, nsP3 and nsP4 (e.g. alphaviral non-structural proteins nsPl, nsP2, nsP3 and nsP4 as in SEQ ID NO 42).

[0019] The disclosure also provides methods of obtaining the recombinant replicon RNA and the specific materials required or otherwise related thereto, such as plasmids or alternative template DNA material encoding the recombinant replicon RNAs.

[0020] The disclosure also provides methods of expressing at least one heterologous sequence, comprising introducing a replicon RNA described herein into a cell, wherein the replicon encodes the heterologous sequence (e.g., a heterologous gene product).

[0021] Thus, according to a further aspect, the present disclosure provides a method of expressing one or more heterologous gene product in a cell, comprising: introducing a replicon RNA as described herein; and expressing the one or more heterologous gene product encoded by said replicon in the cell. In some embodiments, one or more of the heterologous gene products is a heterologous protein (e.g. an antigenic protein or therapeutic protein). Alternatively, or additionally, one or more of the heterologous gene products is a heterologous RNA (e.g. siRNA, gRNA).

[0022] In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the method is performed in vitro. In certain embodiments, the method is performed ex vivo. In certain embodiments, the method is performed in vivo.

[0023] In a further aspect, the disclosure provides a DNA template encoding a replicon RNA as described herein.

[0024] In a further aspect, the disclosure provides a nanoparticle comprising a replicon RNA as described herein. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP) or a virus like particle (VLP).

[0025] In a further aspect, the disclosure provides a cell comprising a replicon RNA as described herein. The cell may be a mammalian cell, such as human cell. The cell may lack native expression of the one or more heterologous gene product (e.g. protein) encoded by the replicon RNA. The cell may have defective expression of the one or more heterologous gene product (e.g. protein) encoded by the replicon RNA. The disclosure also relates to a pharmaceutical composition comprising the replicon RNA.

[0026] It follows that, according to a further aspect, the disclosure provides a pharmaceutical composition comprising a replicon RNA as described herein, a nanoparticle as described herein, or a cell as described herein.

[0027] In a further aspect, the disclosure provides a replicon RNA as described herein, a nanoparticle as described herein, a cell as described herein, or a pharmaceutical composition as described herein for use in therapy.

[0028] In a further aspect, the disclosure provides a replicon RNA as described herein, a nanoparticle as described herein, a cell as described herein, or a pharmaceutical composition as described herein, for use in treating a gene expression disorder in a patient. In some embodiments, the patient lacks the endogenous expression of the one or more heterologous gene product (e.g., protein) encoded by the replicon RNA.

[0029] In a further aspect, the disclosure provides a replicon RNA as described herein, a nanoparticle as described herein, a cell as described herein, or a pharmaceutical composition as described herein, for use in a method of inducing an immune response in an individual.

[0030] In a further aspect, the disclosure provides a method of treating a disease or condition in a human subject in need thereof, comprising: administering a pharmaceutical composition comprising a replicon RNA according to any of the preceding claims to the human subject, thereby causing expression of the replicon RNA in one or more cells, tissues, or organs of the human subject. In some embodiments, the replicon RNA is administered in a therapeutically effective amount. In some embodiments, the therapeutically effective amount comprises an amount sufficient to cause expression of an amount of a heterologous protein that reduces or eliminates one or more symptoms of the disease or condition

[0031] Exemplary aspects of the constructs and methods described herein also relate to the following embodiments: 1. A modified 5'UTR derived from the Venezuelan equine encephalitis virus (VEEV) wild-type 5'UTR sequence of SEQ ID NO 1, modified at least by a first nucleotide insertion, such as a ribonucleotide insertion, between position 3 and position 4 of the corresponding wild-type 5'UTR sequence of SEQ ID NO 1.

[0032] 2. The modified 5'UTR of embodiment 1, wherein the first nucleotide insertion is a guanine, adenine or uracil.

[0033] 3. The modified 5'UTR of embodiment 1 or 2, wherein the first nucleotide insertion is an adenine.

[0034] 4. The modified 5'UTR of embodiment 1 or 2, wherein the first nucleotide insertion is a guanine.

[0035] 5. The modified 5'UTR of any one of the previous embodiments, further comprising a second nucleotide insertion between position 10 and 11 of the corresponding wild-type 5'UTR sequence of SEQ ID NO 1.

[0036] 6. The modified 5'UTR of embodiment 5, wherein the second nucleotide insertion is complementary to the first nucleotide insertion.

[0037] 7. The modified 5'UTR of embodiments 5 or 6, wherein the second nucleotide insertion is an adenine or uracil.

[0038] 8. The modified 5'UTR of any one of embodiments 1-7, further comprising one or more of the following modifications relative to the wild-type 5'UTR sequence of SEQ ID NO 1:

[0039] - G4U in combination with A25U;

[0040] - G18A;

[0041] - AGIO, and

[0042] - insertion of 2 nucleotides between position 17 and 18 (17_18insXX) or between position 18 and 19 (18_19insXX), optionally insertion of 2 uridine nucleotides (17_18insUU or 18_19insUU).

[0043] 9. The modified 5'UTR of any one of the preceding embodiments, wherein the modified 5'UTR sequence is selected from any one of SEQ ID NO 2 to 41.

[0044] 10. A replicon RNA comprising: a modified 5'UTR according to any one of embodiments 1 to 9; one or more heterologous sequences encoding one or more heterologous gene products, wherein each heterologous sequence is operably linked to one or more sub-genomic promoter and / or IRES elements, and a 3' UTR.

[0045] 11. The replicon RNA according to embodiment 10, wherein one or more of the heterologous sequences encodes a heterologous protein.

[0046] 12. The replicon RNA according to embodiment 11, wherein the heterologous protein is an antigenic protein.

[0047] 13. The replicon RNA according to embodiment 11, wherein the heterologous protein is a therapeutic protein.

[0048] 14. The replicon RNA according to any one of embodiments 10-13, wherein the replicon RNA is an alphavirus replicon, and / or where the replicon RNA further comprises sequences encoding the biologically active non-structural proteins nsPl, nsP2, nsP3 and nsP4.

[0049] 15. A method of expressing one or more heterologous gene product in a cell, comprising: introducing a replicon RNA of any of embodiments 10 to 15 into a cell; and expressing the one or more heterologous gene product encoded by said replicon in the cell.

[0050] 16. The method of embodiment 15, wherein one or more of the heterologous gene products is a heterologous protein.

[0051] 17. A DNA template encoding a replicon RNA of any one of embodiments 10 to 14.

[0052] 18. A nanoparticle comprising a replicon RNA of any one of embodiments 10 to 14.

[0053] 19. The nanoparticle according to embodiment 18, wherein the nanoparticle is a lipid nanoparticle (LNP).

[0054] 20. A cell comprising a replicon RNA of any one of embodiments 10 to 14, optionally wherein the cell is a mammalian cell, such as human cell.

[0055] 21. The cell according to embodiment 20, wherein the cell has defective or lacks native expression of the one or more heterologous gene product encoded by the replicon RNA.

[0056] 22. A pharmaceutical composition comprising a replicon RNA according to any one of embodiments 10 to 14, a nanoparticle according to embodiment 18 or 19, or a cell according to embodiment 20 or 21. 23. A replicon RIMA according to any one of embodiments 10-14, a nanoparticle according to embodiment 18 or 19, a cell according to embodiment 20 or 21, or a pharmaceutical composition according to embodiment 22 for use in therapy.

[0057] 24. A replicon RNA according to any one of embodiments 10-14, a nanoparticle according to embodiment 18 or 19, a cell according to embodiment 20 or 21, or a pharmaceutical composition according to embodiment 22, for use in treating a gene expression disorder in a patient, optionally where the patient lacks the endogenous expression of the one or more heterologous gene product encoded by the replicon RNA.

[0058] 25. A replicon RNA according to any one of embodiments 10-14, a nanoparticle according to embodiment 18 or 19, a cell according to embodiment 20 or 21, or a pharmaceutical composition according to embodiment 22 for use in a method of inducing an immune response in an individual.

[0059] DESCRIPTION OF FIGURES

[0060] Figure 1 A illustrates the equilibrium between the stem-loop and pseudoknot conformation of the VEEV derived wild-type 5'UTR sequence; B illustrates the pseudoknot conformation adopted by the VEEV derived 5'UTR sequence comprising the insertion between position 3 and 4.

[0061] Figure 2 shows bioluminescent signal 24 (dayl) and 48 (day 2) hours after transfection in Hela cells as described in Example 3: the mean value of three independent repeats are shown + SD.

[0062] Figure 3 shows viability of Hela cells day 1 and day 2 after transfection as described in Example 3: the mean value of two independent repeats are shown as the mean + SD.

[0063] Figure 4 shows bioluminescent signal (panel A and B) and viability (panel C and D) 24 (day 1 - panel A and C) and 48 (day 2 - panel B and D) hours after transfection in RD cells as described in Example 3: the mean value of three independent repeats are shown + SD.

[0064] Figure 5 shows the impact of delivery vehicle for the saRNA on bioluminescent signal (panel A and B) and viability (panel C and D) 24 (day 1 - panel A and C) and 48 (day 2 - panel B and D) hours after transfection in RD cells as described in Example 3. Figure 6 shows the LoglO transformed mean of fluorescent intensity (MFI) ± SD in Hela cells for constructs 1, 2, 3, 4 and non-self-amplifying mRNA reference over a time span of 8 days as described in Example 4: up to 10 000 cells were measured at every time point.

[0065] Figure 7 illustrates in vivo expression of the GOI (luciferase) over 30 days for construct 4 compared to reference construct 1 and conventional mRNA as described in Example 5.

[0066] Figure 8 illustrated in vivo determined serum cytokine levels 24 hours after injection determined by Legendplex as described in Example 5.

[0067] Figure 9 illustrates in vivo expression of the GOI (luciferase) over 30 days for constructs 2, 3 and 4 compared to reference construct 1 as described in Example 6. Figure 10 illustrated in vivo determined serum cytokine levels 24 hours after injection determined by Legendplex as described in Example 6.

[0068] Figure 11 illustrates in vivo expression of the GOI (luciferase) over 30 days for constructs 4, 18, 29, 33 and 40 compared to reference construct 1 as described in Example 7.

[0069] Figure 12 illustrated in vivo determined serum cytokine levels 24 hours after injection determined by Legendplex as described in Example 7.

[0070] DETAILED DESCRIPTION

[0071] The present disclosure is based in part on the surprising finding that the engineered 5'UTR modifications described herein provide new replicons having advantageous transcriptional and translational functionality. As illustrated by the experiments described herein, the 5'UTR modifications described herein lead to a different expression profile of the replicons, in particular leading to sustained (i.e., more durable in time) expression of the gene of interest, typically whilst maintaining peak expression levels similar to the peak expression level of replicons comprising the 5'UTR wild-type counterpart. The more durable expression of the gene of interest by the modified replicons is considered particularly useful, e.g., when envisaging protein replacement therapy. Without wishing to be bound by theory, it is believed that the modifications to the 5'UTR described herein favour the formation of a pseudoknot structure (see figure 1) thereby protecting the 5' end from the anti-viral non-selfsensing apparatus. In its native stem-loop structure, the ultimate 5'-end nucleotides appear in single stranded form and are susceptible to pattern recognition receptors. Whereas others have attempted to modify 5'UTR through single point mutations, the insertion of one nucleotide between position 3 and 4 of the VEEV derived wild-type 5'UTR sequence, unexpectedly provides for different expression profile of the gene of interest carried by the replicon. It is believed that the insertion of a nucleotide between position 3 and 4 of the wild-type sequence (as exemplified in the structure shown in figure 1 B), allows for the 5' terminal end nucleotides to engage (more easily) in base-pair formation with the loop of the 5'UTR stem-loop structure thereby adopting the pseudoknot configuration (see figure 1 A for the structural equilibrium between the stem-loop and pseudoknot structure of a wild-type alphaviral 5'UTR sequence). At the same time, the introduction of a nucleotide between position 3 and 4 disrupts the stem of the wild-type stem-loop structure, further favouring the pseudoknot conformation. The further modification to the 5'UTR in addition to the insertion between position 3 and 4 and as described herein, are believed to further stabilize the pseudoknot configuration.

[0072] Unless defined otherwise, all technical and scientific terms used herein have the meaning as commonly understood to one of ordinary skill in the art. Otherwise, certain terms used herein have the meanings as set forth in the specification.

[0073] It must be noted "a", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. The term "at least", preceding a series of elements is to be understood to refer to every element in the series. Throughout the specification and the claims, unless the context requires otherwise, the word "comprise" and variations thereof will be understood to imply the inclusion of a stated integer, step or component (or group thereof) but not the exclusion of any other integer, step or component. The term "comprising" and variations thereof may be substituted with the term "containing", "including" or sometimes "having".

[0074] When used herein, "consisting of" excludes any integer, element, step or component not specified in the sentence. When used, "consisting essentially of" does not exclude elements, steps or components that do not materially affect the basic and novel characteristics of the subject matter defined by the sentence.

[0075] As used herein, the term "nucleotide" or "nucleotides" refers to the nucleotide bases that are the basic building blocks of nucleic acids. A nucleotide consists of a sugar molecule (either ribose in RNA or deoxyribose in DNA) attached to a phosphate group and a nitrogen-containing base (either adenine, cytosine, guanine or uracil in RNA, or, adenine, cytosine, guanine or thymine in DNA). Unless specified otherwise, the nucleotides for defining the replicon RNA constructs or sequences of the present disclosure are ribonucleotides. Alternatively, DNA templates used for the manufacture of the replicons described herein are deoxyribonucleotides.

[0076] The term "nucleic acids" refers to long chainlike molecules composed of a series of nucleotides including both RIMA and DNA molecules. The term "nucleic acid sequence" refers to the sequence of nucleotides conventionally going from the 5'-end to the 3'- end of the linear nucleic acid. The term "recombinant" or "engineered" nucleic acid as used herein, refers to the fact that the nucleic acid to which it pertains has been altered through human intervention.

[0077] When used herein, "replicon RNA", used interchangeably with "replicon", "selfamplifying RNA" ("saRNA") or "self-replicating RNA" ("srRNA") refers to an RNA molecule that, contrary to conventional mRNA, contains all of the genetic information required for directing its own amplification or self-replication within a permissive cell. Typically, replicon RNA constructs are engineered from genomes of plus-strand RNA viruses. To direct its own amplification, the RNA molecule encodes an enzyme complex for directing its own amplification in vivo in a target cell and contains the necessary elements required for replication and which are recognized and utilized by the encoded polymerases.

[0078] The term "alphavirus" and its derivations have their conventional meaning in the art and includes various species in Alphavirus genus of the Togoviridae family such as Venezuelan equine encephalitis virus (VEEV; e.g., particular strains referred to as Trinidad donkey, TC-83, etc.), Sindbis virus, Western equine encephalitis virus (WEEV), Eastern Equine Encephalitis Virus (EEEV), Chikungunya virus, Everglades virus, Tonate virus, Mucambo virus, Cabassou virus, Mosso das pedras virus, Rio Negro virus, Pixuna virus, Madriaga virus, Highlands J virus, Fort Morgan virus, and other closely related variants. In some embodiments, the replicon elements are derived from an alphavirus selected from VEEV (e.g., particular strains referred to as Trinidad donkey, TC-83, etc.), Sindbis virus, WEEV, EEEV, Chikungunya virus, Everglades virus, Tonate virus, Mucambo virus Cabassou virus, Mosso das pedras virus, Rio Negro virus, Pixuna virus, Madriaga virus, Highlands J virus, and Fort Morgan virus. In some embodiments, the replicon elements are derived from an alphavirus selected from VEEV (e.g., particular strains referred to as Trinidad donkey, TC-83, etc.), WEEV, EEEV, Everglades virus, Tonate virus, Mucambo virus Cabassou virus, Mosso das pedras virus, Rio Negro virus, Pixuna virus, Madriaga virus, Highlands J virus, Fort Morgan virus. In some embodiments, the replicon elements are derived from an alphavirus selected from VEEV (e.g., particular strains referred to as TC-83, etc.), Everglades virus, Tonate virus, Mucambo virus Cabassou virus, Mosso das pedras virus, Rio Negro virus, and Pixuna virus. In a specific embodiment, the replicon elements are derived from VEEV.

[0079] For the purpose of describing the embodiments herein, a nucleic acid sequence (e.g. 5'UTR) which is "derived from" a reference nucleic acid sequence (e.g. SEQ ID NO 1) refers to a nucleic acid which is a variant of the nucleic acid from which it is derived though in essence retaining the same functionality except for the intended modification. A nucleic acid sequence thus "derived from" a reference sequence is therefore largely based on but not necessarily 100% identical to the reference sequence but may incorporate further sequence variations, preferably which do not impact the effect aimed at by introducing the modifications described herein. Thus, further modifications may be introduced, in particular further modifications that do not interfere with one or more of the effects of the modifications described herein on the functionality of the RNA construct comprising the engineered 5'UTR.

[0080] In some embodiments, the 5'UTR is derived from an alphavirus. In further embodiments, the 5'UTR is an alphaviral 5'UTR sequence, such as Venezuelan Equine Encephalitis Virus (VEEV) 5'UTR sequence. As used herein, the term "modified 5'UTR", "engineered 5'UTR" or related terms refer to a 5'UTR derived from an alphavirus genome modified to at least comprise the insertion of a nucleotide between position 3 and 4 of the corresponding wild-type alphavirus 5'UTR.

[0081] In further embodiments, e.g. when further modifications are considered independent from the modifications described herein, the alphaviral 5'UTR sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type sequence encoding VEEV 5'UTR of SEQ ID NO 1. For purposes of the present disclosure, the degree of sequence identity between two 5'UTR (or any other nucleic acid) sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix.

[0082] The term "heterologous" as used herein in reference to a nucleic acid sequence, refers to a sequence that is from a different source than the nucleic acid sequence to which it is juxtaposed or operably linked. For example, a heterologous sequence incorporated in a replicon RNA as described herein, refers to a nucleic acid sequence encoding a protein that is from a different source to the virus (e.g., alphavirus) from which the replicon is derived. A promoter operably linked to a coding sequence to which it is not operably linked in its natural state, is referred to as a "heterologous promoter".

[0083] An "alphavirus replicon" or "alphavirus replicon RNA" is a replicon derived from an alphaviral genome. In some embodiments, the alphaviral replicon is devoid of at least a portion of the sequence encoding the alphaviral structural proteins. In some embodiments, the replicon is further comprises a heterologous nucleic acid, e.g., a gene of interest (GOI), also referred to as heterologous sequence which may be derived from a wide variety of sequences derived from any desired source. For example, in an alphavirus replicon RNA, the alphavirus autologous sequence encoding its structural proteins is replaced by the one or more GOI or heterologous sequence. Each GOI may be placed under the control of a dedicated (sub-genomic) promoter or other control element as known to the person of skill in the art.

[0084] In some embodiments, a replicon RNA contains the following elements in said order (5'>3'): a 5' Cap, a 5'UTR sequence, sequences which encode biologically active non- structural proteins nsPl, nsP2, nsP3 and nsP4 (catalysing the RNA amplification), one or more heterologous sequences each of which may be operably linked to a sub- genomic promoter or IRES, a 3'UTR sequence, and a poly(A) tail.

[0085] In some embodiments, an alphavirus replicon RNA contains the following elements in said order (5'>3'): a 5' Cap, a 5'UTR sequence, sequences which encode biologically active alphavirus non-structural proteins nsPl, nsP2, nsP3 and nsP4 (catalysing the RNA amplification), one or more heterologous sequences each of which may be operably linked to a sub-genomic promoter or IRES, a 3'UTR sequence, and a poly(A) tail.

[0086] The present disclosure provides a modified form of an alphavirus 5'UTR sequence, in particular of the VEEV 5'UTR sequence, i.e. "modified 5'UTR" sequences as described herein. The VEEV 5'UTR in its native form as referred to herein has the sequence of SEQ ID NO 1 (i.e., VEEV wild-type 5'UTR). The modified 5'UTR of the disclosure is modified at least by a first nucleotide insertion (e.g., a ribonucleotide insertion) between position 3 and position 4 of the corresponding wild-type 5'UTR sequence of SEQ ID NO 1.

[0087] Any RNA construct naturally comprising a 5'UTR sequence may benefit from the 5'UTR modifications described herein. Accordingly, the disclosure provides recombinant RNAs wherein the (naturally occurring) 5'UTR is replaced by a modified 5'UTR described herein. The present disclosure also provides replicon RNAs comprising a modified 5'UTR as described herein (i.e. recombinant replicon RIMA), such as alphavirus replicon RNAs. The modified 5'UTR sequences described herein may be incorporated in replicons derived from other viruses (e.g., alphaviruses other than VEEV) to obtain an effect on the replicon functionality similar to that described for VEEV specifically herein. The modified 5'UTR sequences may confer particular characteristics to the overall replicon (e.g., alphavirus replicon) as illustrated in the experiments described herein, e.g., sustained expression of the GOI or eliciting an alternative innate and / or adaptive immune response.

[0088] In some aspects, the replicon elements of a replicon RNA other than the 5'-UTR (such as non-structural proteins), from any virus (e.g. alphavirus) may be used as the basis from which to derive the replicon sequences of the replicon RNAs described herein. Suitable wild-type alphavirus sequences are well-known and are available from sequence depositories, such as the American Type Culture Collection, Rockville, Md. Representative examples of suitable alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC 30 VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo 5 virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCC VR- 1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCC VR-1250 ATCC VR10 1249, ATCC VR-532), Western equine encephalomyelitis (ATCC VR-70, ATCC VR1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375). In a particular embodiment, the alphavirus is a Venezuelan Equine Encephalitis Virus (VEEV). In a more particular embodiment, the alphavirus is a live attenuated Venezuelan Equine Encephalitis Virus (VEEV), such as strain TC-83.

[0089] The modified 5'UTR

[0090] In some aspects, the modified 5'UTR of the constructs described herein comprises at least a nucleotide insertion between position 3 and 4 of the wild-type sequence, such as the wild type sequence according to SEQ ID NO 1, also referred to hereinafter as 3_4insX. For the purpose of describing the embodiments of the present disclosure, X denotes any nucleotide selected from A, U, G and C and may be specified for specific embodiments. In some embodiments, the nucleotide inserted between position 3 and 4 is selected from G, A and U. In some embodiments, the nucleotide inserted between position 3 and 4 is A (described herein as "3_4insA"). In some embodiments, the nucleotide inserted between position 3 and 4 is G (described herein as "3_4insG").

[0091] For the purpose of describing the embodiments provided herein, unless otherwise indicated, similar terms including "5'UTR" as such, or the term "engineered 5'UTR" all relate to the modified 5'UTR of the constructs described herein.

[0092] Said modified 5'UTR may be further engineered by introducing one or more further insertions, one or more deletions, one or more nucleotide substitutions, or a combination thereof.

[0093] For the purpose of describing the modified 5'UTR sequences of the present disclosure, reference is made to the original wild-type 5'UTR sequence from which the engineered sequences are derived to designate the relative positions of the respective modifications. For example, for an exemplary engineered 5'UTR sequence comprising a substitution of the nucleotide at position 15 relative to the wild-type sequence, the substitution is of the nucleotide at position 15 in the wild-type sequence before any modification was made: accordingly, the nucleotide at position 15 relative to the wild-type sequence will be found at position 16 of a 5'UTR modified by the first nucleotide insertion between position 3 and 4 in accordance with the disclosure. By way of further example, in a modified 5'UTR of the constructs described herein further incorporating a second insertion between position 10 and 11, said substitution would be found on position 17 of the engineered 5'UTR.

[0094] In some embodiments, the modified 5'UTR further comprises a second nucleotide insertion between position 10 and 11 relative to the wild-type sequence of SEQ ID NO 1 (10_llinsX). In some embodiments, the second nucleotide insertion is complimentary to the nucleotide first inserted between position 3 and 4 of the wildtype sequence of SEQ ID NO 1. In some embodiments, the second inserted nucleotide insertion is adenine (described herein as "10_llinsA"). In some embodiments, the second inserted nucleotide insertion is uracil (described herein as "10_llinsU").

[0095] In certain embodiments, the nucleotide inserted between position 3 and 4 is A (3_4insA) 3_4insA and the nucleotide inserted between position 10 and 11 is U (10_llinsU). In alternative embodiments, the nucleotide inserted between position 3 and 4 is U (3_4insU) and the nucleotide inserted between position 10 and 11 is A (10_llinsA). In some embodiments, the modified 5'UTR is further modified by nucleotide substitutions at positions 4, 18 and / or 25 relative to the wild-type sequence of SEQ ID NO 1, i.e., G4X (or 4G>X), G18X (or 18G>X) and / or A25X (or 25A>X).

[0096] In some embodiments, the modified 5'UTR comprises a nucleotide substitution at position 4 relative to the wild-type sequence of SEQ ID NO 1, optionally G4U (or 4G>U).

[0097] In some embodiments, the modified 5'UTR comprises a nucleotide substitution at position 18 relative to the wild-type sequence of SEQ ID NO 1, optionally G18A (or 18G>A).

[0098] In some embodiments, the modified 5'UTR comprises a nucleotide substitution at position 18 relative to the wild-type sequence of SEQ ID NO 1, optionally A25U (or 25A>U).

[0099] In some embodiments, the modified 5'UTR comprises a nucleotide substitution at position 4 and 18 relative to the wild-type sequence of SEQ ID NO 1, optionally G4U and G18A. In some embodiments, the modified 5'UTR comprises a nucleotide substitution at position 4 and 25 relative to the wild-type sequence of SEQ ID NO 1, optionally G4U and A25U.

[0100] In some embodiments, the modified 5'UTR comprises a nucleotide substitution at position 18 and 25, optionally G18A and A25U.

[0101] In some embodiments, the modified 5'UTR comprises a nucleotide substitution at position 4, 18 and 25 relative to the wild-type sequence of SEQ ID NO 1, optionally G4U, G18A and A25U.

[0102] In some embodiments, the modified 5'UTR is further modified by nucleotide deletion at position 10, i.e., lOdelG, also referred to as AGIO.

[0103] In some embodiments, the modified 5'UTR further comprises one or more insertions between positions 17 and 18 relative to the VEEV wild-type sequence of SEQ ID NO 1. In some embodiments, the 5'UTR comprises an insertion of 2 nucleotides between positions 17 and 18, such as 2 uracils (17_18insUU).

[0104] In some embodiments, the 5'UTR comprises one or more insertions between position 18 and 19 relative to the VEEV wild-type sequence of SEQ ID NO 1. In some embodiments, the 5'UTR comprises an insertion of 2 nucleotides between positions 18 and 19, such as 2 uracils (18_19insUU). In some embodiments, the 5'UTR comprises a combination of the further 5'UTR modifications in addition to the insertion of a nucleotide between positions 3 and 4 of the wild type alphavirus 5'UTR sequence, combinations such as illustrated by the sequences listed in Table 1. Particularly contemplated are modified 5'UTRs having any of the sequences of SEQ ID No 2-41 (see Table 1). In some embodiments, the 5'UTRs have the sequence of 5, 6, 14, 25, 29 or 36.

[0105] Table 1

[0106] 5' Cap

[0107] The present disclosure provides various replicon RNAs comprising a modified 5'UTR as described herein. In addition to the modified 5'UTR, the RNA replicon may have at least one 5' terminal cap. As used herein, a "5’-cap" is an entity, typically a modified nucleotide entity, which generally "caps" the 5’-end of a mature mRNA. A 5’-cap may typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide. Preferably, the 5’-cap is linked to the 5’-terminus via a 5’-5’-triphosphate linkage. A 5’-cap may be methylated, e.g. m7GpppN, wherein N is the terminal 5’ nucleotide of the nucleic acid carrying the 5’-cap, typically the 5’-end of an RNA. Further examples of 5’cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4' , 5’ methylene nucleotide, l-(beta-D-erythrofuranosyl) nucleotide, 4’-thio nucleotide, carbocyclic nucleotide, 1 ,5-anhydrohexitol nucleotide, L- nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3 '-2 '-inverted abasic moiety, 1 ,4- butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety. Further modified 5'-cap structures which may be used in the context of the present disclosure are capl (methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (methylation of the ribose of adjacent and the 2nd nucleotide downstream of the m7GpppN), cap3 (methylation of the ribose of the adjacent, 2nd and 3rd nucleotide downstream of the m7GpppN), cap4 (methylation of the ribose of the adjacent, 2nd, 3rd and 4th nucleotide downstream of the m7GpppN), ARCA (anti-reverse CAP analogue), modified ARCA (e.g. phosphothioate modified ARCA), inosine, Nl-methyl-guanosine, 2'-fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, and 2-azido-guanosine. A specifically preferred 5'-cap is the CleanCap structure, offered by TriLink BioTechnologies.

[0108] The non-structural proteins

[0109] As defined herein, a replicon RNA is an RNA molecule that, contrary to conventional mRNA, contains all of the genetic information required for directing its own amplification or self-replication within a permissive cell.

[0110] In some embodiments, the replicon RNA is an alphavirus replicon RNA.

[0111] In some embodiments, the replicon RNA comprises sequences encoding the non- structural proteins nsPl, nsP2, nsP3 and nsP4, optionally derived from one or more alphaviral genomes. The non-structural proteins 1-4 (nsP 1-4) form the RNA- dependent RNA polymerase (RDRP), which is responsible for replication of the saRNA thereby producing copies of the saRNA. Multiple copies of the subgenomic RNA are hence produced from each saRNA originally delivered. This leads to translation of many more copies of the heterologous coding sequence when compared to a nonself amplifying RNA.

[0112] In some embodiments, the non-structural proteins 1 to 4 correspond to their native (wild type) counterparts, like for example SEQ ID NO 42. In alternative embodiments, modified forms of the non-structural proteins, for example modified forms of alphaviral non-structural proteins, are used. Modifications of the native nsPs have been described in the art (see for example Li, Y. et al. In vitro evolution of enhanced RNA replicons for immunotherapy. Sci Rep 9, 6932 (2019)), e.g. to increase transcription or translation efficiency, or to create a favourable immune response (innate and / or adaptive). In some embodiments, the replicon RNA comprises the non-structural proteins derived from VEEV. In some embodiments, the non-structural proteins are derived from VEEV and comprise mutation in nsP2, such as Q739L (see Petrakova O et al. Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells. J Virol. 2005 Jun;79(12):7597-608).

[0113] The subgenomic promoter / IRES

[0114] The disclosure provides a replicon RNA comprising a modified 5'UTR as described herein, as well as one or more heterologous sequence each of which is operably linked to one or more promoter and / or IRES, optionally wherein the promoter is a sub-genomic promoter. Each heterologous sequence encodes one or more heterologous gene products.

[0115] In some embodiments, the replicon sequences otherwise encoding the viral structural proteins (e.g., alphaviral structural proteins) are replaced by the one or more heterologous sequence, thereby placing the heterologous sequence under the control of the native viral promoter (e.g., alphaviral promotor). Alternatively, the heterologous sequence may be placed under the control of a heterologous regulatory element such as a heterologous sub-genomic promoter.

[0116] In some embodiments, the one or more heterologous sequence is operably linked to one or more promoter (e.g. sub-genomic promoter). Two or more heterologous sequences may each be operably linked different promoters (e.g., sub-genomic promoters). Alternatively, two or more heterologous sequences may be operably linked to the same promoter (e.g., sub-genomic promoter).

[0117] As used herein, the term "subgenomic promoter" refers to those sequences that constitute a functional element required for production of subgenomic RNA species. A subgenomic promoter is necessary to drive expression of genes using RNA as template; the subgenomic promoter is recognized by an RNA-dependent RNA polymerase, which may be a viral RNA replicase. The promoter itself may be a composite of segments derived from more than one source, naturally occurring, autologous or heterologous to the source of the replicon, or, synthetic. It should be noted that a subgenomic promoter is located in relation to a subgenomic RNA species or a particular gene whose transcription it initiates, and it is functionally recognized by an RNA-dependent RNA polymerase (or viral RNA replicase) when it is contained within an RIMA molecule of the proper (-) polarity. The (-) sense RNA molecule containing the functional copy of the subgenomic promoter, which comprises the functional units of core promoter and activating domain, can be synthesized by RNA- dependent RNA polymerase using a (+) sense RNA molecule as template, or it may have been synthesized by (cellular) RNA polymerase II as a transcript initiated by a polll promoter.

[0118] In some embodiments, the one or more heterologous sequence is operably linked to one or more IRES. Two or more heterologous sequences may each be operably linked to a different IRES. Alternatively, two or more heterologous sequences may be operably linked to the same IRES.

[0119] Thus, an internal ribosome entry site (IRES) sequence may be incorporated into a replicon RNA as described herein to control the expression of a heterologous coding sequence. An IRES is an RNA element that in its native form allows for translation initiation (ribosome assembly) in a cap-independent manner. IRES elements have been used to modulate expression of a gene of interest administered through mRNA.

[0120] In some embodiments, one or more heterologous sequences is operably linked to one or more promoters (e.g. sub-genomic promoter) and one or more heterologous sequences is operably linked to one or more IRES.

[0121] Heterologous sequences (also referred to as gene of interest (GOI)) and methods of use

[0122] Typically, the saRNA comprising a modified 5'UTR as described herein, is considered useful as a means for tailored expression of a product encoded by a heterologous sequence placed under the control of a subgenomic promoter, IRES or equivalent. Accordingly, in certain embodiments the disclosure relates to the use of the replicon RNAs to express the encoded product in a cell.

[0123] Each heterologous sequence of a replicon RNA constructs described herein may encode one or more heterologous gene products. The heterologous gene product may, for example, be a heterologous protein, oligonucleotide or polynucleotide, optionally wherein the oligonucleotide or polynucleotide is RNA (e.g. siRNA, gRNA). In this context, the gene product is heterologous to the virus from which the replicon RNA is derived.

[0124] In some embodiments, one or more of the heterologous sequence encodes a heterologous protein, for example a heterologous protein that is derived from a pathogen (e.g. bacterial, viral, fungal, protozoan or multi-cellular parasitic pathogen), allergen or tumor.

[0125] In some embodiments, the heterologous protein is an antigenic protein. For example, in some embodiments, the replicon RIMA serves as a vector to deliver pathogen derived antigens. Any protein derived antigen known in the art can be considered for delivery by the replicon RNA, the antigen being encoded by the / a heterologous sequence. In some embodiments, the replicon RNA serves as a vector to deliver tumor derived antigens. Any tumor-associated antigen (TAA) or tumor specific antigen (TSA) made available in the art can be considered for the heterologous sequence of the replicon RNAs provided herein.

[0126] In some embodiments, the heterologous protein is a therapeutic protein. Such therapeutic protein may be a protein otherwise endogenic to the individual but which the individual fails to produce at sufficient levels or for which the endogenous protein is expressed in a non-functional form. The replicon RNAs described herein may be used to deliver the (correct) protein in protein replacement therapy.

[0127] 3'UTR

[0128] In some aspects, the disclosure provides a replicon RNA comprising a modified 5'UTR as described herein, as well as a 3’ UTR.

[0129] A 3’-UTR is typically the part of an mRNA, which is located between the protein coding region (i.e., the open reading frame) and the 3’-terminus of the mRNA. A 3’- UTR of an mRNA is not translated into an amino acid sequence. The 3’-UTR sequence is generally encoded by the gene, which is transcribed into the respective mRNA during the gene expression process. In the context of the present disclosure, a 3’- UTR corresponds to the sequence of a replicon, which is located 3’ to the stop codon of the heterologous coding sequence, preferably immediately 3’ to the stop codon of the heterologous coding sequence, and which extends to the 5’-side of the 3’- terminus of the replicon or of the poly(A) tail, preferably to the nucleotide immediately 5’ to the poly(A) tail. The term "corresponds to" means that the 3’-UTR sequence may be an RNA sequence, such as in the replicon sequence used for defining the 3’-UTR sequence, or a DNA sequence, which corresponds to such RNA sequence. Preferably, the 3’-UTR used according to the present disclosure is heterologous to the heterologous coding sequence, such as derived or retained from the alphaviral genome from which the replicon is derived. Even if 3'-UTR’s derived from naturally occurring genomes are preferred, also synthetically engineered UTR's may be used in the context of the present disclosure. Poly(A) tail

[0130] The term "3'-poly(A) tail" or "poly(A) tail" typically refers to a stretch of adenine nucleotides at to the 3'-end of an mRNA. In the context of the present disclosure, the term refers to such poly(A) tail at the 3' end of the 3'UTR of the replicon. It can, in some instances, be as short as 20 nucleotides or comprise up to about 500 adenine nucleotides. In some cases, the length of the 3'-poly(A) tail may be an essential element with respect to the stability of the RNA strand. Said length can be of up to about 400 adenine nucleotides, e.g. from about 20 to about 400, preferably from about 30 to about 400, more preferably from about 30 to about 300, even more preferably from about 30 to about 250. In embodiments, the poly(A) tail has about 30, about 40, about 50 about 60, about 70, about 80, about 100 or about 120 nucleotides. The poly(A) tail may be a variant such as a segmented poly(A) tail, characterized in that it consists of at least two A-containing elements each defined as a nucleotide sequence consisting of 20-60 adenosines separated by a spacer element of differing length (see for example Trepotec Z, Geiger J, Plank C, Aneja MK, Rudolph C. Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life. RNA. 2019 Apr;25(4):507-518) .

[0131] Methods of making

[0132] The replicon RNAs described herein may be obtained by any means known in the art for the manufacture of RNA molecules. Typically, synthetic RNA molecules are manufactured by in vitro transcription (IVT). IVT requires a linear DNA template containing a dedicated promoter to initiate the transcription of the target RNA sequence.

[0133] Thus, in some aspects the present disclosure provides a DNA template encoding a replicon RNA as described herein. The DNA template is typically obtained by linearisation of a plasmid comprising the promoter and target RNA encoding sequence. Alternatively, the template may be obtained using other techniques in the art such as PCR or synthetic DNA fragment synthesis. In some embodiments, the DNA template is provided for use in a method of making the modified replicon RNA described herein, such as IVT. Common promoters for RNA transcription from the DNA template include the T7, SP6, orT3 promoters. The IVT reaction mixture further comprises ribonucleotide triphosphates, the appropriate phage RNA polymerase for the promoter present in the DNA template (e.g. T7, SP6 or T3 RNA polymerases, including their modified variants described in the art), and a buffer system tailored to the RNA polymerase used. An RNase inhibitor may further be included in the reaction mixture. It is within the artisan's skill to determine the exact conditions to be used in the IVT reaction (inter alia depending on the amount of RNA needed for a specific application as well as length and nature of the target RNA molecule). Various RNA synthesis kits are available, some of which optimized for long RNA transcripts. Examples include HiScribe T7 High Yield RNA Synthesis Kit (New England BioLabs).

[0134] Typically, the RNA molecule is protected by a 5'-cap. 5'-capping of RNA may be completed concomitantly during the IVT reaction using the following chemical RNA cap analogs to generate the 5'-guanosine cap structure according to manufacturer protocols: 3'-O-Me-m7G(5')ppp(5') G [the ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs). 5'- capping of modified RNA may also be completed post-transcriptionally using a Vaccinia Virus Capping Enzyme to generate the "Cap 0" structure: m7G(5')ppp(5')G (New England BioLabscap). Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2'-0 methyl-transferase to generate: m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-O-methylation of the 5'-antepenultimate nucleotide using a 2'-0 methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-O-methylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase. Enzymes may be derived from a recombinant source. Alternatively, 5'-capping of polynucleotides may be achieved by co- transcriptional capping using m2 7,3’-OGpppG or m2 7,2’-OGpppG ARCA (CellScript Inc), CleanCap technology such as CleanCap AU (TriLink Biotechnologies LLC), 5’- phosphorothiolate cap analogs (Univ. Warszawski), or by use of alternative cap analogues.

[0135] Methods Of Expression, Nanoparticles and Cells

[0136] In some aspects, the disclosure provides a method of expressing one or more heterologous gene products in a cell, comprising: introducing a replicon RNA as described herein into a cell; and expressing the one or more heterologous gene products encoded by said replicon in the cell. In some embodiments, one or more of the heterologous gene products is a heterologous protein. In some embodiments, one or more of the heterologous gene products is a heterologous RNA (e.g. siRNA, gRNA). The replicon RNAs described herein are amenable for delivery to cells in an in vitro cell culture as well as in vivo to a subject in need thereof by any means known in the art for the delivery of RIMA. Viral particle like packaging has been described. Nonviral formulations including cationic lipids, LNPs, polymers, and protamine sulfate, as well as physical methods such as electroporation may be used to deliver RNA therapeutics.

[0137] The disclosure provides a nanoparticle comprising a replicon RNA as described herein. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the nanoparticle is a virus like particle (VLP). In some embodiments, the replicon RNA is formulated using lipid nanoparticle technology such as described in WO2023078946, W02023078950, WO2023078954, US18 / 705454,

[0138] US18 / 705479, and / or US18 / 705396, which are incorporated herein by reference.

[0139] Methods known in the art of mRNA for cellular transfection and / or (therapeutic) administration of mRNA to an individual are equally considered for the delivery of the modified replicon RNA described herein. These typically include cationic moieties that allow both association of the lipids with the negatively charged RNA, thereby protecting the RNA for RNase activity, as well as efficient uptake in the cell including endosomal escape. At the same time, the inclusion of the nsPl-4 replicon sequence in saRNAs makes them much longer than their conventional mRNA counterparts which is important for formulation. Over the past year several new or dedicated approaches aimed at improving the delivery of saRNAs have been (see Bloom K. et al. (Gene Therapy (2021) 28: 117-129) and Liu Y et al. (Vaccines 2023, 11, 1142) for reviews on recent developments).

[0140] Thus, in some embodiments of the method, the replicon RNA is introduced into the cell by direct transfection.

[0141] The disclosure also provides a cell comprising a replicon RNA as described herein. The cell may be a mammalian cell, such as human cell. The cell may lack native expression of the one or more heterologous gene product (e.g. protein) encoded by the replicon RNA. The cell may have defective expression of the one or more heterologous gene product (e.g. protein) encoded by the replicon RNA.

[0142] Pharmaceutical Compositions and Methods of Use

[0143] The disclosure provides a pharmaceutical composition comprising a replicon RNA as described herein, a nanoparticle comprising a replicon RNA as described herein, or a cell comprising a replicon RNA as described herein. In embodiments where the pharmaceutical composition comprises a cell, one or more heterologous gene product encoded by the replicon RNA is typically expressed in said cell. For instance, the transcription or translation product encoded by the heterologous sequence of the replicon RNA may be expressed in said cell. In embodiments where the heterologous gene product is a protein, the cell may present the protein on the cell surface. In some embodiments, a pharmaceutical composition comprising one or more modified replicon RNA provides in situ release of the translation product of the one or more heterologous sequences.

[0144] Suitably formulated in a pharmaceutical composition, the replicon RNAs described herein can be used in methods of treatment, either prophylactic or therapeutic. Common routes for administration of such pharmaceutical compositions include, e.g., by injection, such as intradermal (ID), subcutaneous (SC), intramuscular (IM), intranodal (IN), and intravenous (IV). In a particular embodiment, the pharmaceutical composition comprising the suitably formulated replicon RNA is administered IM. Other routes, such as intranasal injection, intravaginal injection, and intratumoral injection described for mRNA delivery (such as reviewed by Zeng C, et al. Curr Top Microbiol Immunol. 2022; 440:71-110) are equally considered for the replicon RNAs of the disclosure.

[0145] The replicon RNA pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the replicon RNA into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0146] The replicon RNA pharmaceutical compositions described herein may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) of one or more of the replicon RNAs described herein. In addition to the replicon RIMA component(s), a pharmaceutical composition of the disclosure may further comprise one or more additional pharmaceutically active agents. It is further contemplated that controlled- or sustained-release replicon RNA formulations may be made using known dosage forms and techniques.

[0147] Exemplary dosage forms suitable for parenteral administration (e.g. subcutaneous, intradermal, subdermal, intramuscular, intravenous, intraperitoneal, intra-articular, and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.

[0148] The disclosure also includes methods of treating a disease or condition or for inducing an immune response in a human subject in need thereof, comprising administering a pharmaceutical composition comprising any of the replicon RNAs described herein, thereby causing expression of the replicon RNA in one or more cells, tissues, or organs of the human subject. In some aspects, the replicon RNA is administered in a therapeutically effective amount. In other aspects, the therapeutically effective amount comprises an amount sufficient to cause expression of an amount of a heterologous protein that reduces or eliminates one or more symptoms of the disease or condition. In other aspects, the methods are directed to treating a gene expression disorder in a human subject, optionally where the patient lacks the endogenous expression of the one or more heterologous gene product encoded by the replicon RNA described herein.

[0149] Aspects of the present disclosure are further described by the following non-limiting examples which illustrate exemplary embodiments and are not intended to, nor should they be interpreted to, limit the scope of the inventions described herein, which are defined solely by the claims.

[0150] EXAMPLES

[0151] Example 1 - saRNA production

[0152] Plasmids are constructed encoding saRNA constructs with the following sequence from 5' to 3':

[0153] 5' UTR selected from SEQ ID 1-41, Non-structural proteins 1, 2, 3 and 4 derived from VEEV strain TC-83 (SEQ ID 42), subgenomic UTR (SEQ ID 43),

[0154] GOI selected from luciferase (SEQ ID 44, bioluminescence, RNA-seq and in vivo expression), eBFP (a blue fluorescent protein, SEQ ID 45, flow cytometry time course) or hemagglutinin (HA, SEQ ID 46, in vivo adaptive immunity),

[0155] - 3'UTR (SEQ ID 47), and, a polyA tail (SEQ ID 48). The plasmids are linearized by digestion with BspQI. The linearized plasmid is used as an IVT template with the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs (NEB)). Next, the template DNA is digested using Turbo DNAse for 30 minutes (ThermoFisher). The resulting IVT saRNA is then purified using the Monarch RNA Cleanup Kit (NEB). The constructs used in the experiments are numbered as follows:

[0156] Example 2 - saRNA delivery: Lipofectamine or lipid nanoparticles (LNP)

[0157] For the purpose of testing the saRNAs, they are delivered to the cells using either Lipofectamine, or formulated in lipid nanoparticles (LNPs). Lipofectamine transfection is performed as described for example 3. LNP formulation is performed according to the following protocol: saRNAs are complexed in LNPs using a mix of 35% C12-200 (expression / innate construct 4) or ((2-(4-(2-((2- butyloctanoyl)oxy)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(butane-4,l-diyl) bis(2- butylocta noate) (compound 8-41 as disclosed in WO23078946) (expression / innate constructs 2-4) or ((piperazine-l,4-diylbis(ethane-2,l- diyl))bis(azanetriyl))tetrakis(butane-4, 1-diyl) tetrakis(2-hexyldecanoate)

[0158] (expression / innate construct 18 / 29 / 33 / 40); 20% DOPE; 1.5% DMG-PEG2000; 43.5% cholesterol using an Ignite (Precision NanoSystems). An N / P ratio of 20 (expression / innate construct construct 4 or expression / innate constructs 2-4) is used with a flow rate ratio of 10. LNPs are dialyzed overnight against buffer in Tris-HCI (10 mM, pH 7.4).

[0159] Example 3 - Bioluminescence and viability in Hela or RD cells

[0160] 10 000 Hela cells or 25 000 rhabdomyosarcoma (RD) cells are seeded in clear bottom black wall, 96 well plates. Hela's are grown and seeded in MEM media (supplemented with 10% FBS, 1% Pen / strep, 1% Sodium pyruvate and 1% MEM NEAA), RD's are grown and seeded in DMEM (supplemented with 10% FBS and 1% Pen / strep). After 24 hours the cells are transfected with the saRNAs constructs expressing luciferase using Lipofectamine MessengerMax (ThermoFisher) (10 ng / well for both Hela and RD) at an RNA / lipofectamine ratio of 1 :2 (pg / pl). The transfection happens in OptiMEM media, 4 hours after transfection the OptiMEM containing the saRNA- lipofectamine complex is removed and replaced with 50 pL growth media. At 24 hours and 48 hours after transfection, luciferase activity and viability are determined using the ONE-Glo + Tox Luciferase Reporter and Cell Viability Assay (Promega). Constructs are tested against "cell" treatment, i.e. same protocol in the absence of RNA and lipofectamine, and, "mock" treatment, i.e. same protocol in the absence of RNA but with lipofectamine.

[0161] As illustrated in Figure 2: Constructs 2, 3 and 4 all show significantly higher bioluminescent signal than the parent backbone in construct 1, both 24 hours and 48 hours after transfection. Construct 5 also performs better than construct 1 on day 2. Figure 3 shows there is no impact on viability of the cells. Figure 4 illustrates that in RD cells, in particular construct 2 provides for higher bioluminescent signal compared to reference construct 1, both 24 and 48 hours after transfection. The modifications do not appear to have a clear cytopathic effect. Figure 5 illustrates the impact of delivery vehicle for the saRNA which is tested by comparing lipofectamine transfection with the formulation of the replicon constructs in LNPs (using C12-200 as ionizable cationic lipid) prepared as described under section 2. When transfecting RD cells with construct 4 formulated in the LNP, higher bioluminescence signal is observed compared to reference construct 1 delivered in the LNP formulation. Overall the signal when transfecting with LNP's is much higher compared to the signal following transfection using lipofectamine. Example 4 - Flow cytometry time course Hela cells

[0162] 50 000 Hela cells are seeded in 24 well plates. After 24 hours the cells are transfected with saRNA constructs expressing eBFP using Lipofectamine MessengerMax (ThermoFisher) (50 ng / well) at an RNA lipofectamine ration of 1 :2 (pg / pl) using optiMEM. Every day from day 1 to day 7 after transfection cells are detached using trypsin and washed twice with cell staining buffer (PBS + 2 % FBS + 5 mM EDTA). Cells are then measured by flow cytometry on a Cytoflex (Beckman Coulter). eBFP signal is measured using a 450 / 45 BP filter in combination with an APD detector. Transfection efficiency (% eBFP positive cells / all cells), MFI of all cells and MFI of eBFP positive cells are used as readout.

[0163] As illustrated in figure 6, peak expression of the three 5'UTR modified constructs 2,

[0164] 3 & 4 is seen two days after transfection (compared to an immediate decline in expression for reference construct 1 and for the conventional mRNA construct). All three modified constructs tested show a signal higher than reference construct 1 at all timepoints.

[0165] Example 5 - In vivo - Expression / Innate immunity construct 4

[0166] 8 week old SWISS mice (Janvier Labs) are injected intramuscularly (IM) with 4 pg (injection volume of 50 pL) of saRNA-LNP complexes. Six mice are included per group. Serum is collected from the group sacrificed the first day after injection to determine the cytokine concentrations of IFN-y, CXCL-1 (KC), TNF-o, CCL2 (MCP-1), IL-12, CCL5 (RANTES), IL-1 , CXCL10 (IP10), GM-CSF, IL-10, IFN-0, IFN-o and IL-6 using a murine multiplex ELISA anti-virus response panel (Biolegend). For the other group, on days 0; 1; 3; 5; 7; 10; 15; 20; 25 and 30 after saRNA-LNP injection mice are subcutaneously injected with 200 pL of 15 mg / ml luciferin and after 10 minutes luciferase activity is measured using an IVIS (Perkin-Elmer). Construct 4 is tested in comparison to reference construct 1 and conventional mRNA expression. Figure 7 shows peak expression is achieved at day 1 (24 hours) for all treatment groups followed by a much slower decline in expression in the weeks to follow for construct

[0167] 4 compared to reference constructs (construct 1 and conventional mRNA). Figure 8 illustrates the effect of the tested constructs on cytokine levels.

[0168] Example 6 - In vivo - Expression constructs 2-4

[0169] 8 week old SWISS mice (Janvier Labs) are injected intramuscularly (IM) with 2 pg (injection volume of 50 pL) of saRNA-LNP complexes. Six mice are included per group. Serum is collected on day 1 to determine the cytokine concentrations of IFN- Y, CXCL-1 (KC), TNF-a, CCL2 (MCP-1), IL-12, CCL5 (RANTES), IL-1 , CXCL10 (IP1O), GM-CSF, IL-10, IFN-0, IFN-a and IL-6 using a murine multiplex ELISA antivirus response panel (Biolegend). On days 0; 1; 3; 5; 7; 10; 15; 20; 25 and 30 after saRNA-LNP injection mice are subcutaneously injected with 200 pL of 15 mg / ml luciferin and after 10 minutes luciferase activity is measured using an IVIS (Perkin- Elmer). Construct 2, 3 and 4 are tested in comparison to reference construct 1. Figure 9 shows a comparable and slower decline in expression for constructs 2, 3 and 4 compared to reference construct (construct 1). Figure 10 illustrates the effect of the tested constructs on cytokine levels.

[0170] Example 7 - In vivo - Expression constructs 18 / 29 / 33 / 40

[0171] 8 week old SWISS mice (Janvier Labs) are injected intramuscularly (IM) with 1 pg (injection volume of 50 pL) of saRNA-LNP complexes. Six mice are included per group. Serum is collected on day 1 to determine the cytokine concentrations of IFN- y, CXCL-1 (KC), TNF-a, CCL2 (MCP-1), IL-12, CCL5 (RANTES), IL-10, CXCL10 (IP10), GM-CSF, IL-10, IFN-0, IFN-a and IL-6 using a murine multiplex ELISA antivirus response panel (Biolegend). On days 0; 1; 3; 5; 7; 10; 15; 20; 25 and 30 after saRNA-LNP injection mice are subcutaneously injected with 200 pL of 15 mg / ml luciferin and after 10 minutes luciferase activity is measured using an IVIS (Perkin- Elmer). Construct 4, 18, 29, 33 and 40 are tested in comparison to reference construct 1. Figure 11 shows a comparable and slower decline in expression for constructs 4, 18, 29, 33 and 40 compared to reference construct (construct 1). Figure 12 illustrates the effect of the tested constructs on cytokine levels. Innate cytokines, particularly IFN-a are lower in constructs with altered 5'UTRs compared to construct 1.

Claims

CLAIMS1. An alphaviral 5' untranslated region (5'UTR), wherein said 5'UTR comprises a first nucleotide insertion, such as ribonucleotide insertion, between position 3 and position 4.

2. The 5'UTR of claim 1, wherein the alphavirus is VEEV and / or the 5'UTR is derived from SEQ ID No 1.

3. The 5'UTR of claim 1 or 2, wherein the first nucleotide insertion is adenine.

4. The 5'UTR of claim 1 or 2, wherein the first nucleotide insertion is guanine.

5. The 5'UTR of any one of the previous claims, further comprising a second nucleotide insertion between position 10 and 11, said second nucleotide insertion being preferably complementary to the first nucleotide insertion.

6. The 5'UTR of claim 4, wherein the second nucleotide insertion is adenine when the first insertion is uracil, and, uracil when the first insertion is adenine.

7. The 5'UTR of any one of the preceding claims, wherein the 5'UTR sequence is selected from any one of SEQ ID NO 2 to 41.

8. A replicon RNA comprising: a 5'UTR according to any one of claims 1 to 7, and, one or more heterologous sequences encoding one or more heterologous gene products, wherein each heterologous sequence is operably linked to one or more sub-genomic promoter and / or IRES elements.

9. The replicon RNA according to claim 7, wherein one or more of the heterologous sequences encodes a heterologous protein, such as an antigenic protein and / or a therapeutic protein.

10. A method of expressing one or more heterologous gene products in a cell, comprising: introducing a replicon RNA of any of claims 7 or 8 into a cell; and expressing the one or more heterologous gene product encoded by said replicon in the cell.

11. A nanoparticle comprising a replicon RNA of any one of claims 7 to 8, wherein said nanoparticle is preferably a lipid nanoparticle (LNP).

12. A cell comprising a replicon RNA of any one of claims 7 to 8, optionally wherein the cell is a mammalian cell, such as a human cell, wherein said cell preferably has defective or lacks native expression of the one or more heterologous gene products encoded by the replicon RNA.

13. A pharmaceutical composition comprising a replicon RIMA according to any one of claims 7 to 8, a nanoparticle according to claim 10, or a cell according to claim 11.

14. A replicon RNA according to any one of claims 7 or 8, a nanoparticle according to claim 10, a cell according to claim 11, or a pharmaceutical composition according to claim 12, for use in therapy.

15. A replicon RNA according to any one of claims 7 or 8, a nanoparticle according to claim 10, a cell according to claim 11, or a pharmaceutical composition according to claim 12, for use in treating a gene expression disorder in a patient, optionally where the patient lacks the endogenous expression of the one or more heterologous gene product encoded by the replicon RNA.

16. A replicon RNA according to any one of claims 7 or 8, a nanoparticle according to claim 10, a cell according to claim 11, or a pharmaceutical composition according to claim 12, for use in a method of inducing an immune response in an individual.

17. A method of treating a disease or condition in a human subject in need thereof, comprising: administering a pharmaceutical composition comprising a replicon RNA according to any of the preceding claims to the human subject, thereby causing expression of the replicon RNA in one or more cells, tissues, or organs of the human subject.

18. The method of claim 17, wherein the replicon RNA is administered in a therapeutically effective amount.

19. The method of claim 18, wherein the therapeutically effective amount comprises an amount sufficient to cause expression of an amount of a heterologous protein that reduces or eliminates one or more symptoms of the disease or condition.

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