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US-20260125673-A1 - ARTIFICIAL POLYNUCLEOTIDES FOR EXPRESSING PROTEINS

US20260125673A1US 20260125673 A1US20260125673 A1US 20260125673A1US-20260125673-A1

Abstract

A polynucleotide comprising, in the 5′ to 3′ direction, a 5′ untranslated region (5′-UTR) and an open reading frame (ORF), wherein 5′-UTR including at least two tandem repeats of sequence 5′-GCCNCC-3′ operatively linked to the ORF, and wherein N is any nucleotide. A composition comprising a lipid nanoparticle and the polynucleotide and a pharmaceutical composition, and their use in medicine, particularly for use as a vaccine or for use in gene therapy.

Inventors

  • Esther BROSET BLASCO
  • Juan Enrique MARTÍNEZ OLIVÁN

Assignees

  • CERTEST BIOTEC, S.L.

Dates

Publication Date
20260507
Application Date
20231006
Priority Date
20221007

Claims (20)

  1. 1 . An artificial polynucleotide comprising in the 5′ to 3′ direction: a 5′ untranslated region (5′-UTR), and an open reading frame (ORF), wherein the 5′-UTR comprises, at its 3′-end, at least two tandem repeats of sequence 5′-GCCNCC-3′ operatively linked to the ORF, wherein N is any nucleotide.
  2. 2 . The polynucleotide according to claim 1 , wherein the at least two tandem repeats are of sequence 5′-GCCRCC-3′, wherein R is a purine nucleotide.
  3. 3 . The polynucleotide according to claim 1 , wherein the 5′-UTR comprises, at its 3′-end, two tandem repeats of sequence 5′-GCCRCC-3′ operatively linked to the ORF.
  4. 4 . The polynucleotide according to claim 1 , wherein the 5′-UTR comprises, in the 5′ to 3′ direction, a sequence from a 5′-UTR of a transcript of a gene linked to the at least two tandem repeats of sequence 5′-GCCNCC-3′.
  5. 5 . The polynucleotide according to claim 1 , wherein the sequence from a 5′-UTR of a transcript of a gene comprises a Kozak sequence at its 3′-end.
  6. 6 . The polynucleotide according to claim 1 , wherein the sequence from a 5′-UTR of a transcript of a gene is from a gene selected from the group consisting of apolipoprotein A2 (APOA2), hemoglobin subunit beta (HBB), pre T cell antigen receptor alpha (PTCRA), and small nuclear ribonucleoprotein D1 polypeptide (SNRPD1).
  7. 7 . The polynucleotide according to claim 1 , wherein the 5′-UTR comprises a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 41, or a variant thereof at least 85% identical to SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, or SEQ ID NO: 41.
  8. 8 . A polynucleotide according to claim 1 , further comprising one or more of: a 5′-cap structure; a 3′ untranslated region (3′-UTR); and a 3′ tailing sequence.
  9. 9 . The polynucleotide according to claim 1 comprising in the 5′ to 3′ direction: (i) a 5′-cap structure; (ii) a 5′ untranslated region (5′-UTR); (iii) an open reading frame (ORF); (iv) a 3′ untranslated region (3′-UTR); and (v) a 3′ tailing sequence.
  10. 10 . The polynucleotide according to claim 1 comprising in the 5′ to 3′ direction: (i) a 5′-cap structure; (ii) a 5′ untranslated region (5′-UTR); (iii) an open reading frame (ORF); (iv) a 3′ untranslated region (3′-UTR); and (v) a 3′ tailing sequence.
  11. 11 . The polynucleotide according to claim 1 comprising in the 5′ to 3′ direction: (i) a 5′-cap structure; (ii) a 5′ untranslated region (5′-UTR); (iii) an open reading frame (ORF); (iv) a 3′ untranslated region (3′-UTR); and (v) a 3′ tailing sequence.
  12. 12 . A polynucleotide according to claim 1 which is an RNA polynucleotide.
  13. 13 . A polynucleotide according to claim 1 , wherein the polynucleotide comprises at least one chemical modification.
  14. 14 . A DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide as defined in claim 1 .
  15. 15 . A pharmaceutical composition comprising: a lipid nanoparticle; a polynucleotide as defined in claim 1 or a DNA construct as defined in claim 14 ; and at least one pharmaceutically acceptable excipient and/or carrier.
  16. 16 .- 18 . (canceled)
  17. 19 . The polynucleotide according to claim 1 , wherein the 5′-UTR comprises, at its 3′-end, two tandem repeats of sequence 5′-GCCRCC-3′ operatively linked to the ORF, wherein the two tandem repeats form the sequence SEQ ID NO: 3.
  18. 20 . The polynucleotide according to claim 1 , wherein the sequence from a 5′-UTR of a transcript of a gene comprises a non-consensus Kozak sequence at its 3′-end.
  19. 21 . The polynucleotide according to claim 1 comprising in the 5′ to 3′ direction: (i) a 5′-cap-1 structure; (ii) a 5′ untranslated region (5′-UTR) of sequence SEQ ID NO: 9 or SEQ ID NO: 41; (iii) an open reading frame (ORF) encoding a polypeptide; (iv) a 3′ untranslated region (3′-UTR) of sequence SEQ ID NO: 18 or SEQ ID NO: 19; and (v) a 3′ tailing sequence of sequence SEQ ID NO: 39.
  20. 22 . A polynucleotide according to claim 1 which is a messenger RNA (mRNA).

Description

This application claims the benefit of European Patent Application EP22382946.6 filed on Oct. 7, 2022. TECHNICAL FIELD The present invention belongs to the field of polynucleotides, in particular artificial polynucleotides encoding polypeptides. The polynucleotides of the present invention are particularly useful for genetic vaccination. BACKGROUND ART Genetic vaccination and gene therapy represent two of the most promising and quickly developing therapeutic areas of modern medicine. They are both based on the delivery of polynucleotides-such as DNA or RNA molecules-into a patient's cells or tissue for producing a polypeptide that confers a therapeutic benefit. The use of RNA in gene therapy and vaccination is generally considered safer than the use of DNA, since RNA does not involve the risk of being stably integrated into the genome of the transfected cell. In addition, RNA is much easier to degrade in vivo, resulting in a relatively short half-life in contrast with DNA. Thus, compared to DNA-based therapies, RNA-based therapies have a lower risk of generating undesired anti-RNA antibodies by the host that could reduce therapy efficacy and produce serious side effects. Therefore, RNA is considered in many cases as the molecule of choice for gene medical therapies. One of the main limitations of RNA-based therapies is their limited protein production efficiency. Because RNAs have a relatively short half-life, it is crucial that they provide exceptionally high translation rates in order to produce sufficient amounts of the desired protein before the RNAs are degraded. Optimization of UTRs to improve protein production has been attempted by rational design of 5′UTRs; however, the advances achieved so far are still very limited. Thus, despite the efforts made in recent years, there is still a need for polynucleotides, in particular RNAs, with high translation rates suitable for use in genetic vaccination and gene therapy. SUMMARY OF INVENTION The present inventors have developed a new nucleic acid sequence that markedly enhances the translation efficiency of mRNA transcripts when it is inserted at the 3′-end of the 5′ UTR. This new sequence allows engineering artificial transcripts with very high protein production yields that may be useful for a wide variety of applications, including nucleic acid therapeutics or industrial recombinant protein production. As shown in the examples below, the present inventors found that inserting only one repeat of the sequence 5′-GCCACC-3′ at the 3′-end of a transcript's 5′-UTR did not affect its protein production capacity, either in vitro or in vivo (see FIGS. 1 and 2, R2 vs R1). However, when two tandem repeats of the said sequence were inserted, a remarkable increase in protein production was observed both in vitro (see FIG. 1, R3 vs R1) and in vivo (see FIG. 2, R3 vs R1), which was completely unexpected in view of the complete absence of effect observed when only one repeat was present. Notably, the synergistic effect provided by the presence of the two tandem repeats allowed for an increase of around 400% in protein expression in vivo after 24 hours with respect to transcripts not containing the two tandem repeats (FIG. 2, R3 vs R1 or R2), and also transcripts containing the regulatory elements of commercial RNAs (FIG. 2, R3 vs R4 or R6) or previously disclosed RNAs (FIG. 2, R3 vs R5). Thus, from the data provided below it is apparent that the polynucleotides provided by the present invention represent an important new tool to overcome the limitations of current artificial RNAs, in particular, their low protein production efficiency. Also, the higher translation efficiency of the polynucleotides of the invention may allow reducing the therapeutic dose of RNA required for genetic vaccination, which may help to reduce the associated secondary effects. In a first aspect, the invention provides an artificial polynucleotide comprising, in the 5′ to 3′ direction, a 5′ untranslated region (5′-UTR), and an open reading frame (ORF), wherein the 5′-UTR comprises, at its 3′-end, at least two tandem repeats of sequence 5′-GCCNCC-3′ operatively linked to the ORF, wherein N is any nucleotide. In a second aspect, the invention provides a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide as defined in the first aspect. In a third aspect, the invention provides an expression vector comprising the DNA construct as defined in the second aspect. In a fourth aspect, the invention provides a cell comprising the polynucleotide as defined in the first aspect, a DNA construct as defined in the second aspect, or an expression vector as defined in the third aspect. In a fifth aspect, the invention provides a composition comprising a lipid nanoparticle and a polynucleotide as defined in the first aspect, a DNA construct as defined in the second aspect, or an expression vector as defined in the third aspect. In a sixth aspect, the invention provides a p