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EP-4735604-A2 - INHIBITORY RNA TARGETING HUNTINGTIN EXPRESSION

EP4735604A2EP 4735604 A2EP4735604 A2EP 4735604A2EP-4735604-A2

Abstract

The present invention features RNA polynucleotide constructs comprising sequences targeting Huntingtin mRNA, polynucleotide constructs comprising a sequence encoding for such RNA constructs, and primary microRNA scaffolds. Constructs comprising sequences targeting Huntingtin mRNA and/or encoding for sequences targeting Huntingtin mRNA can be used, for example, for inhibiting mutant HTT expression and/or treating Huntington disease.

Inventors

  • CALI, Christopher
  • CARGNIN, Francesca
  • HALEY, Benjamin, J.
  • SAVOLA, JUHA
  • LI, QUANYI
  • LI, Mathew, Maoxiang
  • PRICE, Philip, Lee
  • RAMSBURG, ELIZABETH

Assignees

  • Spark Therapeutics, Inc.
  • GENENTECH, INC.

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. CLAIMS I/we claim: 1. An RNA polynucleotide comprising a targeting RNA sequence at least 80% identical to the sequence of any of SEQ ID NOs: 1-19; provided that if the targeting sequence is at least 80% identical to SEQ ID NO: 18, then the RNA polynucleotide is either (a) a pri-amiRNA comprising the targeting sequence embedded in a scaffold selected from an S155e scaffold, a S- 26a scaffold, and S-33 scaffold; or (b) the RNA polynucleotide comprises the sequence of SEQ ID NOs: 117 or 118.
  2. 2. The RNA polynucleotide of claim 1, wherein the targeting RNA sequence is at least 90% identical with any of SEQ ID NOs: 1-3, 5, 6, and 16-19.
  3. 3. The RNA polynucleotide of claim 2, wherein the targeting RNA sequence comprises the sequence of any of SEQ ID NOs: 1-3, 5, 6, and 16-19.
  4. 4. The RNA polynucleotide of claims 2 or 3, wherein the RNA polynucleotide further comprises a second RNA sequence, wherein the second RNA sequence is substantially complementary to the targeting RNA sequence.
  5. 5. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a pri-amiRNA comprising a pri-miRNA scaffold, a guide sequence and a passenger sequence, wherein the guide sequence comprises the targeting sequence and the passenger sequence comprises the second RNA sequence.
  6. 6. The RNA polynucleotide of claim 5, wherein the scaffold is an S155e scaffold, S26a scaffold, a S33 scaffold or S155 scaffold.
  7. 7. The RNA polynucleotide of claim 5, wherein the RNA polynucleotide comprises a sequence at least 90% identical to any of SEQ ID NOs: 51-62, 64-68, and 78-87.
  8. 8. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises the sequence of any of SEQ ID NOs: 51-62, 64-68, and 78-87.
  9. 9. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide consists of the sequence of any of SEQ ID NOs: 51-62, 64-68, and 78-87.
  10. 10. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a pre-miRNA comprising a guide sequence and a passenger sequence, wherein the guide sequence comprises the targeting sequence and the passenger sequence comprises the second RNA sequence.
  11. 11. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is a shRNA comprising a guide sequence and a passenger sequence, wherein the guide sequence comprises the targeting sequence and the passenger sequence comprises the second RNA sequence.
  12. 12. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises a sequence at least 90% identical to any of SEQ ID NOs: 88-96, 98-101, and 111-118.
  13. 13. The RNA polynucleotide of claim 12 wherein the RNA polynucleotide comprises the sequence of any of SEQ ID NOs: 88-96, 98-101, and 111-118.
  14. 14. The RNA polynucleotide of claim 4, wherein the RNA polynucleotide is an inhibitory RNA duplex comprising a guide sequence and a passenger sequence, wherein the guide sequence comprises the targeting sequence and the passenger sequence comprises the second RNA sequence.
  15. 15. The RNA polynucleotide of claim 14, wherein the inhibitory RNA duplex has a guide stand to passenger strand combination selected from group consisting of: a) a guide strand comprising the sequence of SEQ ID NO: 1 and a passenger strand comprising a sequence at least 80% identical to the sequence of any of SEQ ID NOs: 20, 21, and 22; b) a guide strand comprising the sequence of SEQ ID NO: 2 and a passenger strand comprising a sequence at least 80% identical to the sequence of any of SEQ ID NOs: 23, 24 and 25; c) a guide strand comprising the sequence of SEQ ID NO: 3 and a passenger strand comprising a sequence at least 80% identical to the sequence of any of SEQ ID NOs: 26, 27 and 28; d) a guide strand comprising the sequence of SEQ ID NO: 4 and a passenger strand comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 29; e) a guide strand comprising the sequence of SEQ ID NO: 5 and a passenger strand comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 30; f) a guide strand comprising the sequence of SEQ ID NO: 6 and a passenger strand comprising a sequence at least 80% identical to the sequence of any of SEQ ID NOs: 31, 32 and 33; g) a guide strand comprising the sequence of SEQ ID NO: 7 and a passenger strand comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 34; h) a guide strand comprising the sequence of SEQ ID NO: 8 and a passenger strand comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 35; i) a guide strand comprising the sequence of SEQ ID NO: 9 and a passenger strand comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 36; j) a guide strand comprising the sequence of SEQ ID NO: 10 and a passenger strand comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 37; k) a guide strand comprising the sequence of SEQ ID NO: 11 and a passenger strand comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 38; l) a guide strand comprising the sequence of SEQ ID NO: 12 and a passenger strand comprising a sequence at least 80% identical to the sequence of o SEQ ID NO: 39; m) a guide strand comprising the sequence of SEQ ID NO: 13 and a passenger strand comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 40; n) a guide strand comprising the sequence of SEQ ID NO: 14 and a passenger strand comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 41; o) a guide strand comprising the sequence of SEQ ID NO: 15 and a passenger strand comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 42; p) a guide strand comprising the sequence of SEQ ID NO: 16 and a passenger strand comprising a sequence at least 80% identical to the sequence of any of SEQ ID NOs: 43, 44 and 45. q) a guide strand comprising the sequence of SEQ ID NO: 17 and a passenger strand comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 46; r) a guide strand comprising the sequence of SEQ ID NO: 19 and a passenger strand comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 47; and s) a guide strand comprising the sequence of SEQ ID NO: 18 and a passenger strand comprising a sequence at least 80% identical to the sequence of any of SEQ ID NOs: 48, 49, and 50.
  16. 16. The RNA polynucleotide of claim 1, wherein the targeting sequence is a guide sequence at least 90% identical to SEQ ID NO: 18 and the guide sequence is embedded an S26a scaffold or a S33 scaffold.
  17. 17. The RNA polynucleotide of claim 1, wherein the RNA polynucleotide comprises the sequence of SEQ ID NOs: 117 or 118.
  18. 18. The RNA polynucleotide of claim 17, wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 86 or 87.
  19. 19. An optionally modified inhibitory RNA comprising (a) a guide strand able to hybridize to the target sequence of any of SEQ ID NOs: 119-137; and (b) a substantially complementary passenger sequence; wherein one or more nucleotides of the guide strand and the passenger strand are optionally modified RNA.
  20. 20. A polynucleotide comprising a nucleic acid sequence encoding the RNA polynucleotide of any one of claims 1-19.

Description

Attorney Docket Number: 065830.11208/19WO1 INHIBITORY RNA TARGETING HUNTINGTIN EXPRESSION CROSS REFERENCE TO RELATED APPLICATION [0001] The present application claims priority to U.S. Provisional Application No.63/511,187, filed June 30, 2023, U.S. Provisional Application No.63/591,868, filed October 20, 2023, U.S. Provisional Application No.63/557,370, filed February 23, 2024, and U.S. Provisional Application No.63/654,508 filed May 31, 2024, the disclosures of each of the provisional applications are incorporated by reference herein in their entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY [0002] The contents of the electronic sequence listing (065830.19WO.xml; size: 294,358 bytes; and date of creation: June 20, 2024) is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION [0003] Huntington disease is a neurodegenerative disease caused by an abnormal CAG expansion in exon 1 of the huntingtin gene. CAG expansion produces a mutant huntingtin protein having an increased polyglutamine tract. Huntington disease symptoms include motor impairment, cognitive impairment and psychiatric disorders. A large number of different inhibitory RNA have been proposed to treat Huntington disease. (See e.g., Prister et al., Mol. Ther. Nucleic Acids. (2017) Jun 16;7:324-334; Aguiar et al., Transl. Neurodegener. (2017) Nov 27;6:30; Miniarikova et al., Mol. Ther. Nucleic Acids (2016) Mar 22;5(3):e297; Paul et al., Cells (2020) Jul 15;9(7):1698; Wang et al., Hum. Gene. Ther.2022 Jan;33(1-2):37-60; U.S. Patent No.7,947,658; U.S. Patent No.10,457,940; U.S. Patent No.10,767,180; U.S. Patent No. 10,774,327; U.S. Patent No.10,174,321; U.S. Patent Application Publication No. 2020/0155624; U.S. Patent Application Publication No.2020/0377887; International Publication No. WO 2021/127455; and International Publication No. WO 2021/016505.) BRIEF SUMMARY OF THE INVENTION [0004] The present invention features RNA polynucleotide constructs comprising sequences targeting HTT mRNA, along with nucleotides sequences that can be used, for example, as scaffolds for the targeting sequences; and encoding nucleic acid. RNA polynucleotide constructs include inhibitory RNA polynucleotides comprising a sequence targeting Huntingtin mRNA, such as artificial primary microRNA (pri-amiRNA), pre-microRNA (pre-amiRNA), short hairpin RNA (shRNA), artificial microRNA (amiRNA) and optionally modified amiRNA. Constructs comprising sequences targeting HTT mRNA and/or encoding sequences can be used, for example, in methods for inhibiting mutant HTT expression and/or treating Huntington disease. [0005] A HTT mRNA “targeting” sequence is substantially complementary to a HTT mRNA target sequence. HTT mRNA target sequences described herein include those of any of SEQ ID Nos: 119-137. Preferred targeting sequences can serve as guide sequence for inhibitory RNA. Examples of targeting sequences described herein include sequences at least 80% identical to any of SEQ ID NOs: 1-19. [0006] Reference to “inhibitory” RNA polynucleotide indicates the polynucleotide comprises a sequence targeting an RNA that can inhibit RNA activity. Inhibition of RNA activity results in a decrease in protein expression from the targeted mRNA. Inhibitory RNA includes, for example, amiRNA and amiRNA precursors such as pre-amiRNA, sh-RNA, and pri-amiRNA [0007] Thus, a first aspect of the present invention describes an RNA polynucleotide comprising an RNA sequence targeting HTT mRNA. The RNA polynucleotide comprises a targeting RNA sequence at least 80% identical with the sequence of any of SEQ ID NOs: 1-19; provided that if the targeting sequence is at least 80% identical to SEQ ID NO: 18, then the RNA polynucleotide is either (a) a pri-amiRNA comprising the targeting sequence embedded in a scaffold selected from a S155e scaffold, a S26a scaffold, or a S33 scaffold, or (b) the RNA polynucleotide comprises the sequence of SEQ ID NOs: 117 or 118. [0008] A second aspect of the present invention is directed to an optionally modified inhibitory RNA duplex comprising (a) a guide strand able to hybridize to the target sequence of any of SEQ ID NOs: 119-137; and (b) a substantially complementary passenger sequence; wherein one or more nucleotides of the guide strand and the passenger strand are optionally modified RNA. [0009] A third aspect of the present invention is directed to a polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide comprising a HTT mRNA targeting sequence. [0010] A fourth aspect of the present invention is directed to an expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide comprising a HTT mRNA targeting sequence and one or more expression control elements operably coupled to the encoding nucleic acid sequence. [0011] A fifth aspect of the present invention is directed to recombinant viral vector nucleic acid comprising (a) an expression cassette comprising a nucleic acid sequence encod