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KR-20260068065-A - Zinc clip peptide, encoded nucleic acid, method and use

KR20260068065AKR 20260068065 AKR20260068065 AKR 20260068065AKR-20260068065-A

Abstract

This specification discloses polypeptides used for treating diseases associated with pathogenic genomic repeat sequences, such as neurological disorders. It also discloses nucleic acid molecules and vectors encoding such polypeptides. It also discloses therapeutic uses and methods for treating such diseases; in particular, it discloses therapeutic uses and methods for treating Friedreich's ataxia (FRDA). Furthermore, it discloses methods for actively delivering therapeutic molecules to target cells for an extended period in vivo or in vitro, as well as related peptides and nucleic acids.

Inventors

  • 이살란, 마크
  • 미엘차레크, 미하우

Assignees

  • 임피리얼 컬리지 이노베이션스 리미티드

Dates

Publication Date
20260513
Application Date
20240726
Priority Date
20230726

Claims (20)

  1. A poly-zinc clamp peptide capable of binding to a nucleic acid target sequence within a 5'-GAA-3' trinucleotide repeat sequence, a frameshift variant thereof (i.e., 5'-AGA-3' or 5'-AAG-3'), or a nucleic acid sequence complementary thereto, and a polynucleotide encoding a polypeptide comprising a transcription activation domain.
  2. Polypeptide comprising a nucleic acid target sequence within a 5'-GAA-3' trinucleotide repeat sequence, a poly-zinc clamp peptide capable of binding to a frameshift variant thereof (i.e., 5'-AGA-3' or 5'-AAG-3') or a nucleic acid sequence complementary thereto, and a transcription-activating domain.
  3. In claim 1 or claim 2, A polynucleotide or polypeptide wherein the above-mentioned poly-zinc clamp peptide comprises 6 to 32, 6 to 18, or 6 to 12 zinc clamp domains and/or comprises 6, 9, 11, or 12 zinc clamp domains.
  4. In claim 1 or claim 3, or claim 2 or claim 3, (i) at least six adjacent zinc clamp domains of the poly-zinc clamp peptide comprise a recognition helix sequence comprising SEQ ID NO. 10; (ii) at least six adjacent zinc clamp domains of the poly-zinc clamp peptide comprise a recognition helix sequence comprising SEQ ID NO. 11; SEQ ID NO. 12; or SEQ ID NO. 13; (iii) at least 6, at least 9, or at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprise a recognition helix sequence including SEQ ID NO. 11; (iv) at least 6, at least 9, or at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprise a recognition helix sequence comprising SEQ ID NO. 12; or (v) A polynucleotide or polypeptide in which at least 6, at least 9, or at least 11 adjacent zinc clamp domains of the above poly-zinc clamp peptide comprise a recognition helical sequence comprising SEQ ID NO. 13.
  5. Any one of claims 1, 3 and 4, or any one of claims 2 to 4, A polynucleotide or polypeptide wherein the poly-zinc clamp peptide comprises at least 11 zinc clamp domains, and at least 11 adjacent zinc clamp domains (F1 to F11) of the poly-zinc clamp peptide comprise a recognition helical sequence comprising SEQ ID NO. 14 or SEQ ID NO. 15, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helical sequence comprising SEQ ID NO. 14, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helical sequence comprising SEQ ID NO. 15.
  6. Any one of claims 1 and 3 to 5, or any one of claims 2 to 5, (i) zinc clamp domains F1, F3, F5, F7, F9 and F11 have a recognition helix sequence according to SEQ ID NO. 14, and zinc clamp domains F2, F4, F6, F8 and F10 have a recognition sequence according to SEQ ID NO. 15; (ii) zinc clamp domains F1, F3, F4, F6, F8 and F10 have a recognition sequence according to SEQ ID NO. 14, and zinc clamp domains F2, F5, F7, F9 and F11 have a recognition sequence according to SEQ ID NO. 15; or (iii) a polynucleotide or polypeptide in which zinc clamp domains F1, F3, F5, F6, F8 and F10 have recognition sequences according to SEQ ID NO. 14 and zinc clamp domains F2, F4, F7, F9 and F11 have recognition sequences according to SEQ ID NO. 15.
  7. Any one of claims 1, 3 and 4, or any one of claims 2 to 4, The above-mentioned poly-zinc clamp peptide comprises at least 11 zinc clamp domains, and each of the at least 11 adjacent zinc clamp domains (F1 to F11) of the poly-zinc clamp peptide comprises a recognition helix sequence independently selected from any one of SEQ ID NOs 16 to 25, wherein, (i) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 16, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 17; (ii) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 16, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 19; (iii) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 16, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 21; (iv) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 16, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 23; (v) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 18, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 17; (vi) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 18, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 19; (vii) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 18, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 21; (viii) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 18, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 23; (ix) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 21, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 20; (x) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 21, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 22; (xi) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 21, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 24; (xii) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 18, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 25; (xiii) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 23, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 20; (xiv) at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 23, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 22; (xv) At least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 23, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helix sequence comprising SEQ ID NO. 24; (xvi) A polynucleotide or polypeptide wherein at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helical sequence comprising SEQ ID NO. 23, and at least one of the at least 11 adjacent zinc clamp domains of the poly-zinc clamp peptide comprises a recognition helical sequence comprising SEQ ID NO. 25.
  8. Any one of claims 1 and 3 to 7, or any one of claims 2 to 7, A polynucleotide or polypeptide wherein the above-mentioned poly-zinc clamp peptide comprises 9, 10, 11, or 12 zinc clamp domains forming a zinc clamp array according to any one of the following patterns:
  9. Any one of claims 1 and 3 to 8, or any one of claims 2 to 8, A polynucleotide or polypeptide wherein the above-mentioned poly-zinc clamp comprises a sequence selected from one of SEQ ID NOs 102 to 104 or a sequence having at least 90%, at least 95%, or at least 98%, or at least 99% identity with respect to said sequence.
  10. Any one of claims 1 and 3 to 9, or any one of claims 2 to 9, A polynucleotide or polypeptide comprising an activation domain selected from the VP64 domain (SEQ No. 125), the herpes simplex virus (HSV) VP16 domain (SEQ No. 124), or the human or mouse p65-RelA activation domain (SEQ No. 122 or SEQ No. 123).
  11. Any one of claims 1 and 3 to 10, or any one of claims 2 to 10, A polynucleotide or polypeptide wherein the polypeptide comprises a single or double nuclear localization signal (NLS) sequence; optionally, the nuclear localization signal is selected from SV40 NLS (SEQ No. 107), mouse primase p58 NLS (SEQ No. 109), human protein KIAA2022 NLS (SEQ No. 108); double SV40 NLS (SEQ No. 113), double human KIAA2022 NLS (SEQ No. 114) or double mouse primase p58 NLS (SEQ No. 115).
  12. A polynucleotide encoding a polypeptide comprising a nucleic acid target sequence within the frataxin gene promoter of SEQ ID NO. 67, or a poly-zinc clamp peptide capable of binding to a sequence complementary to said sequence, and a transcription activation domain.
  13. A polypeptide comprising a nucleic acid target sequence within the frataxin gene promoter of SEQ ID NO. 67, a poly-zinc clamp peptide capable of binding to a sequence complementary to said sequence, and a transcriptional activation domain.
  14. In claim 12, or claim 13, A polynucleotide or polypeptide in which the above-mentioned poly-zinc clamp peptide is capable of binding to a target sequence within positions 1 to 700 of SEQ ID NO. 67, within positions 1 to 500 of SEQ ID NO. 67, within positions 1 to 400 of SEQ ID NO. 67, or within positions 20 to 380 of SEQ ID NO. 67.
  15. In claim 12 or claim 14, or claim 13 or 14, A polynucleotide or polypeptide in which the poly-zinc clamp peptide is capable of binding to a target sequence within any one of SEQ ID NOs 68 to 75 or to a sequence complementary to said sequence, and in particular, the poly-zinc clamp peptide is capable of binding to SEQ ID NOs 72, 73, 74 or 75 or to a sequence complementary thereto.
  16. Any one of claim 12, 14, or 15, or any one of claims 13 to 15, A polynucleotide or polypeptide wherein the poly-zinc clamp peptide comprises six or more zinc clamp domains, and in particular, the poly-zinc clamp peptide has six zinc clamp domains, nine zinc clamp domains, or eleven zinc clamp domains.
  17. In any one of claim 12 or 14 to 16, or any one of claims 13 to 16, A polynucleotide or polypeptide wherein the above-mentioned poly-zinc clamp peptide comprises six zinc clamp domains, and each zinc clamp domain (F1 to F6) comprises the following recognition helix sequence: (i) F1, sequence number 28; F2, sequence number 31; F3, sequence number 34; F4, sequence number 37; F5, sequence number 40; and F6, sequence number 43; (ii) F1, sequence number 31; F2, sequence number 34; F3, sequence number 37; F4, sequence number 40; F5, sequence number 43; and F6, sequence number 46; (iii) F1, sequence number 34; F2, sequence number 37; F3, sequence number 40; F4, sequence number 43; F5, sequence number 46; and F6, sequence number 49; (iv) F1, sequence no. 37; F2, sequence no. 40; F3, sequence no. 43; F4, sequence no. 46; F5, sequence no. 49; and F6, sequence no. 52; (v) F1, sequence number 29; F2, sequence number 32; F3, sequence number 35; F4, sequence number 38; F5, sequence number 41; and F6, sequence number 44; (vi) F1, sequence number 32; F2, sequence number 35; F3, sequence number 38; F4, sequence number 41; F5, sequence number 44; and F6, sequence number 47; (vii) F1, sequence number 35; F2, sequence number 38; F3, sequence number 41; F4, sequence number 44; F5, sequence number 47; and F6, sequence number 50; (viii) F1, sequence number 38; F2, sequence number 41; F3, sequence number 44; F4, sequence number 47; F5, sequence number 50; and F6, sequence number 53; (ix) F1, sequence number 30; F2, sequence number 33; F3, sequence number 36; F4, sequence number 39; F5, sequence number 42; and F6, sequence number 45; (x) F1, sequence number 33; F2, sequence number 36; F3, sequence number 39; F4, sequence number 42; F5, sequence number 45; and F6, sequence number 48; (xi) F1, sequence number 36; F2, sequence number 39; F3, sequence number 42; F4, sequence number 45; F5, sequence number 48; and F6, sequence number 51; or (xii) F1, sequence number 39; F2, sequence number 42; F3, sequence number 45; F4, sequence number 48; F5, sequence number 51; and F6, sequence number 54.
  18. In any one of claim 12 or 14 to 17, or any one of claims 13 to 17, A polynucleotide or polypeptide wherein the above-mentioned poly-zinc clamp peptide comprises seven zinc clamp domains, and each zinc clamp domain (F1 to F7) comprises the following recognition helix sequence: (i) F1, sequence number 28; F2, sequence number 31; F3, sequence number 34; F4, sequence number 37; F5, sequence number 40; F6, sequence number 43; and F7, sequence number 46; (ii) F1, sequence number 31; F2, sequence number 34; F3, sequence number 37; F4, sequence number 40; F5, sequence number 43; F6, sequence number 46; and F7, sequence number 49; (iii) F1, sequence number 34; F2, sequence number 37; F3, sequence number 40; F4, sequence number 43; F5, sequence number 46; F6, sequence number 49; and F7, sequence number 52; (iv) F1, sequence number 29; F2, sequence number 32; F3, sequence number 35; F4, sequence number 38; F5, sequence number 41; F6, sequence number 44; and F7, sequence number 47; (v) F1, sequence number 32; F2, sequence number 35; F3, sequence number 38; F4, sequence number 41; F5, sequence number 44; F6, sequence number 47; and F7, sequence number 50; (vi) F1, sequence number 35; F2, sequence number 38; F3, sequence number 41; F4, sequence number 44; F5, sequence number 47; F6, sequence number 50; and F7, sequence number 53; (vii) F1, sequence number 30; F2, sequence number 33; F3, sequence number 36; F4, sequence number 39; F5, sequence number 42; F6, sequence number 45; and F7, sequence number 48; (viii) F1, sequence number 33; F2, sequence number 36; F3, sequence number 39; F4, sequence number 42; F5, sequence number 45; F6, sequence number 48; and F7, sequence number 51; or (ix) F1, sequence number 36; F2, sequence number 39; F3, sequence number 42; F4, sequence number 45; F5, sequence number 48; F6, sequence number 51; and F7, sequence number 54.
  19. In any one of claim 12 or 14 to 18, or any one of claims 13 to 18, A polynucleotide or polypeptide wherein the above-mentioned poly-zinc clamp peptide comprises eight zinc clamp domains, and each zinc clamp domain (F1 to F8) comprises the following recognition helix sequence: (i) F1, sequence number 28; F2, sequence number 31; F3, sequence number 34; F4, sequence number 37; F5, sequence number 40; F6, sequence number 43; F7, sequence number 46; and F8, sequence number 49; (ii) F1, sequence number 31; F2, sequence number 34; F3, sequence number 37; F4, sequence number 40; F5, sequence number 43; F6, sequence number 46; F7, sequence number 49; and F8, sequence number 52; (iii) F1, sequence number 29; F2, sequence number 32; F3, sequence number 35; F4, sequence number 38; F5, sequence number 41; F6, sequence number 44; F7, sequence number 47; and F8, sequence number 50; (iv) F1, sequence no. 32; F2, sequence no. 35; F3, sequence no. 38; F4, sequence no. 41; F5, sequence no. 44; F6, sequence no. 47; F7, sequence no. 50; and F8, sequence no. 53; (v) F1, sequence number 30; F2, sequence number 33; F3, sequence number 36; F4, sequence number 39; F5, sequence number 42; F6, sequence number 45; F7, sequence number 48; and F8, sequence number 51; or (vi) F1, sequence number 33; F2, sequence number 36; F3, sequence number 39; F4, sequence number 42; F5, sequence number 45; F6, sequence number 48; F7, sequence number 51; and F8, sequence number 54.
  20. In any one of claim 12 or 14 to 19, or any one of claims 13 to 19, A polynucleotide or polypeptide wherein the above-mentioned poly-zinc clamp peptide comprises nine zinc clamp domains, and each zinc clamp domain (F1 to F9) comprises the following recognition helix sequence: (i) F1, sequence number 28; F2, sequence number 31; F3, sequence number 34; F4, sequence number 37; F5, sequence number 40; F6, sequence number 43; F7, sequence number 46; F8, sequence number 49; and F9, sequence number 52; (ii) F1, sequence number 29; F2, sequence number 32; F3, sequence number 35; F4, sequence number 38; F5, sequence number 41; F6, sequence number 44; F7, sequence number 47; F8, sequence number 50; and F9, sequence number 53; or (iii) F1, sequence number 30; F2, sequence number 33; F3, sequence number 36; F4, sequence number 39; F5, sequence number 42; F6, sequence number 45; F7, sequence number 48; F8, sequence number 51; and F9, sequence number 54.

Description

Zinc clip peptide, encoded nucleic acid, method and use The present invention relates to novel zinc finger peptides and nucleic acids having desirable characteristics, as well as methods and uses of such peptides and nucleic acids. In particular, the present invention relates to novel zinc finger coding nucleic acids or zinc finger peptides for therapeutic use and methods in the treatment of Friedreich's ataxia (FRDA). Neurological disorders are diseases that affect the central nervous system (brain and spinal cord), the peripheral nervous system (peripheral nerves and cranial nerves), and the autonomic nervous system (parts distributed across both the central and peripheral nervous systems). More than 600 types of neurological diseases have been identified in humans, and these diseases collectively affect all bodily functions, including coordination, communication, memory, learning, eating, and in some cases, death. While many tissues and organs in animals possess the ability to heal, the nervous system generally does not. Consequently, neurological disorders are often characterized by symptoms that progressively worsen; they begin as mild problems that are detectable and diagnosable, but gradually become more severe, frequently leading to the death of the affected individual. Although the exact causes or triggers of many neurological disorders have not yet been identified, the causes of some diseases have been well-identified and studied. For some of these diseases, 'effective' treatments exist that alleviate symptoms and/or extend survival. However, despite intensive research efforts, there is still no cure for most neurological disorders, particularly for the most severe ones. Therefore, new therapeutics and treatments for neurological disorders are required. Current knowledge regarding neurological disorders suggests that they can be caused by many diverse factors, including (though not limited to) inherited genetic abnormalities, problems with the immune system, damage to the brain or nervous system, or diabetes. One of the known causes of neurological disorders is nucleic acid repeat sequences, such as the CAG repeat in the htt gene causing Huntington's disease (HD) (Walker (2007) Lancet 369(9557): 218-228; and Kumar et al. Pharmacol. Rep. 62(1): 1-14)), and the GGGGCC repeat in the C9ORF72 gene of amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) (DeJesus-Hernandez et al . (2011), Neuron , 72: 245-56); and is a genetic abnormality that causes pathological expansion of the GAA repeat (PMID: 30905359) in intron 1 of the frataxin gene in Friedreich's ataxia. Friedreich's ataxia (FRDA) is an autosomal recessive disorder characterized by progressive ataxia, speech, hearing and visual impairments, cardiomyopathy, diabetes, and skeletal muscle abnormalities (PMID: 29053830). This disease is a rare genetic disorder occurring in approximately 1 in 40,000 people, and there is currently no cure (PMID: 30905359). The disease is caused by the expansion of triplet GAA nucleotides to up to 900 repeat units within intron 1 of the frataxin locus. Frataxin plays a crucial role in various cellular processes, and the expansion of GAA repeats significantly reduces frataxin mRNA transcript levels (up to 90%), subsequently leading to the down-regulation of the frataxin gene product. In particular, a decrease in frataxin expression causes degeneration of nerve fibers in the spinal cord and peripheral nervous system, making them thin and leading to serious disease (PMID: 30905359). Homozygous FRDA patients exhibit all symptoms, whereas heterozygous carriers are asymptomatic and account for up to 2% of the general population (PMID: 8596916). To date, available treatments for these and similar conditions have focused on managing symptoms rather than addressing the underlying causes. For example, physical therapy and/or speech therapy can assist with normal physical function and activity; walking aids may be used, and surgery may be necessary to correct physical conditions such as scoliosis or foot deformities. Additionally, medications are commonly used to treat heart disease and diabetes, which typically accompany these conditions. In 2023, omaveloxolone (Skyclaris) was approved by the U.S. Food and Drug Administration (FDA) as the first treatment for Friedreich's ataxia. However, it would be highly desirable to have alternative and more effective therapeutic molecules and treatments for diseases such as FRDA and related disorders or symptoms caused by extended GAA repeats. Accordingly, the present invention aims to overcome or at least improve one or more problems identified in the prior art. The inventors have confirmed that improved or normal/wild-type function can be restored by upregulating the suppressed mutant gene allele involved in the onset of disease symptoms. Accordingly, in a general aspect, the present invention provides a novel zinc clamp peptide and a nucleic acid molecule encoding it that