JPWO2020219726A5 - - Google Patents

Claims (28)

A fusion protein comprising a zinc finger protein (ZFP) domain and a transcriptional repressor domain, wherein the ZFP domain is targeted to a target region in an intron segment between exons 1a and 1b on a mutant allele of the human C9orf72 gene. A fusion protein that binds and wherein the target region comprises more than 30 tandem repeats of G ₄ C ₂ (SEQ ID NO: 1). 2. The fusion protein of claim 1 , which suppresses transcription of repeat-containing mRNA from the mutant allele and does not suppress transcription of wild-type mRNA from the gene. The ZFP domain binds to a sense sequence in the target region, where the sense sequence comprises the hexanucleotides GGGGCC (SEQ ID NO: 1), GGGCCG (SEQ ID NO: 2), GGCCGG (SEQ ID NO: 3), GCCGGG (SEQ ID NO: 4), CCGGGG 3. The fusion protein of claim 1 or 2, comprising 1 to 3 tandem repeats of (SEQ ID NO: 5) or CGGGGC (SEQ ID NO: 6). 4. The fusion protein of any one of claims 1-3, which suppresses sense transcription from a mutant C9orf72 allele in human cells. 5. The fusion protein of claim 4, which represses sense transcription from the C9orf72 1a promoter and does not repress sense transcription from the C9orf72 1b promoter. The ZFP domain binds to an antisense sequence in the target region, where the antisense sequence comprises the hexanucleotides GGCCCC (SEQ ID NO:7), GCCCCG (SEQ ID NO:8), CCCCGG (SEQ ID NO:9), CCCGGC (SEQ ID NO:10) , CCGGCC (SEQ ID NO: 11) or CGGCCC (SEQ ID NO: 12). 7. The fusion protein of any one of claims 1-6 , which suppresses antisense transcription from a mutant C9orf72 allele in human cells. 8. The fusion protein of any one of claims 1-7 , which suppresses both sense and antisense transcription from the mutant C9orf72 allele in human cells. repressing sense and/or antisense transcription from the mutant C9orf72 allele by at least about 30%, 40%, 75%, 90% or 95%, optionally not repressing sense transcription from the C9orf72 1b promoter, claim 9. The fusion protein of any one of 1-8 . The ZFP domain is
including 6 zinc fingers,
binds to the target sequences shown in Table 1 and/or comprises the six zinc finger sequences of the ZFP transcription factors shown in Table 1;
10. The fusion protein of any one of claims 1-9 , wherein the fusion protein optionally comprises one or more mutations to residues outside the recognition helix region as shown in Table 1. 11. The fusion protein of any one of claims 1-10 , wherein the transcriptional repressor domain comprises the KRAB domain amino acid sequence from human KOX1. 12. The fusion protein of any one of claims 1-11 , wherein the ZFP domain is linked to the transcriptional repressor domain via a peptide linker. 13. A nucleic acid construct comprising a coding sequence for the fusion protein of any one of claims 1-12, wherein the coding sequence is operably linked to a transcriptional regulatory element. 14. A nucleic acid construct according to claim 13, wherein the transcriptional regulatory element is a mammalian promoter that is constitutively active or inducible in brain cells , optionally wherein the promoter is the human synapsin I promoter. 15. A nucleic acid construct according to claim 13 or 14 , wherein the construct is a viral construct, optionally wherein the viral construct is a recombinant adeno-associated viral construct. 16. A host cell comprising the nucleic acid construct of any one of claims 13-15. 17. The host cell of claim 16, which is a human cell. 18. A host cell according to claim 17, wherein the human cell is a neuron or pluripotent stem cell, wherein the stem cell is optionally an embryonic stem cell or an induced pluripotent stem cell (iPSC). 16. A recombinant virus comprising the nucleic acid construct of claim 15 , optionally a recombinant adeno-associated virus (rAAV) . 20. A pharmaceutical composition comprising the nucleic acid construct of any one of claims 13-15 or the recombinant virus of claim 19 and a pharmaceutically acceptable carrier. A fusion protein according to any one of claims 1 to 12, a fusion protein according to any one of claims 13 to 15, for use in a method of inhibiting transcription of a mutant allele of the C9orf72 gene in human cells. the nucleic acid construct of claim 19, the recombinant virus of claim 19 or the pharmaceutical composition of claim 20 , wherein
wherein the mutant allele comprises the G ₄ C ₂ (SEQ ID NO: 1) repeat region expanded into the intron segment between exons 1a and 1b, and
A fusion protein, nucleic acid construct, recombinant virus or pharmaceutical composition wherein the method comprises introducing the fusion protein, nucleic acid construct, recombinant virus or pharmaceutical composition into a cell. 22. The fusion protein, nucleic acid construct, recombinant virus or pharmaceutical composition of claim 21, wherein the human cells are neurons, glial cells, ependymal cells or neuroepithelial cells. wherein the cells are in the brain or spinal cord of a patient suffering from a C9orf72-related disorder optionally selected from amyotrophic lateral sclerosis (ALS) and C9 familial frontotemporal dementia (C9FTD) 23. The fusion protein, nucleic acid construct, recombinant virus or pharmaceutical composition of paragraphs 21 or 22. for use in a method of treating a patient suffering from a C9orf72-related disorder optionally selected from amyotrophic lateral sclerosis (ALS) and C9 familial frontotemporal dementia (C9FTD) 21. A fusion protein according to any one of claims 1 to 12, a nucleic acid construct according to any one of claims 13 to 15, a recombinant virus according to claim 19 or a pharmaceutical composition according to claim 20. 25. The fusion protein, nucleic acid construct, recombinant virus or pharmaceutical composition of any one of claims 21-24, wherein the fusion protein is introduced via a recombinant virus expressing the fusion protein . 26. The fusion protein, nucleic acid construct, recombination of claim 25, wherein the recombinant virus is adeno-associated virus (AAV), optionally of serotype 9 or pseudotype AAV2/9 or AAV2/6/9. virus or pharmaceutical composition . 27. The fusion of claim 25 or 26, wherein the recombinant virus is administered to the patient by intracerebroventricular, intrathecal, intracranial, retro-orbital (RO), intravenous, intranasal and/or intracisternal routes. Protein, nucleic acid construct, recombinant virus or pharmaceutical composition . Two or more fusion proteins according to any one of claims 1 to 12 are introduced, optionally wherein the coding sequences for the two or more fusion proteins are on the same recombinant viral vector. 28. The fusion protein, nucleic acid construct, recombinant virus or pharmaceutical composition of any one of 21-27.