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EP-4735015-A1 - HTT REPRESSORS AND USES THEREOF

EP4735015A1EP 4735015 A1EP4735015 A1EP 4735015A1EP-4735015-A1

Abstract

Disclosed herein are improved methods and compositions for diagnosing, preventing and/or treating Huntington's Disease. Among other things, provided herein is a gene therapy construct encoding a non-naturally occurring codon-optimized transcription factor (ZFP-TF) comprising a zinc-finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein the ZFP-TF expression is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), an EFS, or an EF1alpha promoter.

Inventors

  • MOORE, SIMON
  • PROETZEL, GABRIELE
  • DUONG, KHANH
  • IWATA, HIDEHISA

Assignees

  • Takeda Pharmaceutical Company Limited

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A gene therapy construct comprising a non-naturally occurring codon optimized transcription factor (ZFP-TF) comprising a zinc-finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein the ZFP comprises a recognition helix region designated ZFP46025 or ZFP45723, and wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene.
  2. 2. A gene therapy construct comprising a non-naturally occurring codon-optimized transcription factor (ZFP-TF) comprising a zinc-finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein the ZFP-TF comprises a nucleotide sequence having at least 85% identity to any one of SEQ ID NO: 11-22 or SEQ ID NO: 24-29, and wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene.
  3. 3. The ZFP-TF of claim 1 or 2, comprising a nucleotide sequence having 90%, 95% or greater identity to any one of SEQ ID NO: 11-22 or SEQ ID NO: 24-29.
  4. 4. The ZFP-TF of claim 3, comprising 100% identity to any one of SEQ ID NO: 11-22 or SEQ ID NO: 24-29.
  5. 5. The ZFP-TF of any one of claims 1-4, wherein the ZFP-TF expression is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EFl alpha promoter.
  6. 6. The ZFP-TF of any one of the preceding claims, wherein the recognition helix region comprises the amino acid sequence of one of SEQ ID NO: 1-5 or SEQ ID NO: 7-9.
  7. 7. The ZFP-TF of any one of the preceding claims, wherein the target site comprises a CAG repeats domain of the mHTT gene.
  8. 8. The ZFP-TF of claim 6, wherein the target site recognizes a sequence comprising 70%, 75%, 80%, 85%, 90%, 95% or greater identity to SEQ ID NO: 6.
  9. 9. The ZFP-TF of claim 7, wherein the target site recognizes a sequence comprising 100% identity to SEQ ID NO: 6.
  10. 10. The ZFP-TF of any one of the preceding claims, further comprising a sequence encoding a nuclear localization sequence (NLS).
  11. 11. The ZFP-TF of claim 10, wherein the NLS is SV40.
  12. 12. The ZFP-TF of any one of the preceding claims, further comprising inverted terminal repeats (ITRs) flanking the promoter.
  13. 13. The ZFP-TF of any one of the preceding claims, further comprising a human growth hormone (hGH) poly adenylation signal.
  14. 14. The gene therapy construct of any one of the preceding claims, wherein the gene therapy construct is delivered using a viral vector.
  15. 15. The gene therapy construct of claim 14, wherein the viral vector is adeno-associated virus (AAV), lentivirus, adenovirus or a virus-like particle (VLP).
  16. 16. The gene therapy construct of any one of the preceding claims, wherein the gene therapy construct is delivered using a lipid nanoparticle (LNP) or liposome.
  17. 17. A recombinant rAAV vector comprising a gene therapy construct encoding a non- naturally occurring transcription factor (ZFP-TF) comprising a zinc-finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein the ZFP-TF expression is driven by a phosphoglycerate kinase 1 (PGK), a ubiquitin C (UBC), an EFS, or an EFl alpha promoter.
  18. 18. An rAAV vector comprising a gene therapy construct comprising a non-naturally occurring codon optimized transcription factor (ZFP-TF) comprising a zinc-finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein the ZFP comprises a recognition helix region designated ZFP46025 or ZFP45723, and wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene.
  19. 19. An rAAV vector comprising a gene therapy construct comprising a non-naturally occurring codon-optimized transcription factor (ZFP-TF) comprising a zinc-finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein the ZFP-TF comprises a nucleotide sequence having at least 85% identity to any one of SEQ ID NO: 11-22 or SEQ ID NO: 24-29, and wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene.
  20. 20. The rAAV vector of any one of claims 17-19, wherein the rAAV vector is AAV1, AAV2, AAV5, AAV7, AAV9, or AAVrhlO.

Description

HTT REPRESSORS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [1] The instant application claims priority to U.S. Provisional Patent Application No. 63/511,437 filed on June 30, 2023, the contents of which are herein incorporated by reference in entirety, for all purposes. INCORPORATION BY REFERENCE OF SEQUENCE LISTING [2] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 19, 2024, is named MIL-037WOl_SL.xml and is 50,328 bytes in size. BACKGROUND [3] Huntington’s Disease (HD), also known as Huntington’s Chorea, is a progressive disorder of motor, cognitive and psychiatric disturbances. The mean age of onset for this disease is 35-44 years, although in about 10% of cases, onset occurs prior to age 21, and the average lifespan post-diagnosis of the disease is 15-18 years. Prevalence is about 3 to 7 among 100,000 people of western European descent. [4] Huntington’s Disease is an example of a trinucleotide repeat expansion disorder and was first characterized in the early 1990s (see Di Prospero and Fischbeck (2005) Nature Reviews Genetics 6:756-765). These disorders involve the localized expansion of unstable repeats of sets of three nucleotides and can result in loss of function of the gene in which the expanded repeat resides, a gain of toxic function, or both. Trinucleotide repeats can be located in any part of the gene, including non-coding and coding gene regions. Repeats located within the coding regions typically involve either a repeated glutamine encoding triplet (CAG) or an alanine encoding triplet (CGA). Expanded repeat regions within noncoding sequences can lead to aberrant expression of the gene while expanded repeats within coding regions (also known as codon reiteration disorders) may cause mis-folding and protein aggregation. [5] The exact cause of the pathophysiology associated with the aberrant proteins is often not known. Typically, in the wild-type genes that are subject to trinucleotide expansion, these regions contain a variable number of repeat sequences in the normal population, but in the afflicted populations, the number of repeats can increase from a doubling to a log order increase. In HD, repeats are inserted within the N terminal coding region of the gene encoding the large cytosolic protein Huntingtin (HTT). Normal HTT alleles contain 15-24 CAG repeats (“CAG” repeats disclosed as SEQ ID NO: 23), while alleles containing 36 or more repeats can be considered potentially HD causing alleles and confer risk for developing the disease. Alleles containing 36-39 repeats are considered incompletely penetrant, and those individuals harboring those alleles may or may not develop the disease (or may develop symptoms later in life) while alleles containing 40 repeats or more are considered completely penetrant. In fact, no persons containing HD alleles with this many repeats have been reported to be asymptomatic. Those individuals with juvenile onset HD (<21 years of age) are often found to have 60 or more CAG repeats. [6] In addition to an increase in CAG repeats, it has also been shown that HD can involve +1 and +2 frameshifts within the repeat sequences such that the region will encode a polyserine polypeptide (encoded by AGC repeats in the case of a +1 frameshift) track rather than poly-glutamine (Davies and Rubinsztein (2006) Journal of Medical Genetics 43:893-896). [7] In HD, the mutant HTT (mHTT) allele is usually inherited from one parent as a dominant trait. Any child bom of a HD patient has a 50% chance of developing the disease if the other parent was not afflicted with the disorder. In some cases, a parent may have an intermediate HD allele and be asymptomatic while, due to repeat expansion, the child manifests the disease. In addition, the HD allele can also display a phenomenon known as anticipation, wherein increasing severity or decreasing age of onset is observed over several generations due to the unstable nature of the repeat region during spermatogenesis. [8] Furthermore, trinucleotide expansion in HTT leads to neuronal loss in the medium spiny gamma-aminobutyric acid (GABA) projection neurons in the striatum, with neuronal loss also occurring in the neocortex. Medium spiny neurons that contain enkephalin and that project to the external globus pallidum are more involved than neurons that contain substance P and project to the internal globus pallidum. Other brain areas greatly affected in people with Huntington’s disease include the substantia nigra, cortical layers 3, 5, and 6, the CAI region of the hippocampus, the angular gyrus in the parietal lobe, Purkinje cells of the cerebellum, lateral tuberal nuclei of the hypothalamus, and the centromedialparafascicular complex of the thalamus (Walker (2007) Lancet 369:218-228). [9] The role of the normal HTT protein is poorly understood, but it may be involved in neuroge