HTT repressor and use thereof

Abstract

Disclosed herein are improved methods and compositions for diagnosing, preventing and / or treating Huntington's disease. Provided herein, inter alia, 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 transcription repression domain wherein the expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EF1 [alpha] promoter.

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 511,437, filed June 30, 2023, the contents of which are incorporated by reference in their entirety for all purposes.

[0002] INCORPORATION BY REFERENCE OF SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on June 19, 2024, is named MIL-037WO1_SL.xml and is 50,328 bytes in size. BACKGROUND

[0003] Huntington’s disease (HD), also known as Huntington’s Chorea, is a progressive motor, cognitive, and psychiatric disorder. The average age of onset is 35-44 years, but approximately 10% of cases have onset before age 21, and the average life expectancy after diagnosis is 15-18 years. In the Western European-derived population, the prevalence is 3 to 7 per 100,000.

[0004] Huntington’s disease is an example of a trinucleotide repeat expansion disorder, first characterized in the early 1990s (see Di Prospero and Fischbeck (2005) Nature Reviews Genetics 6:756-765). These disorders involve the local amplification of unstable repeat sequences of three nucleotides and can result in loss of function, gain of toxic function, or both, of the gene in which the expanded repeat resides. The trinucleotide repeat sequence can reside in any portion of the gene, including non-coding and coding regions of the gene. Repeat sequences located within coding regions often contain repeated glutamine-encoding triplets (CAG) or alanine-encoding triplets (CGA). Expansion of repeat sequence regions in non-coding sequences leads to aberrant expression of the gene, while expansion of repeats in coding regions, also known as codon repeat disorders, can lead to misfolding and protein aggregation.

[0005] The precise cause of the pathophysiology associated with the abnormal protein is often unknown. Typically, in wild-type genes that undergo trinucleotide expansion, these regions contain a variable number of repeats in normal populations, but in affected populations, the number of repeats can double, or even increase logarithmically. In HD, the repeat sequence is inserted within the N-terminal coding region of the gene that encodes the larger cytosolic protein, Huntingtin (HTT). Normal HTT alleles contain 15-24 CAG repeat sequences ("CAG" repeat sequences disclosed in SEQ ID NO: 23), while alleles containing 36 or more repeats can be considered as potentially pathogenic HD alleles, and confer risk of disease. Alleles containing 36-39 repeats are considered incompletely penetrant, and individuals carrying these alleles can or can not develop the disease (or can develop symptoms later in life), while alleles containing 40 or more repeats are considered fully penetrant. In fact, no one carrying an HD allele with so many repeats has been reported to be asymptomatic. Those individuals with juvenile onset HD (<21 years of age) are typically found to have 60 or more CAG repeats.

[0006] In addition to the increase in CAG repeat sequences, HD can also involve +1 and +2 slippage in the repeat, which results in this region encoding a chain of polyserine polypeptides (encoded by AGC repeats in the case of +1 slippage) rather than polyglutamine (Davies and Rubinsztein (2006) Journal of Medical Genetics 43:893-896).

[0007] In HD, the mutant HTT (m HTT ) allele is typically inherited as a dominant trait from one parent. If the parent is not affected with HD, then a child born to an HD patient has a 50% chance of developing the disease. In some cases, a parent can carry an intermediate HD allele, but be asymptomatic, while the child exhibits the disease due to expansion of the repeat sequence. Furthermore, HD alleles can also exhibit a phenomenon known as anticipation, in which an increase in severity or an earlier age of onset is observed over several generations due to instability of the repeat region during spermatogenesis.

[0008] In addition, trinucleotide expansion in HTT leads to neuronal loss of medium spiny gamma-aminobutyric acid (GABA) projection neurons in the striatum, and neuronal loss also occurs in the neocortex. Medium spiny neurons containing enkephalin and projecting to the external globus pallidus are more heavily involved than neurons containing substance P and projecting to the internal globus pallidus. Other regions of the brain that are more heavily affected in Huntington's disease patients include the substantia nigra; layers 3, 5, and 6 of the cortex; the hippocampal CA1 region; the parietal angular gyrus; Purkinje cells of the cerebellum; the lateral tuberal nucleus of the hypothalamus; and the centromedial parafascicular complex of the thalamus (Walker (2007) Lancet 369:218-228).

[0009] The role of normal HTT protein is not fully understood, but it can be involved in neurogenesis, apoptotic cell death, and vesicular transport. In addition, there is evidence that wild-type HTT stimulates the production of brain-derived neurotrophic factor (BDNF), which is a pro-survival factor for striatal neurons. Studies have shown that the progression of HD in a mouse model of HD is associated with decreased expression of BDNF (Zuccato et al. (2005) Pharmacological Research 52(2): 133-139), and delivery of BDNF or glial cell line-derived neurotrophic factor (GDNF) in a mouse model of HD via gene delivery mediated by a recombinant adeno-associated viral (rAAV) vector can protect striatal neurons (Kells et al. (2004) Molecular Therapy 9(5): 682-688).

[0010] Currently, there are very limited diagnostic and therapeutic options for HD. In terms of diagnosis, (mutant) HTT (mHTT) levels correlate significantly with disease burden scores, and the concentration of soluble mHTT species increases as the disease progresses. However, low abundance mHTT is difficult to quantify in the central nervous system of patients, which both limits research into the role of mHTT in the neuropathobiology of HD in vivo and hinders the demonstration of the effects of drugs that reduce HTT through target engagement. See, e.g., Wild et al. (2014) J Neurol Neurosurg Psychiatry 85: e4.

[0011] Current therapies include tetrabenazine (Xenazine) and deutetrabenazine (Austedo), which are approved by the Food and Drug Administration for the suppression of involuntary jerking and writhing (chorea) symptoms associated with Huntington’s disease. However, these drugs have no effect on the progression of the disease and are accompanied by side effects including sleepiness, restlessness, and the risk of exacerbating or inducing depression or other mental illnesses. Antipsychotic drugs such as haloperidol and fluphenazine also suppress movement and can help treat chorea. However, these drugs are also known to exacerbate involuntary contractions (dystonia), restlessness, and sleepiness. Other drugs, such as olanzapine (Zyprexa) and aripiprazole (Abilify), have fewer side effects but are also known to exacerbate symptoms in some patients.

[0012] However, there remains a need for methods for the diagnosis, treatment, and / or prevention of Huntington’s disease, including therapeutic modalities that are capable of widespread delivery to the brain. SUMMARY

[0013] Disclosed herein are improved methods and compositions for the diagnosis, prevention, and / or treatment of Huntington’s disease. Described herein are m HTTNon-naturally occurring zinc finger proteins (ZFPs) that bind to the CAG repeat domain of the gene, including ZFPs comprising the recognition helix region designated as ZFP46025 or ZFP45723, and codon-optimized variants of ZFPs. The invention provides, inter alia, a gene therapy construct comprising a zinc finger protein, such as ZFP46025 or ZFP45723, and codon-optimized variants of ZFPs. The inventors generated hundreds of codon-optimized sequences using the ATUM tool, and then screened these sequences for various parameters, such as removal of potential splice sites, cryptic promoters, long repeat sequences, and having reduced CpGs (e.g., less than 6 CpGs), among others. Through careful experimentation, the inventors discovered codon-optimized sequences that express at higher levels and / or have higher activity than the parent ZFPs, thereby providing improved methods and compositions for treating Huntington’s disease. The ZFPs of the invention are expressed under the control of promoters that have been optimized for favorable in vivo expression profiles, such as phosphoglycerate kinase 1 (PGK) and ubiquitin C (UBC). Without wishing to be bound by any particular theory, it is contemplated that the use of exemplary PGK or UBC promoters, as well as other promoters, such as, but not limited to, EFS or EF1a promoters, can prevent overexpression of the ZFPs, thereby avoiding eliciting an immune response or silencing. Furthermore, the invention provides pharmaceutical compositions comprising a virus (e.g., an AAV class, such as AAV5, AAV9, e.g., AAV comprising a blood-brain barrier-penetrating capsid, i.e., a BBB-penetrating AAV) or non-viral (e.g., lipid nanoparticle, liposome-based) delivery of the gene therapy constructs described herein. In some embodiments, delivery using a BBB-penetrating AAV allows intravenous administration of the ZFP-TFs of the invention into the brain, thereby reducing the challenges and risks of direct CNS administration. Also provided herein are methods and compositions for altering (e.g., modulating) the expression of alleles of the gene to prevent or treat Huntington’s disease, including m HTT HTT repressor (repressing the expression of the m HTT transcript, and thereby also the mHTT protein). The compositions described herein (e.g., the m HTT repressor) will provide therapeutic benefit to a subject, such as by reducing cell death, reducing apoptosis, increasing cell function (metabolism), and / or reducing motor deficits in the subject. The invention provides a method of treating Huntington’s disease by administering a composition comprising the gene therapy constructs described herein. The invention provides a pharmaceutical composition comprising a gene therapy construct, as well as the use of the pharmaceutical compositions comprising ZFP-TFs described herein for treating Huntington’s disease.

[0014] ​The methods and compositions of the present disclosure provide numerous advantages in gene therapy, such as improved expression or reduced immunogenicity and improved safety, and provide improved methods for treating Huntington’s disease. In some aspects, provided herein is 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 expression of the ZFP-TF is driven by a promoter, including but not limited to, a PGK or UBC promoter, including but not limited to an EFS or EF1a promoter.

[0015] In some embodiments, the ZFP comprises a recognition helix region designated as ZFP46025 or ZFP45723. In some embodiments, the ZFP comprises a recognition helix region designated as ZFP46025. In some embodiments, the ZFP comprises a recognition helix region designated as ZFP45723.

[0016] In some embodiments, the ZFP is codon optimized.

[0017] In some embodiments, the ZFP comprises a nucleotide sequence that is at least 60% identical to any one of SEQ ID NOs: 10-29.

[0018] In some embodiments, the ZFP comprises a nucleotide sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more identical to any one of SEQ ID NOs: 10-29.

[0019] In some embodiments, the ZFP-TF comprises a nucleotide sequence that is 100% identical to any one of SEQ ID NOs: 10-29.

[0020] In some aspects, provided herein is 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 as ZFP46025 or ZFP45723, and wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene.

[0021] In some aspects, provided herein is 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 repressor domain, wherein the ZFP-TF comprises a nucleotide sequence that is at least 85% identical to any one of SEQ ID NOs: 11-22 or SEQ ID NOs: 24-29, and wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene.

[0022] In some embodiments, the ZFP-TF comprises a nucleotide sequence that is 90%, 95% or more identical to any one of SEQ ID NOs: 11-22 or SEQ ID NOs: 24-29. In some embodiments, the ZFP-TF comprises a nucleotide sequence that is 90% identical to any one of SEQ ID NOs: 11-22 or SEQ ID NOs: 24-29. In some embodiments, the ZFP-TF comprises a nucleotide sequence that is 95% identical to any one of SEQ ID NOs: 11-22 or SEQ ID NOs: 24-29. In some embodiments, the ZFP-TF comprises a nucleotide sequence that is more than 90% identical to any one of SEQ ID NOs: 11-22 or SEQ ID NOs: 24-29. In some embodiments, the ZFP-TF comprises a nucleotide sequence that is between 90% and 100% identical to any one of SEQ ID NOs: 11-22 or SEQ ID NOs: 24-29.

[0023] In some embodiments, the ZFP-TF is 100% identical to any one of SEQ ID NOs: 11-22 or SEQ ID NOs: 24-29.

[0024] In some embodiments, expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EF1a promoter. In some embodiments, expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK) promoter. In some embodiments, expression of the ZFP-TF is driven by a ubiquitin C (UBC) promoter. In some embodiments, expression of the ZFP-TF is driven by an EFS promoter. In some embodiments, expression of the ZFP-TF is driven by an EF1a promoter.

[0025] In some embodiments, the recognition helix region of the ZFP-TF comprises an amino acid sequence of one of SEQ ID NOs: 1-5 or SEQ ID NOs: 7-9.

[0026] In some embodiments, the target site comprises the CAG repeat domain of the mHTT gene.

[0027] In some embodiments, the target site recognizes a sequence having 70%, 75%, 80%, 85%, 90%, 95%, or more identity to SEQ ID NO: 6. In some embodiments, the target site recognizes a sequence having 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or more identity to SEQ ID NO: 6. In some embodiments, the target site recognizes a sequence having 70% identity to SEQ ID NO: 6. In some embodiments, the target site recognizes a sequence having 75% identity to SEQ ID NO: 6. In some embodiments, the target site recognizes a sequence having 80% identity to SEQ ID NO: 6. In some embodiments, the target site recognizes a sequence having 85% identity to SEQ ID NO: 6. In some embodiments, the target site recognizes a sequence having 90% identity to SEQ ID NO: 6. In some embodiments, the target site recognizes a sequence having 95% identity to SEQ ID NO: 6.

[0028] In some embodiments, the target site recognizes a sequence having 100% identity to SEQ ID NO: 6.

[0029] In some embodiments, the ZFP-TF further comprises a sequence encoding a nuclear localization sequence (NLS).

[0030] In some embodiments, the NLS is SV40.

[0031] In some embodiments, the ZFP-TF further comprises an inverted terminal repeat sequence (ITR) flanking the promoter. In some embodiments, the ZFP-TF further comprises an inverted terminal repeat sequence (ITR) flanking the PGK promoter. In some embodiments, the ZFP-TF further comprises an inverted terminal repeat sequence (ITR) flanking the UBC promoter. In some embodiments, the ZFP-TF further comprises an inverted terminal repeat sequence (ITR) flanking the EFS promoter. In some embodiments, the ZFP-TF further comprises an inverted terminal repeat sequence (ITR) flanking the EF1a promoter.

[0032] In some embodiments, the ZFP-TF further comprises a human growth hormone (hGH) polyadenylation signal.

[0033] In some embodiments, the gene therapy construct is delivered using a viral vector.

[0034] In some embodiments, the viral vector is an adeno-associated virus (AAV), a lentivirus, or an adenovirus. In some embodiments, the viral vector is an adeno-associated virus (AAV). In some embodiments, the viral vector is a lentivirus. In some embodiments, the viral vector is an adenovirus. In some embodiments, the viral vector is a virus-like particle (VLP).

[0035] In some embodiments, the gene therapy construct is delivered using a lipid nanoparticle (LNP) or a liposome. In some embodiments, the gene therapy construct is delivered using a lipid nanoparticle (LNP). In some embodiments, the gene therapy construct is delivered using a liposome.

[0036] In some aspects, provided herein is 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 expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK) or ubiquitin C (UBC) promoter. In some aspects, provided herein is 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 expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK) promoter. In some aspects, provided herein is 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 expression of the ZFP-TF is driven by a ubiquitin C (UBC) promoter. In some aspects, provided herein is 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 expression of the ZFP-TF is driven by an EFS promoter. In some aspects, provided herein is 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 expression of the ZFP-TF is driven by an EF1a promoter.

[0037] In some aspects, provided herein is a 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 as ZFP46025 or ZFP45723, and wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene.

[0038] In some aspects, provided herein is a 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 that is at least 85% identical to any one of SEQ ID NOs: 11-22 or SEQ ID NOs: 24-29, and wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene.

[0039] In some embodiments, the rAAV vector is an AAV1, AAV2, AAV5, AAV7, AAV9, or AAVrh10 vector. In some embodiments, the rAAV vector is AAV1. In some embodiments, the rAAV vector is AAV2. In some embodiments, the rAAV vector is AAV5. In some embodiments, the rAAV vector is AAV7. In some embodiments, the rAAV vector is AAV9. In some embodiments, the rAAV vector is AAVrh10.

[0040] In some embodiments, the rAAV vector comprises a capsid protein that penetrates the blood brain barrier (BBB).

[0041] In some embodiments, the rAAV vector is VCAP-101, VCAP-102, 9P801, VCAP-100, VCAP-103, PAL1A, PAL1B, PAL1C, PAL2, CereAAV, Dyno bCAP1, AAV.CAP-B10, AAV.CAP-B20, AAV2-BR1N, AAV2-BR1, STAC-BBB ® or AAV-TT, or AAV-BI-hTFR1.

[0042] In some embodiments, provided herein is a lipid nanoparticle comprising a gene therapy construct described herein.

[0043] In some embodiments, provided herein is a pharmaceutical composition comprising a rAAV vector or a lipid nanoparticle.

[0044] In some embodiments, provided herein is a method of modulating expression of a mutant Huntington’s disease (mHTT) allele, comprising administering a pharmaceutical composition provided herein.

[0045] In some aspects, provided herein is a method of modulating expression of a mutant Huntington’s Disease HTT (mHTT) allele, the method comprising administering a rAAV or a lipid nanoparticle comprising one or more gene therapy constructs 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 expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EF1a promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein following administration, expression of the mutant (mHTT) allele is reduced. In some aspects, provided herein is a method of modulating expression of a mutant Huntington’s Disease HTT (mHTT) allele, the method comprising administering a rAAV or a lipid nanoparticle comprising one or more gene therapy constructs 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 expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK) promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein following administration, expression of the mutant (mHTT) allele is reduced. In some aspects, provided herein is a method of modulating expression of a mutant Huntington’s Disease HTT (mHTT) allele, the method comprising administering a rAAV or a lipid nanoparticle comprising one or more gene therapy constructs 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 expression of the ZFP-TF is driven by a ubiquitin C (UBC) promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein following administration, expression of the mutant (mHTT) allele is reduced.In some aspects, provided herein is a method of modulating expression of a mutant Huntington’s Disease HTT (mHTT) allele, the method comprising administering a rAAV or a lipid nanoparticle comprising one or more gene therapy constructs 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 expression of the ZFP-TF is driven by an EFS promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein following administration, expression of the mutant (mHTT) allele is reduced. In some aspects, provided herein is a method of modulating expression of a mutant Huntington’s Disease HTT (mHTT) allele, the method comprising administering a rAAV or a lipid nanoparticle comprising one or more gene therapy constructs 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 expression of the ZFP-TF is driven by an EF1a promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein following administration, expression of the mutant (mHTT) allele is reduced.

[0046] In some aspects, provided herein is a method of treating Huntington’s disease, comprising administering to a subject in need thereof a rAAV or a lipid nanoparticle comprising one or more gene therapy constructs 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 expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EF1a promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein one or more symptoms associated with Huntington’s disease are reduced or alleviated following administration. In some aspects, provided herein is a method of treating Huntington’s disease, comprising administering to a subject in need thereof a rAAV or a lipid nanoparticle comprising one or more gene therapy constructs 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 expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK) promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein one or more symptoms associated with Huntington’s disease are reduced or alleviated following administration. In some aspects, provided herein is a method of treating Huntington’s disease, comprising administering to a subject in need thereof a rAAV or a lipid nanoparticle comprising one or more gene therapy constructs 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 expression of the ZFP-TF is driven by a ubiquitin C (UBC) promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein one or more symptoms associated with Huntington’s disease are reduced or alleviated following administration. In some aspects, provided herein is a method of treating Huntington’s disease, comprising administering to a subject in need thereof a rAAV or a lipid nanoparticle comprising one or more gene therapy constructs 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 expression of the ZFP-TF is driven by an EFS promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein one or more symptoms associated with Huntington’s disease are reduced or alleviated following administration.In some aspects, provided herein is a method of treating Huntington’s disease, comprising administering to a subject in need thereof an rAAV or a lipid nanoparticle comprising one or more gene therapy constructs 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 expression of the ZFP-TF is driven by an EFla promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein one or more symptoms associated with Huntington’s disease are reduced or alleviated following administration.

[0047] In some embodiments, the ZFP comprises a recognition helix region designated as ZFP46025 or ZFP45723.

[0048] In some embodiments, the ZFP is codon-optimized.

[0049] In some embodiments, the ZFP-TF comprises a nucleotide sequence that is at least 85% identical to any one of SEQ ID NOs: 11-22 or SEQ ID NOs: 24-29.

[0050] In some aspects, provided herein is a method of treating Huntington’s disease, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition described herein.

[0051] In some embodiments, the one or more symptoms is cell death.

[0052] In some embodiments, the one or more symptoms is apoptosis.

[0053] In some embodiments, the one or more symptoms is a motor defect.

[0054] In some embodiments, the administering is intrathecal, intracerebroventricular, intranasal, or intravenous administration. In some embodiments, the administering is intrathecal administration. In some embodiments, the administering is intracerebroventricular administration. In some embodiments, the administering is intranasal administration. In some embodiments, the administering is intravenous administration.

[0055] In some embodiments, the administering is by focused ultrasound.

[0056] In some embodiments, the administering is to the brain.

[0057] In some embodiments, administration to the brain is administration to any one of the striatum, cortex, caudate, putamen, thalamus, or globus pallidus region. In some embodiments, administration to the brain is administration to the striatum. In some embodiments, administration to the brain is administration to the cortex. In some embodiments, administration to the brain is administration to the caudate region. In some embodiments, administration to the brain is administration to the putamen. In some embodiments, administration to the brain is administration to the thalamus. In some embodiments, administration to the brain is administration to the globus pallidus region. In some embodiments, administration to the caudate region is administration to the globus pallidus region. In some embodiments, administration is administration to at least one region of the brain. In some embodiments, administration is administration to two or more regions of the brain. In some embodiments, administration is administration to three or more regions of the brain. In some embodiments, administration is administration to four or more regions of the brain. In some embodiments, administration is administration to five or more regions of the brain. The skilled artisan will appreciate that administration is administration to any number of regions listed in any order and any combination thereof. As non-limiting examples, in some embodiments, administration is administration to the striatum, cortex, and caudate region. In some embodiments, administration is administration to the cortex, caudate, and putamen region. In some embodiments, administration is administration to the caudate, putamen, and thalamus region. In some embodiments, administration is administration to the putamen, thalamus, or globus pallidus region. In some embodiments, administration is administration to the thalamus, globus pallidus, and striatum region. In some embodiments, administration is administration to the globus pallidus, striatum, and cortex region.

[0058] In some embodiments of the method, administration is systemic.

[0059] In some embodiments of the method, administration is to the central nervous system (CNS).

[0060] In some aspects, provided herein is a method of treating Huntington’s disease, comprising administering to a subject in need thereof a rAAV or a lipid nanoparticle comprising one or more gene therapy constructs described herein, wherein the rAAV is a BBB-penetrating rAAV, wherein the administration is intravenous administration, and wherein one or more symptoms associated with Huntington’s disease are reduced or alleviated following administration.

[0061] These and other aspects and embodiments are examples of the disclosure and are not limiting, as will be apparent to those of skill in the art in light of the entire disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1A 、 Figure 1B and Figure 1C : Figure 1AA summary of zinc finger proteins (ZFPs) is depicted, including a nuclear localization signal, a zinc finger (ZF) domain, and a KRAB transcriptional repressor domain from the KOX1 protein. Figure 1B The amino acid sequence of ZFP46025 is shown, with the NLS, ZF, and KRAB domains outlined. Figure 1C The amino acid sequence of ZFP45723 is shown, with the NLS, ZF, and KRAB domains outlined.

[0063] Figure 2A , Figure 2B , Figure 2C and Figure 2D : Figure 2A is a diagram of an expression cassette containing a hybrid chicken beta-actin (CBh) promoter, a human growth hormone (hGH) polyadenylation signal (polyA), and a transgene including: a ZFP46025 variant (parental sequence and codon-optimized variant sequence), a T2A self-cleaving peptide, and eGFP. Figure 2B A Western blot of HEK293 lysates probed with antibodies targeting ZFP and GAPDH is shown. Figure 2C Quantification of the average Western blot band intensity normalized to GAPDH intensity is shown, with numerical values annotated above each bar. Figure 2D Quantification of ZFP protein expression as measured by analysis of GFP signal intensity from HEK293 cells following transient transfection is shown.

[0064] Figure 3A , Figure 3B and Figure 3C : Figure 3A is a diagram of an expression cassette containing a hybrid chicken beta-actin (CBh) promoter, a human growth hormone (hGH) polyadenylation signal (polyA), and a transgene with either the parental sequence or a codon-optimized variant of ZFP46025. Figure 3B A Western blot of HEK293 lysates probed with antibodies targeting ZFP and GAPDH is shown. Figure 3C Quantification of the average Western blot band intensity normalized to GAPDH intensity is shown, with numerical values annotated above each bar.

[0065] Figure 4A , Figure 4B , Figure 4C and Figure 4D : Figure 4A is a diagram of a recombinant genome with an ITR-flanked human ubiquitin-c (UBC) promoter, a transgene for a ZFP46025 variant, a human growth hormone (hGH) polyadenylation signal, and a 1 kb stuffer sequence.Figure 4B This is a graph showing the PCR mRNA quantification results relative to GAPDH-normalized ZFP46025, with values ​​marked above each bar. Figure 4C The results of immunocytochemistry-based protein quantification of the ZFP46025 product are shown, with values ​​marked above each bar. Figure 4D This is a graph showing the PCR mRNA quantification results of wild-type and mutant HTT alleles relative to GAPDH normalization, with the values ​​marked above each bar.

[0066] Figure 5A , Figure 5B , Figure 5C and Figure 5D : Figure 5A It is a diagram of the recombinant genome, in which the ITR is flanked by the human phosphoglycerate kinase 1 (PGK) promoter, the transgene of the ZFP46025 variant, the human growth hormone (hGH) polyadenylation signal, and a 1 kb filler sequence. Figure 5B This is a graph showing the PCR mRNA quantification results relative to GAPDH-normalized ZFP46025, with values ​​marked above each bar. Figure 5C This is a graph showing the immunocytochemical-based protein quantification results of the ZFP46025 product, with the values ​​marked above each bar. Figure 5D This is a graph showing the PCR mRNA quantification results of wild-type and mutant HTT alleles relative to GAPDH normalization, with the values ​​marked above each bar.

[0067] Figure 6A , Figure 6B , Figure 6C and Figure 6D : Figure 6A It is a diagram of the recombinant genome, in which the ITR is flanked by the human phosphoglycerate kinase 1 (PGK) promoter, the transgene of the ZFP46025 variant, the human growth hormone (hGH) polyadenylation signal, and a 1 kb filler sequence. Figure 6B This is a graph showing the PCR mRNA quantification results relative to GAPDH-normalized ZFP46025, with values ​​marked above each bar. Figure 5C This is a graph showing the immunocytochemical-based protein quantification results of the ZFP46025 product, with the values ​​marked above each bar. Figure 5D This is a graph showing the PCR mRNA quantification results of wild-type and mutant HTT alleles relative to GAPDH normalization, with the values ​​marked above each bar.

[0068] Figure 7A , Figure 7B and Figure 7C : Figure 7AIt is a diagram of an expression cassette containing a hybrid chicken β-actin (CBh) promoter, a human growth hormone (hGH) polyadenylation signal (polyA), and a transgene with a parental sequence or codon-optimized variant of ZFP45723. Figure 7B The method involves using antibodies targeting the KOX region of ZFP or GAPDH to detect the protein blot of HEK293 lysate. Figure 7C The quantitative results of the average protein blot band intensity normalized relative to GAPDH intensity are shown, with numerical values ​​annotated above each bar.

[0069] Figure 8A , Figure 8B and Figure 8C : Figure 8A It is a diagram of the recombinant genome, in which the ITR is flanked by the human phosphoglycerate kinase 1 (PGK) promoter, the transgene of the ZFP45723 variant, the human growth hormone (hGH) polyadenylation signal, and a 1 kb filler sequence. Figure 8B The results of PCR mRNA quantification relative to GAPDH-normalized ZFP46025 are shown, with values ​​marked above each bar. Figure 8C The results of immunocytochemistry-based protein quantification of the ZFP46025 product are shown, with values ​​marked above each bar.

[0070] Figure 9A , Figure 9B and Figure 9C : Figure 9A This analysis of vector genomes (VGs) in the striatum of AAV-treated Q175 mice was performed using qPCR. Figure 9B This shows RNA analysis of transgenic RNA expression in the striatum of AAV-treated Q175 mice. Figure 9C This study presents an assessment of the reduction in mHTT RNA in the striatum of Q175 mice after intrastriatal administration of AAV9, calculated in comparison to a mediator control.

[0071] Figure 10A to Figure 10G : Figure 10A This analysis of vector genomes (VGs) in the striatum and cortex of AAV-treated Q175 mice was performed using qPCR. Figure 10B RNA analysis showing the expression of transgenic RNA in the striatum and cortex of AAV-treated Q175 mice. Figure 10C This study presents an assessment of the reduction in mHTT RNA in the striatum and cortex of Q175 mice following intravenous (IV) administration of AAV9. Figure 10D A graph showing the quantitative levels of soluble mHTT in the cerebral cortex, determined using the MSD immunoassay. Figure 10EA graph showing the level of soluble mHTT in the striatum of the brain, determined using the MSD immunoassay. Figure 10F A graph showing the level of aggregated mHTT in the cerebral cortex as determined by the MSD immunoassay. Figure 10G A graph showing the quantitative levels of aggregated mHTT in the striatum, determined using the MSD immunoassay.

[0072] Figure 11 A plot depicting quantitative ZFP expression shows that the expression levels of codon-optimized sequences SEQ ID NO: 15 (NH035) and SEQ ID NO: 16 (NH035) are lower than those of the non-codon-optimized parent (SEQ ID NO: 10) NH014AAV. Detailed Implementation

[0073] This document discloses compositions for detecting Huntington's disease (HD), monitoring disease progression, treating and / or preventing Huntington's disease (HD), and extensive CNS delivery methods for said compositions. In some aspects, the compositions and methods described herein use AAV carriers (e.g., BBB-penetrating AAVs) to deliver mHTT inhibitors, thereby enabling functional m HTT The inhibitor can diffuse beyond the delivery site. HTT Restrictors (e.g., m) HTT Regulatory transcription factors, such as m-type transcription factors containing zinc finger proteins (ZFP TFs), HTT Regulatory transcription factors will alter the CNS, thereby reducing or eliminating the effects and / or symptoms of HD, for example by reducing the accumulation of HTT in HD neurons, by increasing the energy of HD neurons (e.g., increasing ATP levels), by reducing apoptosis in HD neurons, and / or by reducing motor deficits in HD subjects.

[0074] This document describes a gene therapy construct comprising a non-naturally occurring transcription factor (ZFP-TF) comprising ZFPs operatively linked to a transcriptional repression domain (e.g., KRAB, KOX, etc.), and optionally comprising other elements, such as a nuclear localization signal (NLS) and / or a promoter (e.g., a constitutive promoter, such as the PGK promoter or the UBC promoter) for driving the expression of a ZFP-TF coding sequence (e.g., the ZFP-TF comprising a ZFP designated as ZFP46025 or ZFP45723, additionally comprising a sequence encoding a transcriptional repression domain and optionally comprising a sequence encoding an NLS and / or a promoter driving ZFP-TF expression). In some embodiments, the promoter is flanked by an inverted terminal repeat (ITR). In some embodiments, the gene construct also comprises a human growth hormone polyadenylation signal.

[0075] In some implementations, this document provides one or more ZFP-TFs whose nucleotide sequences are shown in Table 3.

[0076] This document describes a zinc finger protein transcription factor (ZFP-TF) comprising a zinc finger protein (ZFP), the ZFP being designated as ZFP46025 or ZFP45723, or encoded by a sequence of ZFP46025 or ZFP45723 or a codon-optimized variant thereof, as shown in Table 3. Furthermore, one or more polynucleotides encoding one or more ZFP-TFs described herein are described, wherein the one or more polynucleotides may encode one or more identical and / or different ZFP-TFs, optionally wherein the one or more polynucleotides comprise one or more rAAV vectors, for example, rAAVs comprising sequences encoding one or more ZFP-TFs, the ZFP-TFs comprising a ZFP designated as ZFP46025 or ZFP45723, or wherein the rAAV vectors comprise polynucleotides having sequences shown in Table 3, optionally wherein the one or more rAAV vectors also comprise other elements, such as sequences encoding nuclear localization signals (NLS), and optionally comprise promoters driving ZFP-TF expression, such as constitutive promoters (e.g., PGK, UBC, EFS, or EF1α).

[0077] This document also describes a pharmaceutical composition comprising one or more ZFP-TFs, one or more polynucleotides, and / or one or more rAAV vectors, as described herein. A method for altering the expression of the HTT gene, such as a mutant HTT (mHTT gene), in the cells of a subject, such as neurons in the brain, optionally in the striatum, is also provided, the method comprising administering to the subject's cells one or more ZFP-TFs, one or more polynucleotides, one or more rAAV vectors, and / or the pharmaceutical composition described herein.

[0078] Methods for treating and / or preventing Huntington's disease (HD) in subjects of need are also provided, the methods comprising administering to the subject of need one or more ZFP-TFs, one or more polynucleotides, one or more rAAV carriers, and / or pharmaceutical compositions as described herein, optionally wherein said one or more ZFP-TFs, polynucleotides, rAAV carriers, and / or pharmaceutical compositions are bilaterally administered to the striatum of the subject. One or more ZFP-TFs, one or more polynucleotides, one or more rAAV carriers, and / or pharmaceutical compositions as described herein are also provided for inhibiting mutant strains in subjects of need. HTT (m HTTThe use of the expression of ) . Treatment and / or prevention of HD may involve reducing mHTT aggregates and / or motor deficits in the subject. Furthermore, in any method or use described herein, any dose of one or more ZFP-TFs, one or more polynucleotides, one or more rAAV carriers and / or pharmaceutical compositions may be delivered to the brain of a subject, optionally bilaterally to the striatum of the subject, said dose including but not limited to 1 × 10⁻⁶ per striatum. 7 Up to 1× 10 15 The dose of one (or any value between) vector genomes (vg).

[0079] Therefore, on one hand, engineered (non-naturally occurring) mHTT repressors are provided. These repressors may contain regulatory HD alleles (e.g., m...). HTT Systems expressing transcription factors (e.g., zinc finger proteins). Engineered zinc finger proteins are non-naturally occurring zinc finger proteins whose DNA-binding domains (e.g., recognition helices or RVDs) have been altered (e.g., through selection and / or rational design) to bind to pre-selected target sites. Any zinc finger protein described herein may include 1, 2, 3, 4, 5, 6 or more zinc fingers, each having a recognition helice that binds to a target subsite in a selected sequence (e.g., a gene). In some embodiments, the repressor comprises a DNA-binding domain (ZFP) operatively linked to a transcriptional repression domain, thereby producing a non-naturally occurring transcription factor (ZFP-TF repressor). Optionally, the ZFP-TF repressor includes other components, including but not limited to nuclear localization signals (NLS). In some embodiments, these non-naturally occurring TFs (e.g., ZFP-TFs) include protein-protein interaction domains (or “dimerization domains”) that can polymerize when bound to DNA.

[0080] In some embodiments, the zinc finger protein (ZFP) described herein can be operatively linked to a regulatory domain (or functional domain) as part of a fusion protein. In some embodiments, the functional domain is, for example, a transcriptional activation domain, a transcriptional repression domain, and / or a nuclease (cleavage) domain. By selecting the activation or repression domain for use with a DNA-binding domain, these molecules are used to activate or repress gene expression. In some embodiments, the present invention provides a molecule comprising a target m described herein fused with a transcriptional repression domain for downregulating the expression of mutant HTT. HTTThe ZFP is described. In some embodiments, a fusion protein is provided comprising a ZFP targeting the wild-type HTT allele fused to a transcriptional activation domain capable of upregulating the wild-type HTT allele. In some embodiments, the activity of the regulatory domain is regulated by an exogenous small molecule or ligand such that no interaction with the cellular transcriptional apparatus occurs in the absence of the exogenous ligand, while in other embodiments, the exogenous small molecule or ligand prevents the interaction. These exogenous ligands control the degree of interaction between the ZFP-TF and the transcriptional apparatus. The regulatory domain can be operatively linked to any portion of one or more ZFPs, including between one or more ZFPs, outside one or more ZFPs, and any combination thereof. Any fusion protein described herein can be formulated into a pharmaceutical composition.

[0081] In another aspect, a polynucleotide is provided that encodes one or more of the DNA-binding proteins and / or fusion molecules (e.g., non-naturally occurring transcription factors) described herein. In some embodiments, the polynucleotide is carried in a virus (e.g., AAV or Ad or HSV-1, or VLP; Sheridan, Nature Biotechnology ,40, pp. 809–811 (2022); Gurevich, Nature Medicine , 28, pp. 780–788 (2022)) on vectors and / or non-viral means (e.g., plasmids or mRNA vectors or aptamers). Non-limiting examples of non-viral means include vectors, liposomes, nanoparticles, other lipid-containing complexes (including lipid nanoparticles (LNPs)), other macromolecular complexes, inorganic nanoparticles, synthetic modified mRNA, unmodified mRNA, small molecules, non-bioactive molecules (e.g., gold particles), polymeric molecules (e.g., dendritic polymers), naked DNA, phages, transposons, episomes, plasmid vectors, phage vectors, granules, plasmids, artificial chromosomes, etc. Host cell and / or pharmaceutical compositions comprising these polynucleotides (e.g., rAAV vectors) or non-viral means are also provided, said pharmaceutical compositions comprising the polynucleotides, proteins and / or host cells described herein. In some embodiments, the polynucleotide comprises at least one sequence shown in Table 3. Compositions comprising one or more of these polynucleotides are also provided.

[0082] In some embodiments, the polynucleotide encoding DNA-binding proteins and / or non-naturally occurring transcription factors (e.g., ZFP-TF) is mRNA. In some embodiments, the mRNA may undergo chemical modification (see, for example, Kormann et al. (2011)). Nature Biotechnology29(2):154-157). In other embodiments, the mRNA may contain an ARCA cap (see U.S. Patent Nos. 7,074,596 and 8,153,773). In other embodiments, the mRNA may contain a mixture of unmodified and modified nucleotides (see U.S. Patent Publication No. 2012 / 0195936).

[0083] In another aspect, a gene delivery vector is provided, the gene delivery vector comprising one or more polynucleotides described herein. In some embodiments, the vector is an adenoviral vector (e.g., Ad5 / F35 vector); a lentiviral vector (LV), including integration-competent or integration-deficient lentiviral vectors; an AAV vector (AAV), also known as a recombinant adeno-associated virus vector (rAAV); HSV-1 or VLP, such as a vector pseudotyped with VSV-G or other envelope proteins, or a hybrid vector comprising different AAV elements and mammalian bocaviruses (BoV) and prokaryotic phage elements. (Fakhiri, 2021, Molecular Therapy, 29 (12): 3359-82.) The blood-brain barrier (BBB) ​​exists between cerebral vascular cells and brain cells, preventing the transport and exchange of substances. Due to the presence of the BBB, direct administration of pharmaceutical compositions to the brain remains challenging and poses risks to patients. In some embodiments, the AAV carrier is AAV1, AAV2, AAV5, AAV7, AAV9, or AAVrh10, or other BBB-penetrating AAV carriers (e.g., described in PCT disclosures WO2022221400A2, WO2023091948A1, and WO2020014471A1, which are incorporated herein by reference in their entirety). Exemplary BBB-penetrating AAV vectors include, but are not limited to, VCAP-101, VCAP-102, 9P801, VCAP-100, VCAP-103, PAL1A, PAL1B, PAL1C, PAL2, CereAAV, Dyno bCAP1, AAV.CAP-B10, AAV.CAP-B20, AAV2-BR1N, AAV2-BR1, and STAC-BBB. ® Or AAV-TT or AAV-BI-hTFR1, etc. (Stanton et al., Cell Press Med 4, 31-50; Goertsen et al., Nat. Neuroscience , 2022, 25(1):106-115; Tordo et al., 2018, Brain . 2018, 141(7): 2014–2031).

[0084] The AAV vector contains one or more ZFP-TF polynucleotides (one or more of SEQ ID NO:10-29) as shown in Table 3.

[0085] In addition, pharmaceutical compositions are provided comprising nucleic acids and / or proteins (e.g., ZFPs) and / or fusion molecules (e.g., non-naturally occurring transcription factors containing ZFPs). For example, some compositions comprise a combination of a nucleic acid with a pharmaceutically acceptable carrier or diluent, the nucleic acid containing a sequence encoding one of the ZFPs described herein, the sequence being operatively linked to a regulatory sequence that allows the nucleic acid to be expressed in cells. In some embodiments, the encoded ZFP is specific to the HD HTT allele. In some embodiments, the pharmaceutical composition comprises a regulator of HD m HTT ZFP alleles and ZFPs regulating neurotrophic factors. Proteins encoded by the gene therapy constructs disclosed herein include one or more ZFPs and pharmaceutically acceptable carriers or diluents.

[0086] In some embodiments, the pharmaceutical composition comprises one or more polynucleotides from Table 3 for inhibiting HTT. In some embodiments, the pharmaceutical composition comprising an AAV carrier described herein contains 1 × 10⁻⁶ nucleotides. 8 With 5 × 10 16 Between vg (or any value between them), or even more preferably at 1 × 10 8 With 1 × 10 16 Between vg (or any value between them), or even more preferably at 1 × 10 13 With 5 × 10 15 AAV-ZFP-TF between (or any value between) vg. In some embodiments, the AAV carrier is administered at a dose of 1 × 10⁻⁶ in each of the striatum, cortex, caudate nucleus, putamen, thalamus, or globus pallidus regions of the brain. 11 With 1 × 10 15 Vg values ​​between (or any values ​​between), for example, including but not limited to 1e8, 1e9, 1e10, 1e11, 1e12, 1e13, 1e14, or 1e15 Vg for each striatum, cortex, caudate nucleus, putamen, thalamus, and globus pallidus. In some embodiments, the administered dose is 1 × 10⁻⁶ Vg. 11 vg / kg and 1 × 10 15 The dose is between 1 g / kg and administered via intravenous injection. In some embodiments, the dosage is 1 × 10⁻⁶. 12 vg / kg and 1 × 10 14The dose is between vg / kg and administered intravenously. In some embodiments, the dose is between approximately 5e13 × 5e15 vg and administered via intravenous injection (e.g., based on a body weight of 70 kg).

[0087] Intrastriatal administration may be administered to a single hemisphere, or preferably bilaterally (at the same or different doses). In some embodiments, delivery is performed by non-viral means, such as lipid nanoparticles or liposomes. An isolated cell is also provided, the isolated cell comprising any of the proteins, polynucleotides, and / or compositions described herein.

[0088] On the other hand, this document describes a method for altering the expression of the HTT gene in cells (e.g., neurons, in vitro or in vivo in the brain of a subject, such as the striatum, cortex, caudate nucleus, putamen, thalamus, or globus pallidus), said method comprising administering to cells one or more of the gene therapy constructs, pharmaceutical compositions, and / or cells containing ZFP-TF as described herein. In some embodiments, administration (e.g., administration of the pharmaceutical composition containing AAV ZFP-TF as described herein) is performed before and / or after the onset of disease symptoms at any dose (e.g., at 1 × 10⁻⁶). 7 With 5 × 10 15 The administration is performed between (or any values ​​between) AAV vg. In some embodiments, the administration is a single dose or repeated at any interval, and the doses of the repeated doses may be the same or different. In some embodiments, the HTT gene contains at least one wild-type and / or mutant HTT allele. In some embodiments, HTT expression is repressed, for example, where mutant HTT (mHTT) expression is preferentially repressed compared to wild-type expression. Following one or more administrations of the ZFP-TF described herein, HTT repression (including selective repression of mHTT) can last for days, weeks, months, or years. In some embodiments, after a single administration, selective repression of mHTT (compared to wild-type HTT) will last for 6 months or longer.

[0089] On the other hand, this document provides methods for treating and / or preventing Huntington's disease using the methods and compositions (proteins, polynucleotides, and / or cells) described herein. In some embodiments, these methods involve compositions in which the polynucleotides and / or proteins are delivered using viral vectors (including virus-like particles (VLPs)), non-viral vectors, and / or combinations thereof. In some embodiments, the viral vector is an AAV, such as a BBB-penetrating AAV. In some embodiments, non-viral delivery is performed using lipid nanoparticles (LNPs). The pharmaceutical compositions can also be delivered to the subject using standard techniques. In some embodiments, these methods involve compositions comprising stem cell populations containing ZFP or modified with the ZFN of the present invention. The subject may contain at least one mutant and / or wild-type HTT allele.

[0090] In another aspect, this document also describes a method for delivering one or more inhibitors of HTT (e.g., mHTT) to the brain of a subject using an rAAV (e.g., AAV9 or other AAV serotypes, such as AAV1, AAV2, AAV5, AAV7, AAV9, or AAVrh10; e.g., an AAV containing a capsid that penetrates the blood-brain barrier) carrier. In some embodiments, the inhibitor is delivered using AAV9. In some embodiments, the inhibitor is delivered using AAV5. In some embodiments, the inhibitor is delivered using a blood-brain barrier-penetrating AAV, i.e., a BBB-penetrating AAV. Exemplary BBB-penetrating AAV vectors include, but are not limited to, VCAP-101, VCAP-102, 9P801, VCAP-100, VCAP-103, PAL1A, PAL1B, PAL1C, PAL2, CereAAV, Dyno bCAP1, AAV.CAP-B10, AAV.CAP-B20, AAV2-BR1N, AAV2-BR1, and STAC-BBB. ® Or AAV-TT or AAV-BI-hTFR1, etc. (e.g., PCT publications WO2022221400A2, WO2023091948A1 and WO2020014471A1, which are incorporated herein by reference in their entirety; Stanton et al., Cell Press Med 4, 31-50; Goertsen et al., Nat. Neuroscience, 2022, 25(1):106-115).

[0091] In some implementations, delivery of one or more HTT inhibitors (such as ZFP-TF disclosed herein) into the brain is achieved through non-viral means, such as lipid nanoparticles or liposomes.

[0092] Delivery can be made to any brain region, such as one or more of the striatum, cortex, caudate nucleus, putamen, thalamus, or globus pallidus (e.g., putamen; intrastriatal injection, including stereotactic striatal injection), and can be delivered by any suitable means, including the use of a cannula (e.g., intracranial injection). Administration to the brain (e.g., the striatum, cortex, caudate nucleus, putamen, thalamus, or globus pallidus region) can be to one hemisphere or bilaterally (e.g., with the same or different doses in bilateral administration). In some embodiments, delivery is achieved by direct injection into the intrathecal space. In some embodiments, delivery is achieved by intraventricular (ICV) injection. In some embodiments, delivery is achieved by intracerebral microperfusion (ICM). In some embodiments, delivery is achieved by intrathecal injection. In other embodiments, delivery is achieved by intravenous injection. The rAAV vector delivers the repressor broadly to the subject's brain, including via anterograde and retrograde axonal transport to brain regions not directly to which the vector was administered (e.g., to the striatum), and induces delivery to other structures such as the forebrain, posterior cortex, substantia nigra, thalamus, etc. In some embodiments, one or more gene therapy constructs comprising ZFP-TFs from Table 3 (or pharmaceutical compositions comprising ZFP-TF-containing gene therapy constructs) are delivered to the subject. Any one of the repressors shown in Table 3 or combinations thereof (e.g., any combination of 1, 2, 3, 4, or 5 repressors) may be used.

[0093] Therefore, in other respects, this article describes a method for preventing and / or treating HD in a subject, the method comprising administering a mutant to the subject. HTT (m HTTAt least one repressor of the allele. The repressor may be administered in a polynucleotide form, for example, in a protein form using a viral vector (e.g., AAV) and / or a non-viral vector (e.g., plasmid and / or mRNA), and / or via a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising one or more polynucleotides, one or more AAV vectors, one or more LNP compositions, one or more fusion molecules, and / or one or more cellular components, as described herein). In some embodiments, the repressor is administered to the subject's CNS (e.g., striatum or other regions). The inhibitors can provide multiple therapeutic benefits, including, but not limited to, reducing mHTT RNA in blood and / or CSF; reducing mHTT protein levels in blood and / or CSF; reducing the formation of mHTT aggregates in HD neurons of subjects with HD (including reducing mHTT aggregates without affecting nuclear aggregates); reducing cell death in individual neurons or groups of neurons (e.g., individual HD neurons or groups of HD neurons); and / or reducing motor deficits (e.g., grasping, chorea, balance problems, etc.) in HD subjects; improving total motor score (TMS); improving the Composite Unified Huntington's Disease Rating Scale (cUHDRS); and improving total functional capacity (TFC). In some embodiments, the expression of mutant HTT is inhibited by administering one or more proteins to the subject and / or delivering the polynucleotides (or pharmaceutical compositions containing these proteins and / or polynucleotides) listed in Table 3 to the subject.

[0094] In any of the methods described herein, the repressor of the mutant HTT allele can be a ZFP-TF, such as a fusion protein comprising a ZFP that specifically binds to the mutant HTT allele and a transcriptional repression domain (e.g., KOX, KRAB, etc.). In some embodiments, the ZFP-TF comprises a ZFP having a recognition helical region as shown in Table 1, including ZFP-TF repressors encoded by the polynucleotides shown in Table 3. In any of the methods described herein, the repressor can be delivered to a subject (e.g., the brain) in the form of a protein, a polynucleotide, or any combination of a protein and a polynucleotide. In some embodiments, the repressor is delivered using an AAV vector (e.g., AAV5, AAV9, or BBB-penetrating AAV). In some embodiments, the repressor is a fusion protein. In other embodiments, the repressor is delivered in RNA form. In other embodiments, the repressor is delivered using any combination of expression constructs described herein, such as a repressor (or a portion thereof) on one expression construct (e.g., AAV, such as AAV5, AAV9, etc.) and a repressor (or a portion thereof) on another different expression construct (rAAV or other viral or non-viral constructs).

[0095] Furthermore, in any of the methods described herein, the repressor can be delivered at any concentration (dose) to provide the desired effect. As shown herein, HTT repression can be achieved in subjects at exposures as low as 1 VG / cell. In a preferred embodiment, the repressor is delivered using a recombinant adeno-associated virus vector, delivering 10,000–500,000 vector genomes per cell (or any value between them). In other embodiments, the repressor is delivered using a plasmid construct at 150–1,500 ng / 100,000 cells (or any value between them). In other embodiments, the repressor is delivered in mRNA form at 0.003–1,500 ng / 100,000 cells (or any value between them). In some embodiments, the AAV dose is calculated based on the subject. For example, the AAV vector described herein may contain 1 × 10⁻⁶ cells. 7 Up to 5 × 10 16 vg (or any value therein), or even more preferably 1 × 10 9 Up to 1 × 10 15 vg (or any value therein), or even more preferably 1 × 10 12 Up to 1 × 10 14 vg (or any value between). In some implementations, the AAV vector is in the form of 1 × 10-1 12 Up to 1× 10 15Administered at a dose of vg (or any value between vg and vg). In some embodiments, administration is in the form of 1 × 10⁻⁶. 11 vg / kg and 1× 10 14 Between vg / kg, 1 × 10 12 vg / kg and 1 × 10 13 The dose is between vg / kg and administered intravenously. In some embodiments, administration is performed by intravenous injection at a dose between approximately 1e15 and 5e15 vg.

[0096] Intrastriatal administration can be applied to a single hemisphere, or preferably bilaterally (at the same or different doses). For example, in some embodiments, the inhibitor is delivered at a dose of about 9e13 vg, or between about 9e10 vg and 5e14 vg, or between about 3e11 vg and 9e13 vg. In some embodiments, the AAV dose is less than 9e10 vg (e.g., 6e8 vg or less), while in other embodiments, the AAV dose is greater than 9e13 vg.

[0097] In any of the methods described herein, the compositions and methods described herein can repress the expression of mutant HTT alleles in one or more HD neurons of a subject by about 70% or more, about 75% or more, about 85% or more, about 90% or more, about 92% or more, or about 95% or more. Furthermore, the compositions and methods described herein can exhibit selectivity for HTT (e.g., mHTT) repression (compared to repression of off-target sites), achieving repression of at least 50%, such as 50%-95%, 60%-80% (or any value between these values), or greater than 85% (compared to controls).

[0098] In other aspects, the invention described herein comprises one or more HTT-regulated transcription factors, such as HTT-regulated transcription factors comprising one or more zinc finger proteins (ZFP TFs). In some embodiments, the HTT-regulated transcription factor can repress the expression of mutant HTT alleles in one or more HD neurons of a subject. The degree of repression of mutant HTT alleles in one or more HD neurons of a subject can reach 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 92% or higher, or 95% or higher compared to untreated (e.g., wild-type) neurons of the subject. In some embodiments, the HTT-regulated transcription factor can be used to implement one or more methods described herein. In some embodiments, the ZFP-TF comprises the amino acid sequence of the mHTT repressor shown in Table 3.

[0099] In some implementation schemes, therapeutic efficacy is assessed using the Unified Huntington's Disease Rating Scale (UHDRS) (Huntington Study Group (1996)). Mov Disord 11(2):136-142) The efficacy was measured by analyzing obvious clinical symptoms. In other embodiments, positron emission tomography (PET) and magnetic resonance imaging (MRI) were used to measure the efficacy on the patient. In some embodiments, treatment with mutant HTT-regulated transcription factors will prevent any further development of obvious clinical symptoms and prevent any further loss of neuronal function. In other embodiments, treatment with mutant HTT-regulated transcription factors will improve clinical symptoms (e.g., motor function determined using known measures such as grasping behavior, rotational rod analysis, etc.) and improve neuronal function.

[0100] A kit is also provided comprising one or more HTT regulators (e.g., repressors) and / or components comprising the HTT regulators described herein and / or polynucleotides encoding the HTT regulators described herein (or components thereof). The kit may also comprise cells (e.g., neurons), reagents (e.g., for detecting and / or quantifying mHTT proteins, for example, in cerebrospinal fluid), and / or instructions for use, including the methods described herein.

[0101] General Principles Unless otherwise indicated, the practice of the methods disclosed herein, as well as the preparation and use of the compositions, employs conventional techniques from molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA, and related fields, all of which are within the scope of skill in this art. These techniques have been explained in detail in the literature. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd ed., Cold Spring Harbor Laboratory Press, 1989 and 3rd ed., 2001; Ausubel et al., *Current Protocols in Molecular Biology*, John Wiley & Sons, New York, 1987 and regularly updated; the series *Methods in Enzymology*, Academic Press, San Diego; Wolffe, *Chromatin Structure and Function*, 3rd ed., Academic Press, San Diego, 1998; *Methods in Enzymology*, Vol. 304, “Chromatin” (edited by PM Wassarman and AP Wolffe), Academic Press, San Diego, 1999; and *Methods in Molecular Biology*, Vol. 119, “Chromatin Protocols” (edited by PB Becker), Humana Press, Totowa, 1999.

[0102] definition The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably and refer to deoxyribonucleotides or ribonucleotide polymers in linear or cyclic conformations, in single-stranded or double-stranded form. For the purposes of this disclosure, these terms should not be construed as limiting the length of the polymer. The terms may cover known analogs of natural nucleotides, as well as nucleotides modified in the base, sugar, and / or phosphate moieties (e.g., the phosphate thioester backbone). Generally, analogs of a particular nucleotide have the same base-pairing specificity; that is, an analog of A will pair with a T base.

[0103] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The term also applies to amino acid polymers, where one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids.

[0104] "Binding" refers to sequence-specific, non-covalent interactions between macromolecules (e.g., between proteins and nucleic acids). Not all binding interaction components need to be sequence-specific (e.g., contacting phosphate residues in the DNA backbone); the interaction as a whole only needs to be sequence-specific. Such interactions are typically defined by a dissociation constant (K). d ) is 10 -6 M -1 Characterized by a lower or even lower K. "Affinity" refers to the strength of the binding: increased binding affinity is associated with a lower K. d Related.

[0105] A "binding protein" is a protein that can bind non-covalently to another molecule. Binding proteins can bind to, for example, DNA molecules (DNA-binding proteins), RNA molecules (RNA-binding proteins), and / or protein molecules (protein-binding proteins). In the case of protein-binding proteins, it can bind itself (to form homodimers, homotrimers, etc.) and / or it can bind one or more different proteins, including one or more molecules. A single binding protein can possess multiple types of binding activity. For example, zinc finger proteins exhibit DNA-binding, RNA-binding, and protein-binding activities.

[0106] A "zinc finger DNA-binding protein" (or binding domain) is a protein, or a domain within a larger protein, that binds to DNA in a sequence-specific manner via one or more zinc fingers, where the zinc finger is an amino acid sequence region within the binding domain that is stabilized by coordination with zinc ions. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP.

[0107] Zinc finger binding domains can be "engineered" to bind to predetermined nucleotide sequences, for example, by engineering the recognition helical region of naturally occurring zinc finger proteins (by altering one or more amino acids). Therefore, engineered zinc finger proteins are non-natural proteins. Non-limiting examples of methods for engineering zinc finger proteins include design and selection. "Designed" zinc finger proteins are proteins that do not exist in nature and whose design / composition is primarily derived from rational criteria. Rational design criteria include applying substitution rules and computer algorithms to process existing ZFP design information and binding data stored in databases. "Selected" zinc finger proteins are proteins that do not exist in nature and whose production is primarily achieved through empirical methods such as phage display, interaction traps, or hybridization selection. See, for example, U.S. Patent Nos. 8,586,526, 6,140,081, 6,453,242, 6,746,838, 7,241,573, 6,866,997, 7,241,574, and 6,534,261; see also International Patent Publication No. WO 03 / 016496.

[0108] The term "sequence" refers to a nucleotide sequence of any length, which can be DNA or RNA; it can be linear, circular, or branched, and can be single-stranded or double-stranded. The term "donor sequence" refers to a nucleotide sequence inserted into the genome. Donor sequences can be of any length, for example, between 2 and 10,000 nucleotides (or any integer value between or greater than this), preferably between about 100 and 1,000 nucleotides (or any integer value between this and this), and more preferably between about 200 and 500 nucleotides.

[0109] "Target site" or "target sequence" refers to the nucleic acid sequence that a binding molecule will bind to, provided that sufficient binding conditions exist.

[0110] "Exogenous" molecules are those that are not normally present in cells but can be introduced into cells through one or more genetic, biochemical, or other methods. "Normal presence in cells" is determined for a specific developmental stage and environmental conditions. Therefore, for example, a molecule present only during embryonic muscle development is an exogenous molecule relative to adult muscle cells. Similarly, a heat shock-induced molecule is an exogenous molecule relative to cells that have not undergone heat shock. Exogenous molecules can include, for example, functional forms of dysfunctional endogenous molecules, or dysfunctional forms of normally functioning endogenous molecules.

[0111] Exogenous molecules can be, in particular, small molecules, such as those produced through combinatorial chemistry, or large molecules, such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, and any modified derivatives of the above molecules, or any complex containing one or more of the above molecules. Nucleic acids include DNA and RNA, and can be single-stranded or double-stranded; linear, branched, or circular; and can be of any length. Nucleic acids include those capable of forming double strands and those capable of forming triple strands. See, for example, U.S. Patent Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA-binding proteins, polymerases, methyltransferases, demethylases, acetyltransferases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, DNA gyrases, and helicases.

[0112] Exogenous molecules can be molecules of the same type as endogenous molecules, such as exogenous proteins or nucleic acids. For example, exogenous nucleic acids can contain infectious viral genomes, plasmids or episomes introduced into cells, or chromosomes that are not normally present in cells. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate coprecipitation, DEAE-glucan-mediated transfer, and viral vector-mediated transfer. Exogenous molecules can also be molecules of the same type as endogenous molecules but derived from a species different from the cell source. For example, human nucleic acid sequences can be introduced into cell lines originally derived from mice or hamsters.

[0113] In contrast, "endogenous" molecules are molecules that are typically present in specific cells under specific developmental stages and environmental conditions. For example, endogenous nucleic acids can include chromosomes; the genomes of mitochondria, chloroplasts, or other organelles; or naturally occurring free nucleic acids. Other endogenous molecules can include proteins, such as transcription factors and enzymes.

[0114] A "fusion" molecule is a molecule in which two or more subunit molecules are linked, preferably by covalent bonds. The subunit molecules can be molecules of the same chemical type or molecules of different chemical types. Examples of the first type of fusion molecules include, but are not limited to, fusion proteins (e.g., fusions between ZFP and one or more activated domains) and fusion nucleic acids (e.g., nucleic acids encoding the fusion proteins described above). Examples of the second type of fusion molecules include, but are not limited to, fusions between nucleic acids and polypeptides formed by triplet formation, and fusions between minor groove binders and nucleic acids. The term also includes systems in which polynucleotide components bind to polypeptide components to form functional molecules.

[0115] Expression of fusion proteins in cells can be achieved by delivering the fusion protein to cells or by delivering a polynucleotide encoding the fusion protein to cells, wherein the polynucleotide is transcribed and the transcript is translated, thereby producing the fusion protein. Protein expression in cells may also involve trans-splicing, peptide cleavage, and peptide linking. Other parts of this disclosure present methods for delivering polynucleotides and peptides to cells.

[0116] "Multimerizing domains" (also known as "dimerizing domains" or "protein-protein interaction domains") are domains incorporated into the N-terminal, C-terminal, or N-terminal and C-terminal regions of a ZFPTF. These domains allow for the multimerization of multiple ZFPTF units, making longer trinucleotide repeat domains, relative to shorter wild-type length regions, more preferably bound by multimerized ZFPTFs. Examples of multimerizing domains include leucine zippers. Multimerizing domains can also be regulated by small molecules, where they only conform appropriately in the presence of small molecules or external ligands to allow interaction with another multimerizing domain. In this way, exogenous ligands can be used to modulate the activity of these domains.

[0117] For the purposes of this disclosure, "gene" includes the DNA region encoding a gene product (see below), as well as all DNA regions that regulate the production of the gene product, whether or not these regulatory sequences are adjacent to the coding and / or transcribed sequences. Therefore, a gene includes, but is not limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix connection sites, and locus control regions.

[0118] "Gene expression" refers to the conversion of information contained in a gene into a gene product. Gene products can be direct transcription products of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozymes, structural RNA, or any other type of RNA) or proteins produced by the translation of mRNA. Gene products also include RNA modified through methods such as capping, polyadenylation, methylation, and editing, as well as proteins modified through methods such as methylation, acetylation, phosphorylation, ubiquitination, ADP ribosylation, myristoylation, and glycosylation.

[0119] The “regulation” of gene expression refers to changes in gene activity. Expression regulation includes, but is not limited to, gene activation and gene repression. Genome editing (e.g., cleavage, alteration, inactivation, random mutation) can be used to regulate expression. Gene inactivation refers to any reduction in gene expression compared to cells excluding the ZFP described herein. Therefore, gene inactivation can be partial or complete.

[0120] A "region of interest" (ROI) is any region of cellular chromatin that requires the binding of exogenous molecules, such as a gene or a non-coding sequence within or near a gene. Binding can be used to target DNA cleavage and / or recombination. For example, RIOs can be located in chromosomes, episomes, organelle genomes (e.g., mitochondria, chloroplasts), or the genome of an infecting virus. RIOs can be located within the coding region of a gene, within a transcribed non-coding region (e.g., leader sequences, tail sequences, or introns), or within a non-transcribed region upstream or downstream of the coding region. The length of an RIO can be as small as one nucleotide pair, as large as 2,000 nucleotide pairs, or any integer value of nucleotide pairs.

[0121] "Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells, and human cells (such as T cells).

[0122] The terms "operative linkage" and "operatively linked" (or "operably linked") are used interchangeably to refer to the juxtaposition of two or more components (e.g., sequence elements) arranged such that both components function normally and allow at least one component to mediate the function of at least one other component. For example, if a transcriptional regulatory sequence (e.g., a promoter) controls the transcriptional level of a coding sequence in response to the presence or absence of one or more transcriptional regulatory factors, then the transcriptional regulatory sequence is operatively linked to the coding sequence. The transcriptional regulatory sequence is typically cis-operatively linked to the coding sequence, but does not need to be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence operatively linked to a coding sequence, even if they are not adjacent.

[0123] In the context of fusion peptides, the term "operationally linked" can refer to a situation where each component performs the same function when linked to another component as it does when not linked. For example, in a fusion peptide that fuses a ZFP with an activation domain, the ZFP and activation domain are in an operationally linked state if the ZFP in the fusion peptide can bind to its target site and / or its binding site, and the activation domain can upregulate gene expression. ZFPs fused to domains that regulate gene expression are collectively referred to as "ZFP-TFs" or "zinc finger transcription factors."

[0124] A “functional fragment” of a protein, polypeptide, or nucleic acid is a protein, polypeptide, or nucleic acid whose sequence differs from the full-length protein, polypeptide, or nucleic acid, but which retains the same function as the full-length protein, polypeptide, or nucleic acid. Functional fragments may have more, fewer, or the same number of residues than the corresponding natural molecule, and / or may contain one or more amino acid or nucleotide substitutions. Methods for determining nucleic acid function (e.g., coding function, ability to hybridize with another nucleic acid) are well known in the art. Similarly, methods for determining protein function are also well known. For example, the DNA-binding function of a polypeptide can be determined, for example, by membrane binding, changes in electrophoretic mobility, or immunoprecipitation assays. DNA cleavage can be determined by gel electrophoresis. See Ausubel et al., ibid. The ability of a protein to interact with other proteins can be determined by co-immunoprecipitation, two-hybrid assays, or genetic and biochemical complementation. See, for example, Fields et al. (1989). Nature 340:245-246; US Patent No. 5,585,245 and International Patent Publication No. WO 98 / 44350.

[0125] "Vector" is a device that can transfer gene sequences to target cells. Generally, "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct that can guide the expression of a gene of interest and transfer the gene sequence to target cells. Therefore, the terminology includes both cloning and expression vectors. The terminology includes viral and non-viral vectors, including but not limited to plasmids, mRNA, AAV (also referred to herein as "recombinant AAV" or "rAAV"), adenovirus vectors (Ad), lentiviral vectors (e.g., IDLV), lipid nanoparticles, liposomes, etc.

[0126] A “reporter gene” or “reporter sequence” is a sequence that produces a protein product that is easily measurable, preferably (but not necessarily) measurable in routine assays. Suitable reporter genes include, but are not limited to: sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance); sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase); and proteins that mediate enhanced cell growth and / or gene amplification (e.g., dihydrofolate reductase). Epitope tags include one or more copies of, for example, FLAG, His, myc, Tap, HA, or any detectable amino acid sequence. “Expression tags” include sequences encoding reporter genes that can be operatively linked to desired gene sequences for monitoring the expression of genes of interest.

[0127] DNA-binding domain The methods described herein utilize compositions, such as HTT-regulated transcription factors, comprising a DNA-binding domain that specifically binds to a target sequence in an HTT gene, particularly to a mutant HTT allele (mHTT) comprising multiple trinucleotide repeat sequences. Any polynucleotide or polypeptide DNA-binding domain may be used in the compositions and methods disclosed herein, such as DNA-binding proteins (e.g., ZFP) or DNA-binding polynucleotides (e.g., guide RNA). In some embodiments, the DNA-binding domain binds to a target site comprising 9 to 28 (or any value between, including 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) consecutive copies of the nucleotide of SEQ ID NO: 6.

[0128] In some implementations, m HTT Regulatory transcription factors or their DNA-binding domains contain zinc finger proteins. The selection of target sites, ZFP, and the design and construction of fusion proteins (and their encoded polynucleotides) are known to those skilled in the art and are described in detail in U.S. Patent Nos. 6,140,081, 5,789,538, 6,453,242, 6,534,261, 5,925,523, 6,007,988, 6,013,453, and 6,200,759; and International Patent Publications Nos. WO 95 / 19431, WO 96 / 06166, WO 98 / 53057, WO 98 / 54311, WO 00 / 27878, WO 01 / 60970, WO 01 / 88197, WO 02 / 099084, WO98 / 53058, WO 98 / 53059, WO 98 / 53060, WO In 02 / 016536 and WO 03 / 016496.

[0129] In some embodiments, ZFPs can selectively bind to either a mutant HTT allele or a wild-type HTT sequence. HTT target sites typically include at least one zinc finger, but can also have multiple zinc fingers (e.g., 2, 3, 4, 5, 6, or more). See, for example, U.S. Patent Nos. 9,234,016, 9,943,565, 8,841,260, and 9,499,597; and U.S. Patent Publications 2015 / 0335708, 2018 / 0200332, 2017 / 0096460, 2017 / 0035839, 2016 / 0296605, and 2019 / 0322711. Typically, ZFPs include at least three zinc fingers. Some ZFPs include four, five, or six zinc fingers, while some ZFPs include 7, 8, 9, 10, 11, or 12 zinc fingers. ZFPs with three zinc fingers typically recognize target sites comprising 9 or 10 nucleotides; ZFPs with four zinc fingers typically recognize target sites comprising 12 to 14 nucleotides; and ZFPs with six zinc fingers can recognize target sites comprising 18 to 21 nucleotides. ZFPs can also be fusion proteins comprising one or more regulatory domains, which can be transcriptional activation domains or transcriptional repression domains. In some embodiments, the fusion protein comprises two ZFP DNA-binding domains linked together. Therefore, these zinc finger proteins can contain 8, 9, 10, 11, 12, or more zinc fingers. In some embodiments, the two DNA-binding domains are connected by an extendable flexible linker, such that one DNA-binding domain contains 4, 5, or 6 zinc fingers, while the second DNA-binding domain contains another 4, 5, or 5 zinc fingers. In some embodiments, the linker is a standard interdigital linker, such that the finger array contains a DNA-binding domain containing 8, 9, 10, 11, 12, or more fingers. In other implementations, the linker is atypical, such as a flexible linker. The DNA-binding domain is fused to at least one regulatory domain and can be viewed as a “ZFP-ZFP-TF” architecture. Specific examples of these implementations can be referred to as “ZFP-ZFP-KOX,” which comprises two DNA-binding domains linked by a flexible linker and fused to a KOX repressor; or as “ZFP-KOX-ZFP-KOX,” in which two ZFP-KOX fusion proteins are fused together via a linker.

[0130] Alternatively, the DNA-binding domain can originate from nucleases. For example, the recognition sequences of homing endonucleases and meganucleases are known, such as I- Sce I、I- Ceu I, PI- Psp I, PI- Sce 、I- Sce IV, I-Csm I、I- Pan I、I- Sce II, I- Ppo I、I- Sce III, I -Cre I、I- Tev I、I- Tev II and I- Tev III. See also U.S. Patent No. 5,420,032; U.S. Patent No. 6,833,252, and Belfort et al. (1997). Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22:1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the catalogue of New England Biolabs. Furthermore, homing endonucleases and meganucleases can be engineered to bind to non-natural target sites. See, for example, Chevalier et al. (2002). Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) Current Gene Therapy 7:49-66; US Patent Publication No. 2007 / 0117128.

[0131] "Biphasic" zinc finger proteins are proteins in which two zinc finger DNA-binding domain clusters are separated by inserted amino acids, allowing the two zinc finger domains to bind to two discontinuous target sites. An example of a biphasic zinc finger binding protein is SIP1, in which four zinc finger clusters are located at the amino terminus of the protein, while three zinc finger clusters are located at the carboxyl terminus (see Remacle et al. (1999)). EMBO Journal 18(18):5073-5084). Each zinc finger cluster in these proteins can bind to a unique target sequence, and the spacing between two target sequences can contain many nucleotides. Biphasic ZFPs can include functional domains, for example, fused to one or two ZFPs. Therefore, it is clear that functional domains can be attached to the outside of one or two ZFPs, or can be located between ZFPs (attached to two ZFPs).

[0132] Table 1 and specific examples of ZFPs targeting HTT are disclosed in U.S. Patent Nos. 9,234,016, 8,841,260, and 6,534,261; U.S. Patent Publications 2017 / 0096460, 2015 / 0056705, 2015 / 0335708, and 2019 / 0322711, all of which are incorporated herein by reference in their entirety for all purposes. The first column of this table is the internal reference name (number) of the ZFP, which corresponds to the name in column 1 of Table 2. “F” refers to a zinc finger, and the number following “F” indicates which zinc finger (e.g., “F1” refers to the first zinc finger).

[0133] Table 1: Zinc finger proteins targeting HTT

[0134] Table 2 discloses the sequences and locations of the target sites for these proteins. Nucleotides in the target sites that are in contact with the ZFP recognition helix are represented by uppercase letters; uncontacted nucleotides are represented by lowercase letters.

[0135] Table 2: Target sites on human and mouse HTT

[0136] The ZFP-TF described herein may also include the ability to recognize one or more mutations outside the helical region (e.g., to the main chain region), including the mutations described in U.S. Patent Publication No. 2018 / 0087072.

[0137] Any suitable promoter can be used to drive ZFP-TF expression. In a preferred embodiment, the phosphoglycerate kinase 1 (PGK) promoter is used. In other preferred embodiments, the ubiquitin C (UBC) promoter is used. For example, the PGK and UBC promoters provide the optimal expression profile for ZFP-TF, preventing toxicity, adverse immune responses, or silencing due to overexpression. Other available promoters for generalized expression include cytomegalovirus (CMV), Rausch's sarcoma virus (RSV), CAG promoter, chicken β-actin (CBh), human β-actin, mammalian elongation factor 1α (EF1α), EFS, simian virus 40 (SV40), ferritin heavy or light chain, HSP90AB1, etc. For expression in the brain or other CNS cells, suitable promoters may include: synaptophysin I for all neurons, CaMKIIα for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. For endothelial cells, suitable promoters may include ICAM. For hematopoietic cells, suitable promoters may include IFNβ or CD45. For muscle tissue, suitable promoters may include human skeletal muscle α-actin, muscle creatine kinase (…). MCK / CKM,Creatine kinase (M type), CK6, MHCK7, desmin promoter, MLC, myosin heavy chain gene (αMHC) promoter, myosin light chain promoter (MLC2v), cardiac troponin T promoter (cTnT), etc.

[0138] In some implementations, the promoter is side-mounted with an AAV ITR. This advantageously eliminates the need for additional promoter elements, thereby saving space in the vector. The freed-up space can be used to drive the expression of other elements, such as guide nucleic acids or selective markers. ITR activity is relatively weak; therefore, it can be used to mitigate potential toxicity from overexpression.

[0139] Fusion Molecules The DNA-binding domain can be fused with any other molecule (e.g., a peptide) for use in the methods described herein. In some embodiments, these methods employ a fusion molecule comprising at least one DNA-binding molecule (e.g., ZFP) and a heterologous regulatory (functional) domain (or a functional fragment thereof).

[0140] In some implementations, the functional domains include transcriptional regulatory domains. Commonly used domains include, for example, transcription factor domains (activators, repressors, co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members, etc.); DNA repair enzymes and their associated and modifying factors; DNA rearrangement enzymes and their associated and modifying factors; chromatin-associated proteins and their modifying factors (e.g., kinases, acetyltransferases, and deacetylases); and DNA modifying enzymes (e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated and modifying factors. See, for example, U.S. Patent No. 2013 / 0253040, which is incorporated herein by reference in its entirety.

[0141] Suitable activation domains include the HSV VP16 activation domain (see, for example, Hagmann et al. (1997)). J. Virol 71:5952-5962) Nuclear hormone receptors (see, for example, Torchia et al. (1998) Curr. Opin. Cell. Biol 10:373-383); p65 subunit of nuclear factor κB (Bitko and Barik (1998) J. Virol 72:5610-5618; and Doyle and Hunt (1997). Neuroreport 8:2937-2942; Liu et al. (1998) Cancer Gene Ther. 5:3-28), or artificially fused functional domains, such as VP64 (Beerli et al. (1998)). Proc. Natl. Acad. Sci. USA 95:14623-33) and degron (Molinari et al. (1999) EMBO J (18:6439-6447). Other exemplary activation domains include Oct 1, Oct-2A, Sp1, AP-2, and CTF1 (Seipel et al. (1992)). EMBO J. 11:4961-4968), and p300, CBP, PCAF, SRC1 PvALF, AtHD2A, and ERF-2. See, for example, Robyr et al. (2000). Mol. Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol. Endocrinol 23:255-275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska(1999) Acta Biochim. Pol 46:77-89; McKenna et al. (1999) J. Steroid Biochem. Mol. Biol 69:3-12; Malik et al. (2000) Trends Biochem. Sci. 25:277-283; and Lemon et al. (1999) Curr. Opin. Genet. Dev 9:499-504. Other exemplary activating domains include, but are not limited to, OsGAI, HALF-1, C1, AP1, ARF-5, ARF-6, ARF-7 and ARF-8, CPRF1, CPRF4, MYC-RP / GP, and TRAB1. See, for example, Ogawa et al. (2000). Gene 245:21-29; Okanami et al. (1996) Genes Cells 1:87-99; Goff et al. (1991) Genes Dev 5:298-309; Cho et al. (1999) Plant Mol. Biol. 40:419-429; Ulmason et al. (1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al. (2000) Plant J 22:1-8; Gong et al. (1999) Plant Mol. Biol. 41:33-44; and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.

[0142] Exemplary repressor domains include, but are not limited to, KRAB A / B, KOX, TGF-β-induced early gene (TIEG), v-erbA, SID, MBD2, MBD3, DNMT family members (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2. See, for example, Bird et al. (1999). Cell 99:451-454; Tyler et al. (1999) Cell 99:443-446; Knoepfler et al. (1999) Cell 99:447-450; and Robertson et al. (2000) Nature Genet 25:338-342. Other exemplary blocking structures include, but are not limited to, ROM2 and AtHD2A. See, for example, Chem et al. (1996). Plant Cell 8:305-321; and Wu et al. (2000) Plant J. 22:19-27.

[0143] Fusion molecules are constructed using cloning and biochemical conjugation methods well known to those skilled in the art. Fusion molecules contain DNA-binding domains and functional domains (e.g., transcriptional activation or repression domains). Fusion molecules also optionally contain nuclear localization signals (e.g., signals from the T antigen in SV40) and epitope tags (e.g., FLAG and hemagglutinin). The fusion proteins (and the nucleic acids encoding them) are engineered to preserve the translational reading frames between the components of the fusion.

[0144] Fusions between a polypeptide component (or a functional fragment thereof) with a non-protein DNA-binding domain (e.g., antibiotics, intercalators, minor groove conjugates, nucleic acids) on the other hand are constructed using biochemical conjugation methods known to those skilled in the art. See, for example, the catalogue of ierce Chemical Company (Rockford, IL). Methods and compositions for fusing minor groove conjugates with polypeptides have been described. Mapp et al. (2000) Proc. Natl. Acad. Sci. USA 97:3930-3935.

[0145] Fusion molecules can be formulated with pharmaceutically acceptable carriers, as is known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 17th edition, 1985; and co-owned international patent publication number WO 00 / 42219.

[0146] The functional components / domains of a fusion molecule can be selected from a variety of different components that can influence gene transcription after the fusion molecule binds to the target sequence through its DNA-binding domain. Therefore, functional components can include, but are not limited to, various transcription factor domains, such as activators, repressors, co-activators, co-repressors, and silencers.

[0147] In some embodiments, the fusion molecule comprises one or more ZFP-TFs (repressors) wherein the ZFPs are operatively linked to a transcriptional repression domain. Non-limiting examples of repression domains include KOX (KRAB) domains, etc. Other elements, such as NLS, may also be included, and any linker may be used between zinc finger domains and / or between the ZFP and the repression domain (and / or any other element). The polynucleotide encoding these ZFP-TF repressors may also include other additional elements, such as promoters, enhancers, insulators, etc., driving ZFP-TF expression.

[0148] Table 3 shows exemplary ZFP-TF polynucleotide sequences containing the ZFP described herein (identified by name in the first column).

[0149] Table 3: Nucleotide sequence of ZFP-TF

[0150] The polynucleotide encoding the repressor described herein can be delivered using any suitable expression vector, including but not limited to viral vectors (e.g., AAV, Ad, HSV1, etc.) and non-viral vectors (e.g., mRNA, plasmids, small loops, etc.). Non-viral delivery mechanisms include lipid nanoparticles, EVs, liposomes, etc. Expression vectors may include other elements, such as nuclear localization signals (NLS) and / or promoters driving repressor expression (e.g., constitutive promoters such as PGK, UBC, EFS, or EF1α promoters). One or more polynucleotides (e.g., expression vectors) in the same or different forms (e.g., viral vectors and / or non-viral vectors) can be delivered to a subject, and said polynucleotides can be formulated into one or more pharmaceutical compositions. The polynucleotides described herein can be maintained in a free (extrachromosomal) form and / or can be stably integrated into cells after delivery.

[0151] In some implementations, the fusion protein comprises a DNA-binding domain and a nuclease domain, thereby creating functional entities that can recognize their predetermined nucleic acid targets through their engineered (ZFP) DNA-binding domains and generate nucleases (e.g., zinc finger nucleases) that cause DNA to be cleaved near the DNA-binding site through nuclease activity.

[0152] The expression of fusion proteins may be controlled by constitutive or inducible promoters. In some embodiments, the promoter self-regulates the expression of the fusion protein, for example by incorporating a high-affinity binding site. See, for example, U.S. Patent No. 9,624,498.

[0153] deliver The proteins and / or polynucleotides (e.g., HTT repressors) described herein, and compositions comprising proteins and / or polynucleotides, can be delivered to target cells by any suitable means, such as by injection of the protein, by mRNA, and / or using expression constructs (e.g., plasmids, lentiviral vectors, AAV vectors, Ad vectors, exosomes, extracellular vesicles) (Herrmann, 2021, Nature Nanotechnology, 16, pp. 748–759 (2021)). In some embodiments, the repressor is delivered using AAV1, AAV2, AAV5, AAV7, AAV9, or AAVrh10. In some embodiments, the repressor is delivered using AAV1. In some embodiments, the repressor is delivered using AAV2. In some embodiments, the repressor is delivered using AAV5. In some embodiments, the repressor is delivered using AAV7. In some embodiments, the repressor is delivered using AAV9. In some embodiments, the repressor is delivered using AAVrh10. In some implementations, the repressor is delivered using an AAV that crosses the blood-brain barrier, i.e., a BBB-penetrating AAV. Exemplary BBB-penetrating AAV vectors include, but are not limited to, VCAP-101, VCAP-102, 9P801, VCAP-100, VCAP-103, PAL1A, PAL1B, PAL1C, PAL2, CereAAV, Dyno bCAP1, AAV.CAP-B10, AAV.CAP-B20, AAV2-BR1N, AAV2-BR1, and STAC-BBB. ®Or AAV-TT or AAV-BI-hTFR1, etc. (e.g., PCT publications WO2022221400A2, WO2023091948A1 and WO2020014471A1, which are incorporated herein by reference in their entirety; Stanton et al., Cell Press Med 4, 31-50; Goertsen et al., Nat. Neuroscience, 2022, 25(1):106-115).

[0154] The methods for delivering proteins containing zinc finger proteins described herein are described in, for example, U.S. Patent Nos. 6,453,242, 6,503,717, 6,534,261, 6,599,692, 6,607,882, 6,689,558, 6,824,978, 6,933,113, 6,979,539, 7,013,219, and 7,163,824, all of which are disclosed herein by reference in their entirety.

[0155] Any vector system may be used, including but not limited to plasmid vectors, retroviral vectors, poxvirus vectors, herpesvirus vectors, adeno-associated virus vectors, virus-like particles (VLPs), etc. In some embodiments, an adeno-associated virus vector is used. In some embodiments, virus-like particles (VLPs) are used. In some embodiments, a lentiviral vector or an adenoviral vector is used. See also U.S. Patent Nos. 8,586,526, 6,534,261, 6,607,882, 6,824,978, 6,933,113, 6,979,539, 7,013,219, and 7,163,824, which are incorporated herein by reference in their entirety. Furthermore, it is apparent that any of these vectors may contain one or more DNA-binding protein-coding sequences. Therefore, when one or more HTT repressors are introduced into a cell, sequences encoding protein components and / or polynucleotide components may be carried on the same vector or different vectors. When multiple vectors are used, each vector may contain sequences encoding one or more HTT repressors or components thereof.

[0156] Conventional viral and nonviral gene transfer methods can be used to introduce nucleic acids encoding engineered HTT repressors into cells (e.g., mammalian cells) and target tissues. These methods can also be used to administer nucleic acids encoding said repressors (or components thereof) to cells in vitro. In some embodiments, the administration of nucleic acids encoding the repressor is for in vivo or in vitro gene therapy purposes. Nonviral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with a delivery medium (such as liposomes). Viral vector delivery systems include DNA and RNA viruses that have episomes or integrated genomes after delivery to cells. For a review of gene therapy procedures, see Anderson (1992). Science 256:808-813; Nabel and Felgner (1993) TIBTECH 11:211-217; Mitani&Caskey (1993) TIBTECH 11:162-166; Dillon (1993) TIBTECH 11:167-175; Miller (1992) Nature 357:455-460; Van Brunt (1988) Biotechnology 6(10):1149-1154; Vigne (1995) Restorative Neurology and Neuroscience 8:35-36; Kremer and Perricaudet (1995) British Medical Bulletin 51(1):31-44; Haddadada et al., Current Topics in Microbiology and Immunology Doerfler and Böhm (eds.) (1995); and Yu et al. (1994). Gene Therapy 1:13-26.

[0157] Non-viral delivery methods for nucleic acids include electroporation, liposome transfection, microinjection, bioballistics, virions, liposomes, immunoliposomes, polycationic or lipid:nucleic acid conjugates, naked DNA, naked RNA, artificial viral particles, and reagent-enhanced DNA uptake. Sonoporosis using, for example, the Sonitron 2000 system (Rich-Mar) can also be used for nucleic acid delivery. In a preferred embodiment, one or more nucleic acids are delivered in the form of mRNA. Using capped mRNA to increase translation efficiency and / or mRNA stability is also preferred. Particularly preferred are ARCA (anti-reverse cap analogue) caps or variants thereof. See U.S. Patent Nos. 7,074,596 and 8,153,773, which are incorporated herein by reference.

[0158] Other exemplary nucleic acid delivery systems include those offered by Amaxa Biosystems (Cologne, Germany), Maxcyte (Rockville, Maryland), BTX Molecular Delivery Systems (Hollston, Massachusetts), and Copernicus Therapeutics (see, for example, U.S. Patent No. 6,008,336). Liposome transfection technology is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and liposome transfection reagents are commercially available (e.g., Transfectam™, Lipofectin™, and Lipofectamine™ RNAiMAX). Cationic and neutral lipids suitable for highly efficient receptor recognition of polynucleotides in liposome transfection include those in Felgner's International Patent Publications WO 91 / 17424 and WO 91 / 16024. Delivery can be to cells (ex vivo administration) or to target tissues (in vivo administration).

[0159] The preparation of lipid:nucleic acid complexes, including targeted liposomes (e.g., immunolipid complexes), is well known to those skilled in the art (see, for example, Crystal (1995)). Science 270:404-410; Blaese et al. Human (1995) Cancer Gene Ther. 2:291-297; Behr et al. (1994) Bioconjugate Chem 5:382-389; Remy et al. (1994) Bioconjugate Chem. 5:647-654; Gao et al. (1995) Gene Therapy 2:710-722; Ahmad et al. (1992) Cancer Res. 52:4817-4820; U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0160] Other delivery methods involve packaging the nucleic acid to be delivered into an EnGeneIC delivery vehicle (EDV). These EDVs are specifically delivered to the target tissue using bispecific antibodies, where one arm of the antibody is specific to the target tissue and the other arm is specific to the EDV. The antibody carries the EDV to the surface of the target cells, where the EDV then enters the cell via endocytosis. Once inside the cell, the contents are released (see MacDiarmid et al. (2009)). Nature Biotechnology 27(7):643).

[0161] The delivery of nucleic acids encoding engineered ZFPs using RNA or DNA virus-based systems leverages highly evolved processes for targeting viruses to specific cells in vivo and transporting viral payloads to the cell nucleus. Viral vectors can be administered directly to patients (in vivo) or used in vitro to treat cells and deliver modified cells to patients (ex vivo). Conventional virus-based systems for ZFP delivery include, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vaccinia virus and herpes simplex virus vectors, and virus-like particles (VLPs) for gene transfer. High transduction efficiency has been observed in many different cell types and target tissues. Vectors containing therapeutic ZFP nucleic acids (e.g., retroviruses, adenoviruses, liposomes, VLPs, etc.) can be administered directly to organisms to transduce cells in vivo. Alternatively, naked DNA can be administered. Administration is any route commonly used to introduce molecules into blood or tissue cells and ultimately bring them into contact with blood or tissue cells, including but not limited to injection, infusion, local application, and electroporation. Suitable methods for applying these nucleic acids are available and well known to those skilled in the art, and while a particular composition can be applied via various routes, a particular route can generally provide a more direct and efficient response than others.

[0162] Adenovirus-based vectors do not require cell division. High titers and high expression levels have been achieved using these vectors. These vectors can be produced in large quantities in relatively simple systems. Adeno-associated virus (“AAV”) vectors have also been used to transduce target nucleic acids into cells, for example, for the in vitro production of nucleic acids and peptides, and for in vivo and in vitro gene therapy procedures (see, for example, West et al. (1987)). Virology 160:38-47; US Patent No. 4,797,368; International Patent Publication No. WO 93 / 24641; Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invest. The construction of recombinant AAV vectors has been described in several publications, including U.S. Patent No. 5,173,414; Tratschin et al. (1985). Mol. Cell. Biol. 5:3251-3260; Tratschin et al. (1984) Mol. Cell. Biol. 4:2072-2081; Hermonat and Muzyczka (1984) PNAS 81:6466-6470; and Samulski et al. (1989) J. Virol. 63:03822-3828.

[0163] Recombinant adeno-associated virus vectors (rAAVs) are a promising alternative gene delivery system. Many AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, AAV10, AAV11, AAV12, AAV13, and AAVrh10, as well as pseudotyped AAVs such as AAV2 / 8, AAV2 / 5, and AAV2 / 6, can also be used according to the present invention. Furthermore, shuttle AAVs or synthetic AAVs with preferred tropism, such as those capable of crossing the blood-brain barrier (BBB) ​​and liver detargeting, can also be used. In some embodiments, a BBB-penetrating AAV capsid is used. In some embodiments, AAV9 is used. In some embodiments, AAV5 is used. In some implementations, the BBB-penetrating AAV is any of the following: VCAP-101, VCAP-102, 9P801, VCAP-100, VCAP-103, PAL1A, PAL1B, PAL1C, PAL2, CereAAV, Dyno bCAP1, AAV.CAP-B10, AAV.CAP-B20, AAV2-BR1N, AAV2-BR1, STAC-BBB ® Or AAV-TT or AAV-BI-hTFR1, etc.

[0164] Packaging cells are used to form viral particles capable of infecting host cells. These cells include 293 cells for packaging adenoviruses and ψ2 or PA317 cells for packaging retroviruses. Viral vectors used in gene therapy are typically produced by production cell lines that package nucleic acid vectors into viral particles. Vectors typically contain the minimum viral sequence required for packaging and subsequent integration into the host; other viral sequences are replaced by expression cassettes encoding the proteins to be expressed. Missing viral functions are provided trans-form by the packaging cell lines. For example, AAV vectors used in gene therapy typically contain only inverted terminal repeat (ITR) sequences from the AAV genome, which are required for packaging and integration into the host genome in the presence of AAV replication proteins. Viral genes are supplemented in trans-form in cell lines containing helper plasmids encoding other AAV genes (i.e., rep and cap) but lacking the ITR sequences. The cell lines are also infected with adenoviruses as helpers. The helper virus promotes AAV genome replication and AAV gene expression in the helper plasmid. Due to the lack of ITR sequences, the helper plasmids are not packaged in large quantities. Adenovirus contamination can be reduced, for example, by heat treatment, as adenoviruses are more sensitive to heat treatment than AAVs. In some implementations, AAVs are produced on a large scale using a baculovirus expression vector system (BEVS). (Sandro et al.) Methods Mol Biol. , 2019;1937:91-99).

[0165] In many gene therapy applications, it is necessary to deliver gene therapy vectors with high tropism to specific tissue types. Therefore, viral vectors can be modified to be tropist for a given cell type by expressing ligands as fusion proteins with viral capsid proteins on the outer surface of the virus. The chosen ligands must have affinity for receptors known to be present on the cell types of interest. For example, filamentous phages can be engineered to display antibody fragments (e.g., FAB or Fv) that have specific binding affinity for virtually any selected cell receptor. While the above description primarily applies to viral vectors, the same principles apply to non-viral vectors. These vectors can be engineered to contain specific uptake sequences that facilitate uptake by specific target cells.

[0166] Gene therapy vectors can be delivered via administration to individual patients, typically systemically. In some embodiments, administration is intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion, intranasal administration (including direct injection into the brain), or local application, as described below.

[0167] In some embodiments, the compositions described herein (e.g., polynucleotides and / or proteins) are delivered directly in vivo. These compositions (cells, polynucleotides, and / or proteins) can be administered directly to the central nervous system (CNS), including but not limited to direct injection into the brain or spinal cord. They can be targeted to one or more regions of the brain, including but not limited to the striatum and / or caudate nucleus, putamen, thalamus, or globus pallidus. In some embodiments, the compositions are delivered to skeletal muscle. As an alternative to or supplement to CNS delivery, the compositions can also be administered systemically. In some embodiments, administration is intravenous. In some embodiments, administration is intraperitoneal. In some embodiments, administration is intracardiac. In some embodiments, administration is intramuscular. In some embodiments, administration is intranasal. In some embodiments, administration is intrathecal. In some embodiments, administration is subcutaneous. In some embodiments, administration is via intracranial infusion. Alternatively, AAV can also be delivered via focused ultrasound. Methods and compositions for delivering the compositions described herein directly to a subject (including direct delivery to the CNS) include, but are not limited to, direct injection via needle assembly (e.g., stereotactic injection). In some embodiments, for example, convection-enhanced delivery is used, creating a pressure gradient at the tip of the infusion catheter to deliver the therapeutic agent directly to the interstitial space of the central nervous system. In some embodiments, a neuronavigation system, such as a robotic navigation system, stereotactic frame, frameless navigation system, ClearPoint system, or other stereotactic neuronavigation system, is employed in conjunction with a suitable imaging system such as MRI or CT to achieve precise delivery. Delivery methods are described, for example, in U.S. Patent Nos. 7,837,668; 8,092,429, relating to the delivery of compositions (including expression vectors) into the brain; and U.S. Patent Publication No. 2006 / 0239966, both of which are incorporated herein by reference in their entirety.

[0168] The intended effective dose may vary from patient to patient and depends on the administration method and site. Therefore, the effective dose can be determined by those skilled in the art. After sufficient time (e.g., typically 4-15 days) for the repressor to express, the level of the therapeutic peptide in serum or other tissues is analyzed and compared to the initial level before administration to determine whether the administered dose is too low, within the appropriate range, or too high. In some embodiments, when using a viral vector, such as AAV, the administered dose is 1 × 10⁻⁶. 8 With 5 × 10 15 Between vg / ml (or any value between), even more preferably between 1 × 10 13 With 1× 10 14 Between vg / ml (or any value between), even more preferably between 1 × 10 12 With 1 × 1013 Between vg / ml (or any value between).

[0169] To enable the direct delivery of ZFP to the human brain using a recombinant adeno-associated virus (rAAV) vector, a dose of 1 × 10⁻⁶ ppm can be used per striatum. 8 -5 × 10 15 vg / mL (or any value between these values, including, for example, 1 × 10⁻⁶) 12 With 1 × 10 14 Any value between vg / mL or 1 × 10 12 With 1 x 10 13 The dose range for the vector genome is any value between vg / mL. The dose range is 1 × 10⁻⁶. 8 Up to 5 × 10 15 vg / kg or vg / striatum (or any value between them), even more preferably at 1 × 10 8 With 1 × 10 14 Vg / kg or Vg / striatum (or any value between), even more preferably 1 × 10 8 With 1 × 10 13 Vg / kg or Vg / striatum (or any value between these values). As described, the dosage may vary for other brain structures and different delivery protocols. Methods for directly delivering the rAAV vector to the brain are known in the art. See, for example, U.S. Patent Nos. 9,089,667, 9,050,299, 8,337,458, 8,309,355, 7,182,944, 6,953,575, and 6,309,634.

[0170] In some implementations, the vector can be delivered to ex vivo cells, such as cells explanted from an individual patient (e.g., hematopoietic stem cells, lymphocytes, bone marrow aspirate, tissue biopsy samples) or universal donor hematopoietic stem cells, and then these cells can be re-implanted into the patient, typically after selecting cells that have been incorporated into the vector.

[0171] Ex vivo cell transfection for diagnostic, research, or gene therapy purposes (e.g., by re-infusing transfected cells into a host organism) is well known to those skilled in the art. In some embodiments, cells are isolated from a target organism, transfected with at least one HTT repressor or a component thereof, and re-infused back into the target organism (e.g., a patient).

[0172] In some implementations, virus-like particles (VLPs) are used to deliver nucleic acids. A virus-like particle (VLP) is a protein complex that resembles a natural viral particle but does not contain the viral genome and therefore cannot replicate. Nevertheless, VLPs can mimic viral antigenicity but are not pathogenic. VLPs consist of one or more natural or artificial protein units, which have multiple copies and can exist naturally (e.g., the empty capsid of extracellular poliovirus) or be produced by recombinant expression of proteins required to generate VLPs (e.g., vesicular virus glycoproteins). Some VLPs are self-assembled from a single type of capsid protein (e.g., L1 of human papillomavirus); some VLPs require several structural proteins (e.g., bluetonguevirus VLP); and some VLPs require a combination of structural and non-structural proteins (e.g., poliovirus VLP). The repeating surface structure and size of 20–200 nanometers make VLPs highly immunogenic, enabling them to efficiently present foreign antigens, such as RNA that can be loaded onto their surface, and to induce strong humoral and cellular immune responses.

[0173] In some embodiments, a non-viral delivery method is employed. In some embodiments, the non-viral nucleic acid carrier is a nanoparticle, which may be inorganic or organic. Nanoparticles are well known in the art. Any suitable nanoparticle design can be used to deliver the gene therapy constructs or encoded ZFP-TF proteins of this disclosure. For example, in some embodiments of this disclosure, organic (e.g., lipids and / or polymers) nanoparticles are suitable as delivery media. Exemplary lipids for nanoparticle formulations are shown in Table 4 (below).

[0174] Table 4 lists exemplary lipids in nanoparticle formulations. Table 5 lists exemplary polymers used in nanoparticle formulations.

[0175] Table 6 summarizes the delivery methods for the polynucleotides encoding ZFP-TF described in this paper.

[0176] In other preferred embodiments, one or more nucleic acids of the HTT repressor are delivered in the form of mRNA. Using capped mRNA to increase translation efficiency and / or mRNA stability is also preferred. Particularly preferred is the use of ARCA (anti-reverse cap analogue) caps or variants thereof. For a discussion of how to isolate and culture cells from patients, see U.S. Patent Nos. 7,074,596 and 8,153,773, which are incorporated herein by reference in their entirety. Various cell types suitable for in vitro transfection are well known to those skilled in the art (see, for example, Freshney et al.). , Culture of Animal Cells, A Manual of Basic Technique (3rd edition, 1994) and the references cited therein.

[0177] In one implementation, stem cells are used in ex vivo cell transfection and gene therapy procedures. The advantage of using stem cells is that they can differentiate into other cell types in vitro, or they can be introduced into mammals (e.g., cell donors) where they will be implanted into the bone marrow. Methods for differentiating CD34+ cells into clinically significant immune cell types in vitro by administering cytokines such as GM-CSF, IFN-γ, and TNF-α are known (see Inaba et al. (1992)). J. Exp. Med. 176:1693-1702).

[0178] Stem cells are isolated using known methods for transduction and differentiation. For example, stem cells are isolated from bone marrow cells by panning bone marrow cells with antibodies that bind to unwanted cells such as CD4+ and CD8+ (T cells), CD45+ (pan-B cells), GR-1 (granulocytes), and Iad (differentiated antigen-presenting cells) (see Inaba et al. (1992)). J. Exp. Med . 176:1693-1702).

[0179] In some embodiments, modified stem cells may also be used. For example, apoptosis-resistant neuronal stem cells may be used as therapeutic compositions, wherein the stem cells also contain the ZFP TF of the present invention. Resistance to apoptosis can be achieved, for example, by knocking out BAX and / or BAK using BAX or BAK-specific ZFNs in stem cells (see U.S. Patent No. 8,597,912) or by using cells with disrupted apoptotic proteases, such as by re-inducing apoptosis protease-6-specific ZFNs. These cells can be transfected with ZFP TFs known to regulate mutant or wild-type HTT.

[0180] Methods for introducing DNA into hematopoietic stem cells are disclosed, for example, in U.S. Patent No. 5,928,638. These methods can be used to introduce transgenes into hematopoietic stem cells (e.g., CD34).+ The vectors for cells include adenovirus type 35.

[0181] Vectors suitable for introducing transgenes into immune cells (e.g., T cells) include non-integrating lentiviral vectors. See, for example, Naldini et al. (1996). Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al. (1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol. 72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222.

[0182] The pharmaceutically acceptable carrier portion is determined by the specific composition being administered and the specific method of administration. Therefore, a variety of suitable pharmaceutical composition formulations are available, as described below (see, for example...). Remington’s Pharmaceutical Sciences (17th edition, 1989).

[0183] As described above, the disclosed methods and compositions can be used with any type of cell, including but not limited to prokaryotic cells, fungal cells, archaea cells, plant cells, insect cells, animal cells, vertebrate cells, mammalian cells, and human cells. Cell lines suitable for protein expression are known to those skilled in the art, including but not limited to COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2 / O-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6, and insect cells (e.g., fall armyworm cells). Spodoptera fugiperda ,Sf)) and fungal cells (e.g. yeast) Saccharomyces Pichia pastoris () Pischia ) and fission yeast ( Schizosaccharomyces Progeny, variants, and derivatives of these cell lines can also be used. In a preferred embodiment, the method and composition are delivered directly to brain cells, such as the striatum.

[0184] application The HTT-binding molecules (e.g., ZFP) and the codon-optimized gene therapy constructs encoding them described in this article can be used in a variety of applications. These applications include therapeutic methods in which... HTTA binding molecule (including a nucleic acid encoding a DNA-binding protein) is applied to the subject (e.g., an AAV, such as AAV5, AAV9, or BBB-penetrating AAV) and used to regulate the expression of target genes (and therefore proteins) in the subject's body. In some embodiments, regulation is in the form of repression, such as repression of mHTT, which leads to the Huntington's disease state. In some embodiments, regulation is in the form of activation when activating or increasing the expression of endogenous cellular genes can improve the disease state. In other embodiments, regulation can be cleavage (e.g., using one or more nucleases), such as making a mutant... HTT Gene inactivation. As mentioned above, for these applications, HTT The molecules, or more commonly, the nucleic acids that encode them, are combined with pharmaceutically acceptable carriers to form drug compositions.

[0185] Individual HTT binding molecules or carriers encoding them, or combinations with other suitable components (e.g., liposomes, nanoparticles, or other components known in the art), can be formulated into aerosol formulations (i.e., they can be “nebulized”) for administration by inhalation. Aerosol formulations can be placed in pressurizable propellants such as dichlorodifluoromethane, propane, nitrogen, etc. Formulations suitable for parenteral administration, such as intravenous, intramuscular, intradermal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injectable solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Compositions can be administered, for example, by intravenous infusion, oral administration, topical administration, intraperitoneal administration, intravesical administration, intracranial administration, intranasal administration, or intrathecal administration. In some embodiments, the composition is administered by intravenous infusion. In some embodiments, the composition is administered orally. In some embodiments, the composition is administered topically. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intravesically. In some embodiments, the composition is administered intracranially. In some embodiments, the composition is administered intranasally. In some embodiments, the composition is administered intrathecally. Formulations of the compound may be provided in single-dose or multi-dose sealed containers (such as ampoules and vials). Injectable solutions and suspensions may be prepared from sterile powders, granules, and tablets of the types previously described.

[0186] The dose administered to the patient should be sufficient to achieve a beneficial therapeutic response in the patient over time. The dose is determined by the efficacy of the specific HTT-binding molecule used and K... d The dosage will be determined by the condition of the target cells and the patient, as well as the patient's weight or body surface area. The dosage will also be determined by the presence, nature, and extent of any adverse side effects that occur in a particular subject in connection with the administration of the specific compound or carrier.

[0187] Beneficial treatment responses can be measured in a variety of ways. For example, improvements can be measured in Huntington's disease-related motor disorders such as involuntary jerks or twisting movements; muscle problems such as stiffness or muscle contractures (dystonia); bradykinesia or abnormal eye movements; gait, posture, and balance abnormalities; physical difficulties in speech or swallowing; and improvements in voluntary motor disorders. Signs of treatment-related improvement in other impairments, such as cognitive impairment and psychiatric disorders, can also be monitored. The UHDRS scale can be used to quantify the clinical characteristics of the disease. Other biomarker measurements can also be used to determine outcomes, including measuring mHTT in the CSF.

[0188] Treatment is particularly important for patients who are asymptomatic, as it offers the opportunity to treat the disease before widespread neurodegeneration occurs in HD. This damage begins before the onset of the aforementioned obvious symptoms. HD pathology primarily involves the toxic effects of mutant HTT on medium-sized polyspinous neurons in the striatum. These medium-sized polyspinous neurons highly express phosphodiesterase 10A (PDE10A), an enzyme that regulates the cAMP and cGMP signaling cascades involved in gene transcription factors, neurotransmitter receptors, and voltage-gated channels (Niccolini et al. (2015)). Brain 138:3016-3029), and it has been demonstrated that PDE10A expression is reduced in HD mice, and the same has been found in human autopsy studies. Furthermore, positron emission tomography (PET) ligands have been developed that are ligands for the PDE10A enzyme (e.g., ligands for positron emission tomography (PET)). 11 C-IMA107, see, for example, Niccolini et al., above; 18 FMNI-659, see, for example, Russell et al. (2014) JAMA Neurol 71(12):1520-1528), and these molecules have been used to evaluate HD patients who have not yet developed symptoms. Studies have shown that PDE10A levels in HD patients change even before symptoms appear. Therefore, the therapeutic efficacy of the compositions of the present invention can be measured by assessing PDE10A levels by PET before, during, and after treatment. “Therapeutic efficacy” may mean improvements in clinical and molecular measurements, or it may mean protection of patients from any further decline in the function of intermediate polyspinous neurons or increased loss of polyspinous neurons, or from the further development of significant clinical manifestations associated with HD.

[0189] The following examples relate to exemplary embodiments of this disclosure, wherein the HTT regulator comprises a zinc finger protein. It should be understood that this is merely illustrative and other methods may also be used. HTTRegulators (e.g., repressors), including but not limited to TALE-TF, CRISPR / Cas systems, other ZFP, ZFN, TALEN, other CRISPR / Cas systems (e.g., Cfp systems), and homing endonucleases (meganucleases) with engineered DNA-binding domains.

[0190] Example Example 1. Materials and Methods Naming of Constructs As shown in Figure 1, the amino acid sequences of ZFP46025 (SEQ ID NO: 37) and ZFP45723 (SEQ ID NO: 38) include the SV40 nuclear localization signal, an array of 4 or 5 zinc finger domains, and a KRAB transcriptional repression domain derived from the human ZNF10 / KOX1 protein. The parental nucleotide sequence is referred to as "ZFP46025_P" or "ZFP45723_P", while the codon-optimized sequence is named by replacing "P" with "co" followed by a number (e.g., ZFP46025_co1, ZFP45723_co3).

[0191] HEK293 cell culture, transfection, and Western blot analysis Approximately 70,000 HEK293 cells (ATCC, catalog number 50-188-446FP) were seeded into each well of a 24-well tissue culture plate and transfected with 0.5 μg of plasmid using Lipofectamine™ 3000 transfection reagent (ThermoFisher, catalog number L3000001) according to the manufacturer's protocol after 48 hours. Approximately 48 hours post-transfection, cells were washed twice with PBS and lysed with RIPA buffer. Total protein content in the supernatant was measured using the Pierce™ Rapid Gold BCA Protein Assay Kit (ThermoFisher, catalog number A53226). Western blot analysis was performed using primary antibodies against the KRAB domain (ThermoFisher, catalog number PA5-110594) and GAPDH (Cell Signaling Technologies, catalog number 97166) at a concentration of 10 μg of total protein. Imaging and analysis of protein blot membranes were performed using the LI-COR Odyssey® system.

[0192] Generation and characterization of rAAV vectors HEK293 cells were triple-transfected with plasmids containing: (1) the AAV2 Rep gene and the AAV9 or AAV.PHP.eB Cap gene; (2) helper plasmids containing the adenovirus genes E2A, E4, and VA; and (3) transgenic plasmids containing AAV2 ITRs with side-linked transgenic expression cassettes. AAV particles were purified using a POROSCaptureSelect AAVX column followed by cesium chloride precipitation, or by two rounds of cesium chloride precipitation. The fractions containing the main intact capsids were collected and subjected to buffer exchange in phosphate-buffered saline containing 0.001% Pluronic F-68. Genomic titer was determined by ddPCR, purity was assessed by SDS-PAGE followed by silver staining or CE-SDS to observe VP1, VP2, and VP3, and endotoxin was assessed by lumulus amebocyte lysate assay (Endosafe). The characterized vectors were aliquoted and stored at -80°C.

[0193] HD Human iPSC-derived Cortical Culture, Transduction, and PCR Analysis Human induced pluripotent stem cells (iPSCs) from HD patients (NINDS Human Cell and Data Repository, #ND36999) with heterozygous polyglutamine (180 repeats) in the Htt gene were differentiated into cortical neuronal progenitor cells (NPCs) using the method reported by Shi et al. (Nat Protoc. 2012 Oct; 7(10):1836-4). Before plate seeding, poly-D-lysine (PDL) 96-well plates (Corning, #356640) were coated with 10 μg / mL laminin (R&Dsystems, #3400-010-02) for more than 3 hours. NPCs were seeded at 20,000 cells per well onto the laminin-PDL-coated 96-well plates. AAVs were transduced at MOIs of 1E6, 5E6, and 1E7 7 days post-seeding and cultured for another 7 days. Total RNA was extracted from each sample using the RNeasy Micro Kit (Qiagen, #74004), followed by cDNA synthesis using the SuperScriptVILO cDNA Synthesis Kit (Invitrogen, #11754250). RT-qPCR was performed using the SsoAdvanced™ UniversalSYBR® Green Supermix (Bio-Rad, #172-5271) and the QuantStudio 12K Flex Real-Time PCR Instrument (Thermofisher). PrimePCR™ SYBR® Green assay: GAPDH was detected using human GAPDH (Bio-Rad, #qHsaCED0038674). Primer sequences for wild-type Htt and mutant Htt, as well as thermal cycling parameters for each RT-qPCR reaction, are shown below.

[0194] Table 7. Primer Sequences

[0195] Table 8. PCR Cycles

[0196] Quantitative analysis of ZFP mRNA by RT-qPCR Total RNA was isolated and subsequent cDNA was synthesized using the above procedure. RT-qPCR was performed using a SsoAdvanced™ UniversalSYBR® Green Supermix (Bio-Rad, #172-5271) and a QuantStudio 12K Flex real-time PCR instrument (Thermofisher). The primer sequences for each reaction and the thermal cycling parameters for each RT-qPCR reaction are shown below.

[0197] Table 9. Primer Sequences

[0198] Table 10. PCR conditions

[0199] Quantification of ZFP protein by immunocytochemistry (ICC) HD-derived NPCs were cultured for 7 days and each AAV was transduced as described above. After 7 days of transduction, the NPCs were fixed in 4% paraformaldehyde (Fujifilmwako, #163-20145) at room temperature for 30 minutes, followed by incubation with Triton X-100 (MPBiomedicals, #807423) for 1 hour. At 4°C, each plate was incubated with an antibody against the KRAB domain (ThermoFisher, #PA5-110594) for more than 12 hours. After washing with D-PBS (Fujifilmwako, #045-29795), each plate was incubated with goat anti-rabbit IgG (H+L) AF488 (Invitrogen, #A11034), washed with D-PBS, and stained with Hoechst 33342 (Invitrogen, #H3570) for nuclear staining. All data were acquired using a CellVoyager 8000 imaging system (Yokogawa). The nuclear region was used as the region of interest (ROI) and delineated using nuclear staining-positive areas. ZFP protein levels in the cell nucleus were measured by detecting ZFP immunochemical signals overlapping with the ROI.

[0200] Example 2: Transfection of HEK293 cells with a codon-optimized variant of ZFP46025 To generate codon-optimized sequences, hundreds of sequences were produced using the ATUM tool (US Patent Nos. 7,561,972, 7,561,973, 8,126,653, and 8,401,798). These sequences were then screened using bioinformatics tools to remove potential splicing sites, cryptic promoters, and long repetitive sequences. From the remaining candidate sequences, sequences containing 6 CpGs or fewer (SEQ ID NO: 11-16) were selected.

[0201] The parent ZFP46025_P (SEQ ID NO 10) has 38 CpGs. Two alternative codon-optimized variants with 4 CpGs (ZFP46025_co1 and ZFP46025_co2) (SEQ ID NO: 11 and 12), ZFP46025_co3 with 6 CpGs (SEQ ID NO: 13), ZFP46025_co4 with 5 CpGs (SEQ ID NO: 14), and ZFP46025_co5 and ZFP46025_co6 with 0 CpGs (SEQ ID NO: 15 and 16) were designed. An expression plasmid containing a hybrid chicken β-actin (CBh) promoter, a human growth hormone (hGH) polyadenylation signal (polyA), and a transgene comprising: ZFP46025_P fused with a T2A self-cleaving peptide and eGFP, or one of six codon-optimized variant sequences. Figure 2A Western blot analysis of transfected HEK293 cells showed that, compared with the parental sequence, the codon-optimized variant had a higher average ZFP protein expression level. Figure 2B and Figure 2C ).

[0202] The codon optimization algorithm was applied again to the two selected sequences SEQ ID NO: 15 and SEQ ID NO: 16 to further improve the expression. Further analysis was performed on the four candidate sequences SEQ ID NO: 17-21.

[0203] Construct an expression plasmid containing a hybrid chicken β-actin (CBh) promoter, a human growth hormone (hGH) polyadenylation signal (polyA), and a transgene, wherein the transgene comprises: SEQ ID NO:15 and SEQ ID NO:16 or one of four codon-optimized variant sequences fused with a T2A self-cleaving peptide and eGFP. Figure 2A GFP signal intensity analysis of transiently transfected HEK293 cells showed that, compared to the parental sequence, some codon-optimized variants had higher average ZFP protein expression levels. Figure 2D ).

[0204] The results showed that some, but not all, codon-optimized sequences were expressed at higher levels than the parental variants. For example, plasmids with SEQ ID Nos. 19 and 22 were observed to be expressed at reduced levels in the HEK293 assay system during transfection experiments.

[0205] Construct an expression plasmid containing the hybrid chicken β-actin (CBh) promoter, human growth hormone (hGH) polyadenylation signal (polyA), and codon-optimized variant sequence as a transgene. Figure 3AWestern blot analysis of transfected HEK293 cells showed that all three substitution sequences of ZFP46025_co5 resulted in increased ZFP expression, while only ZFP46025_co6b and ZFP46025_co6c showed higher expression compared to ZFP46025_co6. Figure 3B and Figure 3C ).

[0206] Overall, transfection in HEK293 cells revealed an unexpected increase in the expression of some codon-optimized sequences compared to wild-type.

[0207] Example 3: Transduction of human iPSC-derived cortical neurons using a ZFP46025 codon-optimized variant ZFP46025_co5 and ZFP46025_co6 were inserted into an AAV expression plasmid containing an AAV2 ITR, which is side-linked to the human ubiquitin-c (UBC) promoter, the human growth hormone (hGH) polyadenylation signal, and a 1 kb filler sequence derived from hSCNB (intron 5) to prevent mispackaging. Figure 4A Human iPSC-derived cortical neurons were transduced with MOIs of 1E5, 1E6, 5E6, and 1E7. The mRNA and protein levels of ZFP46025 were determined by RT-qPCR and ICC, respectively. Figure 4B and Figure 4C Compared to the parental AAV of ZFP46025, ZFP46025_co5 and ZFP46025_co6 showed lower mRNA and protein levels. Allele-specific PCR analysis indicated that at 5E6 and 1E7, the mutant Htt allele mRNA was reduced ( Figure 4D ).

[0208] ZFP46025_co5 and ZFP46025_co6 were inserted into an AAV expression plasmid containing an AAV2 ITR, which is side-linked to the human phosphoglycerate kinase 1 (PGK) promoter, the human growth hormone (hGH) polyadenylation signal, and a 1kb filler sequence derived from hSCNB (intron 5) to prevent mispackaging. Figure 5A Human iPSC-derived cortical neurons were transduced with MOIs of 1E6, 5E6, and 1E7. The mRNA and protein levels of ZFP46025 were determined by RT-qPCR and ICC, respectively. Figure 5B and Figure 5CZFP46025_co5 and ZFP46025_co6 showed mRNA and protein expression levels comparable to the parental ZFP46025. AAV based on the PGK promoter had higher mRNA and protein levels compared to AAV based on the UBC promoter. Allele-specific PCR analysis showed that, at all MOIs, the mRNA of the mutant Htt allele was reduced (…). Figure 5D ).

[0209] ZFP46025_co1 (SEQ ID NO: 11), ZFP46025_co3 (SEQ ID NO: 13), ZFP46025_co4 (SEQ ID NO: 14), ZFP46025_co5b (SEQ ID NO: 18), and ZFP46025_co6b (SEQ ID NO: 21) were inserted into an AAV expression plasmid containing an AAV2 ITR, which is side-linked to a human phosphoglycerate kinase 1 (PGK) promoter, a human growth hormone (hGH) polyadenylation signal, and a 1kb filler sequence derived from hSCNB (intron 5) to prevent mispackaging. Figure 6A Human iPSC-derived cortical neurons were transduced with MOIs of 1E6, 5E6, and 1E7. The mRNA and protein levels of ZFP46025 were determined by RT-qPCR and ICC, respectively. Figure 6B and Figure 6C All codon-optimized ZFP46025 variants showed mRNA and protein expression levels comparable to the parental ZFP46025. Allele-specific PCR analysis indicated that, at all MOIs, mutant Htt allele mRNA was reduced (…). Figure 6D ).

[0210] Example 4: Transfection of HEK293 with an optimized variant of the ZFP45723 codon The parental line ZFP45723_P (SEQ ID NO 23) has 34 CpGs. Six alternative codon-optimized variants (SEQ ID NO: 24 to 29) with 0 CpGs were designed. An expression plasmid containing the hybrid chicken β-actin (CBh) promoter, the human growth hormone (hGH) polyadenylation signal (polyA), and one of the sequences of the parent ZFP45723 or one of the six codon-optimized variants was constructed. Figure 7A ).

[0211] The results showed that Western blot analysis after transfection of HEK293 cells indicated that the codon-optimized variant had similar or reduced protein expression levels compared to the parental sequence. Figure 7B and Figure 7CThis indicates that not all codon optimizations improve expression levels, as constructs containing co3, co4, co5, or co6 show a 50% or greater reduction in expression levels, while constructs containing co1 and co2 show an increase or a similar increase.

[0212] Example 5: Transduction of human iPSC-derived cortical neurons using a ZFP45723 codon optimized variant Human iPSC-derived cortical neurons were transduced using MOIs of 1E6, 5E6, and 1E7. Protein levels of ZFP45723 were determined by ICC. Figure 8B All codon-optimized ZFP45723 (SEQ ID NO: 24-29) showed protein expression comparable to the parental ZFP45723 (SEQ ID NO: 23). Allele-specific PCR analysis showed that, under all MOIs, the mutant Htt allele mRNA was reduced (…). Figure 8C ).

[0213] Example 6: ZFP expression in iPSC-derived neurons after AAV transduction iPSC-derived neural progenitor stem (NPC) cells were differentiated for 7 days, followed by AAV transduction, in which ZFP expression was controlled by the UBC promoter. After 7 days, the cells were fixed, and data analysis was performed to determine ZFP expression. Fixed cells were stained with anti-ZNF10 antibody, and ZFP expression was detected using fluorescent labeling. Cell analysis was performed using a high-content imaging system / CV8000. The figure shows the average fluorescence intensity of each well.

[0214] Figure 11 The results showed that the expression levels of codon-optimized sequences SEQ ID NO: 15 (NH035) and SEQ ID NO: 16 (NH035) were lower than those of the non-codon-optimized parent (SEQ ID NO: 10) NH014 AAV.

[0215] Example 7: Evaluation of several promoters in the Q175 HD mouse model In this embodiment, two ZFPs (46025 and 47523) were packaged into AAV9 with four different promoters: CBh, UBC, EFS (a short form of the EF1α promoter) / CHIMin, and PGK / CHIMin, and their in vivo activity was analyzed in a Q175 HD mouse model (Menalled LB et al., 2012, PLoS One 7: e49838). At 11 weeks of age, bilateral stereotactic striatal injections of AAV9 containing ZFPS were performed. Two doses of AAV were administered at each injection site: 1.2e9 vg and 1.2e10 vg, in a volume of 2 μL. The stereotactic coordinates were: anterior-posterior [AP], +0.8 mm; medial-lateral [ML], ±1.8 mm; dorsal-ventral [DV], -3.0 mm. Eight weeks after AAV administration, mice were sacrificed, and their brains were collected for analysis. Vector genomic quantification and RT-PCR were performed using standard methods and the conditions described in the previous embodiments. Analysis of vector genomic (Vg) DNA ( Figure 9A ), transgenic transcripts ( Figure 9B ) and its effect on reducing mHTT RNA ( Figure 9C ).

[0216] All promoters tested reduced mHTT RNA by more than 30% at high doses.

[0217] Example 8: Evaluation of PGK and UBC promoters in the Q175 HD mouse model after IV administration Three-month-old Q175 HD heterozygous mice were administered buffer, or two doses of AAV-PHP.eB-NH014 (UBC-46025) or AAV-PHP.eB-NH016 (PGK-46025), at either 5e12 vg / kg or 4e13 vg / kg via intravenous (IV) administration of AAV-PHP.eB-NH016 (PGK-46025). The test substance was administered via a single intravenous injection (maximum 200 µl). Briefly, the mice were placed under a heat lamp (approximately 3–5 inches from the top of the cage) for 30 seconds to dilate the lateral tail vein. Mice were secured using a mouse tail illumination device (Braintree Scientific), and the distal injection site was disinfected with 70% isopropanol. The test substance was delivered into the lateral tail vein using a 28G 0.5cc ½-inch U-100 insulin syringe. Four males and four females were injected in the treatment group, while three males and three females were injected in the control group. Three months after application, brain tissue was isolated, and the biodistribution of AAV was analyzed by vector genome (VG) analysis, transgene expression, and the effect on the reduction of mutant huntingtin protein (mHTT) RNA. Figure 10AThe dose-dependent vector genome (DNA) distribution in the cortex and striatum was shown, exhibiting a dose-dependent distribution. Figure 10B Dose-dependent transgene expression in the cortex and striatum was shown, and the results indicated that the two promoters reached similar expression levels. Figure 10C The effects on mHTT were shown, with dose-dependent repression of mHTT detected in both vectors. The PGK promoter outperformed the UBC promoter.

[0218] Soluble and aggregated mutant HTT protein was analyzed by immunohistochemistry (IHC), and mutant HTT protein in the cerebral cortex and striatum was quantified by Meso Scale Discovery (MSD) immunoassay. Positive IHC staining for ZFP was observed in the brain of mice administered a high dose (4e13 vg / kg) of AAV-PHP.eB-NH014 (UBC-46025) and mice receiving two doses (5e12 vg / kg and 4e13 vg / kg) of AAV-PHP.eB-NH016 (PGK-46025). In the high-dose AAV-PHP.eB-NH016 group, ZFP expression was most prominent in neuronal nuclei.

[0219] In mice administered AAV-PHP.eB-NH016, extensive staining was observed throughout the brain, with increasing staining intensity and distribution. In both groups (low-dose and high-dose) of PHP.eB-NH016, positive staining for ZFP was negatively correlated with mHTT staining. In the high-dose AAV-PHP.eB-NH016 group, mHTT was not detected.

[0220] Figure 10D A graph showing the quantitative levels of soluble mHTT in the cerebral cortex determined using the MSD immunoassay. Figure 10E A graph showing the quantitative levels of soluble mHTT in the striatum of the brain, determined using the MSD immunoassay. Figure 10F A graph showing the quantitative levels of aggregated mHTT in the cerebral cortex, determined using the MSD immunoassay. Figure 10G A graph showing the quantitative levels of aggregated mHTT in the striatum, determined using the MSD immunoassay.

[0221] Figures 10D to 10G The results showed that AAV-PHP.eB-NH016 (PGK-46025) effectively reduced mHTT soluble and aggregated proteins in a dose-dependent manner, while AAV-PHP.eB-NH014 (UBC-46025) reduced both soluble and aggregated mHTT at high doses.

[0222] In summary, the exemplary ZFP-TF driven by the PGK and UBC promoters, delivered to mice via the exemplary BBB-penetrating AAV vector, demonstrated dose-dependent in vivo mHTT repression in a mouse model of Huntington's disease.

[0223] Table 11. Summary of Immunohistochemical Scoring

[0224] Legend: N = Normal; Neg = Negative; NP = Not present in the slide; *The first value represents the percentage of positive cells, and the second value is the intensity score; Percentage of positive cells: 0 = 0%; 1 = <1-5%; 2 = 6-25%; 3 = 26-50%; 4 = 51-75%; 5 = 76-100%. Intensity score: 1 = Weak; 2 = Moderate; 3 = Strong; 4 = Very Strong.

[0225] All patents, patent applications and publications mentioned herein are hereby incorporated in their entirety by reference for all purposes.

[0226] While some detailed disclosure has been provided through illustration and examples for ease of understanding, those skilled in the art will understand that various changes and modifications can be made without departing from the spirit or scope of this disclosure. Therefore, the above description and embodiments should not be construed as limiting.

[0227] Implementation plan with numbering 1. A gene therapy construct encoding a non-naturally occurring transcription factor (ZFP-TF), the ZFP-TF comprising a zinc finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK) or ubiquitin C (UBC) promoter.

[0228] 2. The gene therapy construct as described in embodiment 1, wherein the ZFP includes a recognition helical region designated as ZFP46025 or ZFP45723.

[0229] 3. The gene therapy construct as described in embodiment 1 or 2, wherein the ZFP is codon-optimized.

[0230] 4. The gene therapy construct as described in any of the embodiments numbered above, wherein the ZFP comprises a nucleotide sequence having at least 60% identity with any of SEQ ID NO: 10-29.

[0231] 5. The gene therapy construct of embodiment 3 as indicated by number, wherein the ZFP comprises a nucleotide sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher identity with any of SEQ ID NO:10-29.

[0232] 6. The gene therapy construct of embodiment 4 as indicated by number, wherein the ZFP-TF comprises a nucleotide sequence having 100% identity with any of SEQ ID NO: 10-29.

[0233] 7. A gene therapy construct comprising a non-naturally occurring codon-optimized transcription factor (ZFP-TF), wherein the ZFP-TF comprises a zinc finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain. The ZFP includes a recognition spiral region designated as ZFP46025 or ZFP45723, and The ZFP binds to a target site in the mutant HTT (mHTT) gene.

[0234] 8. A gene therapy construct comprising a non-naturally occurring codon-optimized transcription factor (ZFP-TF), the 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 with any 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.

[0235] 9. The ZFP-TF as described in embodiment 7 or 8, wherein the ZFP-TF comprises a nucleotide sequence having 90%, 95% or higher identity with any of SEQ ID NO: 11-22 or SEQ ID NO: 24-29.

[0236] 10. The ZFP-TF as described in embodiment 9, wherein the ZFP-TF is 100% identical to any one of SEQ ID NO: 11-22 or SEQ ID NO: 24-29.

[0237] 11. The ZFP-TF as described in any of the numbered embodiments 7 to 10, wherein the expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EF1α promoter.

[0238] 12. The ZFP-TF as described in any of the embodiments numbered above, wherein the recognition helical region comprises an amino acid sequence of one of SEQ ID NO: 1-5 or SEQ ID NO: 7-9.

[0239] 13. The ZFP-TF as described in any of the aforementioned numbered embodiments, wherein the target site comprises the CAG repeat domain of the mHTT gene.

[0240] 14. The ZFP-TF as described in embodiment 12, wherein the target site identifies a sequence having 70%, 75%, 80%, 85%, 90%, 95% or higher identity with SEQ ID NO: 6.

[0241] 15. The ZFP-TF as described in embodiment 13, wherein the target site identification is a sequence having 100% identity with SEQ ID NO: 6.

[0242] 16. The ZFP-TF as described in any of the aforementioned numbered embodiments, wherein the ZFP-TF further comprises a sequence encoding a kernel localization sequence (NLS).

[0243] 17. The ZFP-TF as described in embodiment 16, wherein the NLS is SV40.

[0244] 18. The ZFP-TF as described in any of the embodiments numbered above, wherein the ZFP-TF further comprises an inverted terminal repeat sequence (ITR) side-connected to the promoter.

[0245] 19. The ZFP-TF as described in any of the embodiments numbered above, wherein the ZFP-TF further comprises a human growth hormone (hGH) polyadenylation signal.

[0246] 20. The gene therapy construct as described in any of the embodiments numbered above, wherein the gene therapy construct is delivered using a viral vector.

[0247] 21. The gene therapy construct as described in embodiment 20, wherein the viral vector is adeno-associated virus (AAV) or virus-like particle (VLP).

[0248] 22. The gene therapy construct as described in any of the embodiments numbered above, wherein the gene therapy construct is delivered using lipid nanoparticles (LNPs) or liposomes.

[0249] 23. A recombinant rAAV vector comprising a gene therapy construct encoding a non-naturally occurring transcription factor (ZFP-TF), the ZFP-TF comprising a zinc finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EF1α promoter.

[0250] 24. An rAAV vector comprising a gene therapy construct comprising a non-naturally occurring codon-optimized transcription factor (ZFP-TF), the ZFP-TF comprising a zinc finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein the ZFP comprises a recognition helical region designated as ZFP46025 or ZFP45723, and wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene.

[0251] 25. An rAAV vector comprising a gene therapy construct comprising a non-naturally occurring codon-optimized transcription factor (ZFP-TF), the 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 with any 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.

[0252] 26. The rAAV carrier as described in any of the numbered embodiments 23 to 25, wherein the rAAV carrier is AAV1, AAV2, AAV5, AAV7, AAV9 or AAVrh10.

[0253] 27. The rAAV vector as described in any of the numbered embodiments 23 to 25, wherein the rAAV vector comprises a capsid protein that penetrates the blood-brain barrier (BBB).

[0254] 28. The rAAV vector as described in embodiment 27, wherein the rAAV vector is VCAP-101, VCAP-102, 9P801, VCAP-100, VCAP-103, PAL1A, PAL1B, PAL1C, PAL2, CereAAV, Dyno bCAP1, AAV.CAP-B10, AAV.CAP-B20, AAV2-BR1N, AAV2-BR1, STAC-BBB ® Or AAV-TT, or AAV-BI-hTFR1.

[0255] 29. A lipid nanoparticle comprising the gene therapy construct described in any of the embodiments listed above.

[0256] 30. A pharmaceutical composition comprising the rAAV carrier or lipid nanoparticles described in any of the embodiments listed in the preceding numbered embodiments.

[0257] 31. A method for regulating the expression of a mutant Huntington's disease (mHTT) allele, the method comprising administering the pharmaceutical composition of embodiment 30.

[0258] 32. A method for regulating the expression of the mutant Huntington's disease HTT (mHTT) allele, the method comprising: The rAAV or lipid nanoparticles are administered, the rAAV or lipid nanoparticles comprising one or more gene therapy constructs encoding a non-naturally occurring transcription factor (ZFP-TF), the ZFP-TF comprising a zinc finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein the expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EF1α promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein, upon administration, the expression of the mutant (mHTT) allele is reduced.

[0259] 33. A method for treating Huntington's disease, the method comprising: Administering rAAV or lipid nanoparticles to subjects in need, the rAAV or lipid nanoparticles comprising one or more gene therapy constructs encoding a non-naturally occurring transcription factor (ZFP-TF), the ZFP-TF comprising a zinc finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EF1α promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein, upon administration, one or more symptoms associated with Huntington's disease are reduced or alleviated.

[0260] 34. The method according to any of the embodiments numbered above, wherein the ZFP includes an identification spiral region designated as ZFP46025 or ZFP45723.

[0261] 35. The method described in any of the embodiments numbered above, wherein the ZFP is codon-optimized.

[0262] 36. The method according to any of the embodiments numbered above, wherein the ZFP-TF comprises a nucleotide sequence having at least 85% identity with any of SEQ ID NO: 11-22 or SEQ ID NO: 24-29.

[0263] 37. A method for treating Huntington's disease, the method comprising administering to a subject in need a therapeutically effective amount of the pharmaceutical composition of embodiment 30.

[0264] 38. The method as described in embodiment 33, wherein one or more symptoms are cell death.

[0265] 39. The method as described in embodiment 33, wherein one or more symptoms are apoptosis.

[0266] 40. The method as described in embodiment 33, wherein one or more symptoms are motor defects.

[0267] 41. The method of any of the embodiments numbered 33 to 40, wherein the administration is intrathecal, intraventricular, intranasal, or intravenous.

[0268] 42. The method of any of the embodiments 33 to 40, wherein the application is performed by focused ultrasound.

[0269] 43. The method of any of the embodiments listed in the preceding numbered embodiments, wherein the administration is administration to the central nervous system (CNS).

[0270] 44. The method described in any of the embodiments listed in the preceding numbered embodiments, wherein the application to the brain is application to one or more of the striatum, cortex, caudate nucleus, putamen, thalamus, or globus pallidus.

[0271] 45. The method described in any of the embodiments listed in the preceding numbered embodiments, wherein the application is systemic.

[0272] 46. ​​The method according to any of the embodiments numbered above, wherein the application is applied to nerve cells.

[0273] 47. A method for treating Huntington's disease, the method comprising: Administering rAAV or lipid nanoparticles to a subject in need, the rAAV or lipid nanoparticles comprising one or more gene therapy constructs according to any one of the preceding claims, wherein the rAAV is a BBB-penetrating rAAV, wherein the administration is intravenous, and wherein, after administration, one or more symptoms associated with Huntington's disease are reduced or relieved.

Claims

1. A gene therapy construct comprising a non-naturally occurring codon-optimized transcription factor (ZFP-TF), wherein the ZFP-TF comprises a zinc finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain. The ZFP includes a recognition spiral region designated as ZFP46025 or ZFP45723, and The ZFP binds to a target site in the mutant HTT (mHTT) gene.

2. A gene therapy construct comprising a non-naturally occurring codon-optimized transcription factor (ZFP-TF), the 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 with any 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. The ZFP-TF as claimed in claim 1 or 2, wherein the ZFP-TF comprises a nucleotide sequence having 90%, 95% or higher identity with any one of SEQ ID NO: 11-22 or SEQ ID NO: 24-29.

4. The ZFP-TF as described in claim 3, wherein the ZFP-TF is 100% identical to any one of SEQ ID NO: 11-22 or SEQ ID NO: 24-29.

5. The ZFP-TF of any one of claims 1 to 4, wherein the expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EF1α promoter.

6. The ZFP-TF as claimed in any of the preceding claims, wherein the recognition helical region comprises an amino acid sequence of one of SEQ ID NO: 1-5 or SEQ ID NO: 7-9.

7. The ZFP-TF as claimed in any of the preceding claims, wherein the target site comprises the CAG repeat domain of the mHTT gene.

8. The ZFP-TF of claim 6, wherein the target site identifies a sequence having 70%, 75%, 80%, 85%, 90%, 95% or higher identity with SEQ ID NO:

6.

9. The ZFP-TF of claim 7, wherein the target site identifier is a sequence having 100% identity with SEQ ID NO:

6.

10. The ZFP-TF as described in any of the preceding claims, wherein the ZFP-TF further comprises a sequence encoding a kernel localization sequence (NLS).

11. The ZFP-TF of claim 10, wherein the NLS is SV40.

12. The ZFP-TF as described in any of the preceding claims, wherein the ZFP-TF further comprises an inverted terminal repeat sequence (ITR) side-connected to the promoter.

13. The ZFP-TF as described in any of the preceding claims, wherein the ZFP-TF further comprises a human growth hormone (hGH) polyadenylation signal.

14. The gene therapy construct as claimed in any of the preceding claims, wherein the gene therapy construct is delivered using a viral vector.

15. The gene therapy construct of claim 14, wherein the viral vector is adeno-associated virus (AAV), lentivirus, adenovirus, or virus-like particle (VLP).

16. The gene therapy construct as claimed in any of the preceding claims, wherein the gene therapy construct is delivered using lipid nanoparticles (LNPs) or liposomes.

17. A recombinant rAAV vector comprising a gene therapy construct encoding a non-naturally occurring transcription factor (ZFP-TF), the ZFP-TF comprising a zinc finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EF1α promoter.

18. An rAAV vector comprising a gene therapy construct comprising a non-naturally occurring codon-optimized transcription factor (ZFP-TF), the ZFP-TF comprising a zinc finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein the ZFP comprises a recognition helical region designated as ZFP46025 or ZFP45723, and wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene.

19. An rAAV vector, the rAAV vector gene therapy construct comprising a non-naturally occurring codon-optimized transcription factor (ZFP-TF), the 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 with any 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. The rAAV vector of any one of claims 17 to 19, wherein the rAAV vector is AAV1, AAV2, AAV5, AAV7, AAV9 or AAVrh10.

21. The rAAV vector of any one of claims 17 to 19, wherein the rAAV vector comprises a capsid protein that penetrates the blood-brain barrier (BBB).

22. The rAAV vector of claim 21, wherein the rAAV vector is VCAP-101, VCAP-102, 9P801, VCAP-100, VCAP-103, PAL1A, PAL1B, PAL1C, PAL2, CereAAV, Dyno bCAP1, AAV.CAP-B10, AAV.CAP-B20, AAV2-BR1N, AAV2-BR1, STAC-BBB ® Or AAV-TT, or AAV-BI-hTFR1.

23. A lipid nanoparticle comprising the gene therapy construct according to any one of the preceding claims.

24. A pharmaceutical composition comprising the rAAV carrier or lipid nanoparticles as described in any of the preceding claims.

25. A method for regulating the expression of a mutant Huntington's disease (mHTT) allele, the method comprising administering the pharmaceutical composition of claim 24.

26. A method for regulating the expression of the mutant Huntington's disease HTT (mHTT) allele, the method comprising: The rAAV or lipid nanoparticles are administered, the rAAV or lipid nanoparticles comprising one or more gene therapy constructs encoding a non-naturally occurring transcription factor (ZFP-TF), the ZFP-TF comprising a zinc finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein the expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EF1α promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein, upon administration, the expression of the mutant (mHTT) allele is reduced.

27. A method for treating Huntington's disease, the method comprising: Administering rAAV or lipid nanoparticles to subjects in need, the rAAV or lipid nanoparticles comprising one or more gene therapy constructs encoding a non-naturally occurring transcription factor (ZFP-TF), the ZFP-TF comprising a zinc finger protein (ZFP) sequence and a sequence encoding a transcriptional repression domain, wherein expression of the ZFP-TF is driven by a phosphoglycerate kinase 1 (PGK), ubiquitin C (UBC), EFS, or EF1α promoter, wherein the ZFP binds to a target site in a mutant HTT (mHTT) gene, and wherein, upon administration, one or more symptoms associated with Huntington's disease are reduced or alleviated.

28. The method of any of the preceding claims, wherein the ZFP includes an identification spiral region designated as ZFP46025 or ZFP45723.

29. The method as described in any of the preceding claims, wherein the ZFP is codon-optimized.

30. The method of any of the preceding claims, wherein the ZFP-TF comprises a nucleotide sequence having at least 85% identity with any one of SEQ ID NO: 11-22 or SEQ ID NO: 24-29.

31. A method of treating Huntington's disease, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 24 to a subject in need.

32. The method of claim 27, wherein one or more symptoms are cell death.

33. The method of claim 27, wherein one or more of the symptoms are apoptosis.

34. The method of claim 27, wherein one or more symptoms are motor defects.

35. The method of any one of claims 27 to 34, wherein the administration is intrathecal, intraventricular, intranasal, or intravenous.

36. The method of any one of claims 27 to 34, wherein the application is performed by focused ultrasound.

37. The method of any of the preceding claims, wherein the application is applied to the brain.

38. The method of any of the preceding claims, wherein the application to the brain is application to one or more of the striatum, cortex, caudate nucleus, putamen, thalamus, or globus pallidus.

39. The method as described in any of the preceding claims, wherein the application is systemic.

40. The method of any of the preceding claims, wherein the application is applied to the central nervous system (CNS).

41. A method for treating Huntington's disease, the method comprising: Administering rAAV or lipid nanoparticles to a subject in need, the rAAV or lipid nanoparticles comprising one or more gene therapy constructs according to any one of the preceding claims, wherein the rAAV is a BBB-penetrating rAAV, wherein the administration is intravenous, and wherein, after administration, one or more symptoms associated with Huntington's disease are reduced or relieved.

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