Zinc finger protein transcription factors for the treatment of prion diseases

Abstract

The present disclosure provides zinc finger fusion proteins that inhibit prion gene expression in the nervous system and methods of using the proteins to treat prion diseases.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 909,725, filed October 2, 2019, and U.S. Provisional Application No. 63 / 023,197, filed May 11, 2020, the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy above was created on October 2, 2020, is named 025297_WO012_SL.txt, and is 197,128 bytes in size. [Background technology]

[0003] Background of the Invention Prion diseases refer to a group of progressive neurodegenerative disorders that affect both humans and animals. These disorders are characterized by the accumulation of misfolded isoforms of the prion protein (PrP Scrapie; PrPSc) that cause the brain to become spongy, accompanied by neuronal loss and gliosis. The term prion stands for proteinaceous infectious particle and refers to the protein-only nature of these pathogenic isoforms. The abnormally shaped protein (PrP Sc ) subsequently converts into the abundantly expressed physiological form of prion protein (cellular PrP; PrP C ), which binds to the disease-causing isoform PrP Sc This phenomenon is known as self-templated. Sc biophysically similar to PrP in terms of solubility, structure, and stability. C (Riesner, Brit Med Bull. (2003) 66:21-33). PrP ScThe proliferation of isoforms is followed by aggregation, which causes neuronal cell death in the central nervous system. Human prion diseases can be hereditary (accounting for 10–15% of cases), sporadic, or acquired, and include Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), and kuru. In humans, prion diseases impair brain function, causing progressive cognitive decline and abnormal movements. Prion diseases are always fatal, typically occurring within months to years of onset.

[0004] Although the exact function of PrP is still controversial in the field, PrP has been hypothesized to play a role in neurogenesis and neuroprotection, circadian rhythms, myelin maintenance, epithelial-mesenchymal transition (EMT), and long-term potentiation (LTP), among many phenotypes. In addition to familial forms, prion diseases can also be sporadic or acquired. Individuals with sporadic prion diseases have no family history of the disease or identifiable mutations in the PRNP gene. Sporadic prion diseases are characterized by the absence of PrP. C PrP Sc Sporadic prion diseases include sporadic CJD (sCJD), sporadic fatal insomnia (sFI), and variably protease-sensitive prionopathy (VPSPr). Acquired prion diseases are caused by the acquisition of PrP from an external source. Sc For example, variant CJD (vCJD) is caused by exposure to PrP from cows with prion disease. Sc It is a type of acquired prion disease resulting from the consumption of beef products containing prion protein. In cattle, this form of the disease is known as bovine spongiform encephalopathy (BSE) or "mad cow disease." Another example of an acquired human prion disease is kuru, which has been identified among the South Fore people of Papua New Guinea. Kuru was transmitted when individuals ate diseased human tissue during cannibalistic funerals.

[0005] Reduction of PrP expression is a therapeutic strategy supported by a number of genetic proof of concept studies, as well as the in vivo efficacy of PrP-lowering antisense oligonucleotides (ASOs), which have been shown to prolong survival in prion-infected mice. See, e.g., Bueler et al., Cell (1993) 73(7):1339-47; Bueler et al., Mol Med Camb Mass. (1994) 1(1):19-30; Fischer et al., EMBO J. (1996) 15(6):1255-64; Mallucci et al., Science (2003) 302(5646):871-4; and Safar et al., J Gen Virol. (2005) 86(Pt 10):2913-23, Minikel et al., Nucleic Acids Res. (2020) 10.1093 / nar / gkaa616. ASOs achieving 50% PrP knockdown have been shown to delay the onset of prion disease in mice by more than two-fold, although higher levels of PrP knockdown may provide additional therapeutic benefit. The distribution of PrP knockdown beyond that achievable with ASOs may also be important for efficacy. Thus, there remains a need for effective treatments of prion diseases by targeting PrP expression. Summary of the Invention

[0006] The present disclosure provides zinc finger protein (ZFP) domains that target sites within or near the mammalian (e.g., human, non-human primate, rodent, or mouse) PRNP gene. The ZFP domains of the present disclosure can be fused to transcription factors to specifically inhibit the mammalian PRNP gene at the DNA level. These fusion proteins, also known as zinc finger protein transcription factors (ZFP-TFs), contain (i) a ZFP domain that specifically binds to a target region of the PRNP gene and (ii) a transcriptional repressor domain that reduces transcription of the gene. These proteins can be used to treat prion diseases.

[0007] In one aspect, the present disclosure provides a fusion protein comprising a zinc finger protein (ZFP) domain and a transcriptional repressor domain, wherein the ZFP domain binds to a mammalian (e.g., human, non-human primate, rodent, or mouse) prion protein gene (PRNP gene). In certain embodiments, the target region of the ZFP-TF is within about 1 kb or 500 bp of the transcription start site (TSS) in the PRNP gene. In certain embodiments, the fusion protein may contain one or more (e.g., 2, 3, 4, 5, or 6) zinc fingers, which, as appropriate, suppress PRNP gene expression by at least about 40%, 75%, 90%, 95%, or 99%, with no or minimal detectable off-target binding or activity. Non-limiting examples of zinc finger domains are shown in Figures 4 and 8A. In certain embodiments, the fusion protein comprises one or more recognition helix sequences shown in the tables of Figures 4 and 8A. In further embodiments, the fusion protein comprises some or all of the recognition helix sequences from a row of the tables of Figures 4 and 8A, with or without the backbone mutations shown. In certain embodiments, the fusion protein comprises an amino acid sequence shown in the tables of Figures 9A or 9B.

[0008] In certain embodiments, the transcriptional repressor domain of the fusion protein can comprise the amino acid sequence of the KRAB domain of KOX1. In the fusion protein, the ZFP domain can be linked to the transcriptional repressor domain via a peptide linker.

[0009] In another aspect, the present disclosure provides a nucleic acid construct comprising a coding sequence for a fusion protein of the present invention, wherein the coding sequence is operably linked to a transcriptional regulatory element, such as a mammalian promoter that is constitutively active or inducible in brain cells (e.g., the human synapsin I promoter). The present disclosure also provides a host cell comprising the nucleic acid construct. The host cell may be, for example, a human cell, and / or a brain cell or a pluripotent stem cell, which may optionally be an embryonic stem cell or an induced pluripotent stem cell (iPSC). The present disclosure also provides a recombinant virus (optionally, a recombinant adeno-associated virus (AAV) of serotype 6 or 9) comprising the nucleic acid construct.

[0010] In yet another aspect, the present disclosure provides a method for inhibiting expression of prion protein (PrP) in mammalian brain cells, comprising introducing a fusion protein of the invention into said cells, optionally by introduction of a nucleic acid construct described herein (e.g., via a recombinant virus), thereby inhibiting expression of PrP in said cells. The mammalian brain cells may be, for example, human, non-human primate, rodent, or murine cells, and / or may be neurons, glial cells, ependymal cells, or neuroepithelial cells. In one embodiment, the cell is in the brain of a patient suffering from or at risk of developing a prion disease, wherein the prion disease is, as appropriate, a familial, sporadic, or acquired prion disease, e.g., Creutzfeldt-Jakob disease (CJD), sporadic CJD, variant CJD, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), sporadic fatal insomnia (sFI), kuru, or variably protease-sensitive prionopathy (VPSPr).

[0011] The present disclosure also provides a method for treating a neurodegenerative disease in a patient, comprising administering to the patient a recombinant AAV encoding the fusion protein of the present invention. The neurodegenerative disease may be a prion disease, and the prion disease may be, as appropriate, a familial, sporadic, or acquired prion disease, such as CJD, sporadic CJD, variant CJD, GSS, FFI, sFI, kuru, or VSPPr. In some embodiments, the disease may be a tauopathy, such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), corticobasal degeneration (CBD), or chronic traumatic encephalopathy (CTE). In other embodiments, the disease may be a synucleinopathy, such as Parkinson's disease (PD), multiple system atrophy (MSA), or dementia with Lewy bodies (DLB).

[0012] In certain embodiments, the AAV encoding the fusion protein is introduced into the patient via intravenous, intrathecal, intracerebroventricular, intracisternal, or intrathalamic injection, or injection into any brain region.

[0013] The present disclosure provides fusion proteins, nucleic acid constructs, and recombinant viruses of the invention for use in the methods described herein, as well as the use of the fusion proteins, nucleic acid constructs, recombinant viruses for the manufacture of medicaments in the methods described herein.

[0014] Other features, objects, and advantages of the present invention will be apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawings]

[0015] [Figure 1]Figure 1 shows the upstream genomic region of the mouse Prnp gene. Small triangles in the cluster below the gene indicate the target genomic regions of 384 ZFP-TFs prepared to target the mouse Prnp gene. Right-pointing triangles indicate that the ZFP-TF binds to the sense strand of the gene. Left-pointing triangles indicate that the ZFP-TF binds to the antisense strand of the gene. 192 of the ZFP-TFs were parent proteins with the standard structure. The other 192 proteins were mutants, one for each parent, incorporating three R-to-Q substitutions at the fourth position N-terminal to the first amino acid of the zinc finger helix of the three zinc fingers of the ZFP-TF. [Figure 2] Figure 2 shows the effect of parental and mutant ZFP-TFs on reducing mouse Prnp mRNA expression in Neuro2a cells harvested 24 hours after transfection with ZFP-TF mRNA. Messenger RNA levels were measured by RT-qPCR. Normalized Prnp expression levels are indicated by the gradient bar "PRNP mRNA." The deepest (red) color indicates a 100% reduction. The lightest (white) color indicates a 0% reduction. RT-qPCR data were normalized to the mean mRNA levels of ATP5B and EIF4A2. [Figure 3] Figure 3A-D is a panel of bar graphs showing the effect of each of 384 ZFP-TFs (#81070-#81390) on reducing mouse Prnp mRNA expression at a dose-ranging time (from left to right: ZFP-TF mRNA doses of 3 ng, 10 ng, 30 ng, 100 ng, 300 ng, and 1000 ng). [Figure 4]Figure 4 is a table showing the recognition helix sequences and genomic sequences bound by 36 selected engineered ZFPs in or near the mouse PRNP gene. For each ZFP, a line displays the genomic target sequence (binding sequence) and DNA-binding recognition helix sequence (i.e., F1-F6) for each zinc finger within the ZFP domain. A "^" in the table indicates that the arginine (R) residue at the fourth position upstream of the first amino acid of the indicated helix has been changed to glutamine (Q). The assigned SEQ ID NO for each sequence is shown in parentheses following the sequence. [Figure 5] FIG. 5 is a panel of bar graphs showing the Prnp repression activity of 36 selected ZFP-TFs in Neuro2a cells, assessed as described above for FIG. 3D. [Figure 6] Figures 6A and 6B show Prnp mRNA levels in primary mouse cortical neurons 7 days after infection with recombinant AAV encoding one of 36 selected ZFP-TFs. The neurons were infected with increasing AAV multiplicities of infection (MOIs) (from left to right: 1E2, 3E2, 1E3, 3E3, 1E4, and 3E4). Figure 6A is a table showing normalized RT-qPCR data (mean Prnp mRNA levels and standard deviations). The data are then depicted in the bar graph shown in Figure 6B. RT-qPCR data were normalized to the mean mRNA levels of ATP5B, EIF4A2, and GAPDH. [Figure 7A]Figure 7A shows a series of volcano plots showing the off-target activity of 36 tested mouse Prnp ZFP-TFs in mouse primary neurons. The volcano plots summarize microarray data showing transcriptome changes in mouse primary neurons 7 days after AAV6 transduction. In the volcano plots, green circles (to the right of each volcano plot) represent significantly up-regulated off-target genes (FDR P<0.05), and red circles represent significantly down-regulated off-target genes (FDR P<0.05). Yellow circles represent microarray probe sets covering both the mouse Prnp and Prnd (located downstream of Prnp) gene loci. Prnd is barely expressed in mouse cortical neurons, and therefore minimal changes in Prnp expression were detected. [Figure 7B] FIG. 7B is a table showing the number of off-target genes dysregulated for mouse Prnp ZFP-TFs tested in mouse primary cortical neurons corresponding to the volcano plot in FIG. 7A. [Figure 8A] Figure 8A is a table showing the recognition helix sequences and genomic sequences bound by 12 selected engineered ZFPs in or near the human PRNP gene. For each ZFP, a line displays the genomic target sequence (binding sequence) and DNA-binding recognition helix sequence (i.e., F1-F6) for each zinc finger within the ZFP domain. A "^" in the table indicates that the arginine (R) residue at the fourth position upstream of the first amino acid of the indicated helix has been changed to glutamine (Q). The assigned SEQ ID NO for each sequence is shown in parentheses below the sequence. [Figure 8B]Figure 8B is a panel of figures showing the 12 ZFP-TFs from Figure 8A targeting human PRNP in human iPSC-derived neurons. The y-axis is PRNP mRNA expression normalized to the geometric mean of three housekeeping genes (ATP5B, EIF4A2, and GAPDH) and assessed 31 days after transduction of iPSC-derived neurons with AAV6 encoding different ZFP-TFs. The amount of AAV6 used is shown on the x-axis, with AAV6 doses increasing from left to right (1E3, 3E3, 1E4, 3E4, 1E5, and 3E5). Bars represent the mean of four technical replicates, and error bars represent standard deviation. A magnified version of the titration scale is shown at the bottom of the figure. [Figure 8C] FIG. 8C is a table showing the repression of human PNRP in human iPSC-derived neurons. [Figure 8D] Figure 8D is a series of volcano plots showing the off-target activity of 12 human PRNP ZFP-TFs tested in human iPSC-derived neurons. The volcano plots summarize microarray data showing transcriptome changes in human iPSC-derived neurons 19 days after AAV6 transduction. In the volcano plots, green circles (to the right of each volcano plot) represent significantly up-regulated off-target genes (FDR P<0.05), and red circles represent significantly down-regulated off-target genes (FDR P<0.05). Yellow circles represent microarray probe sets detecting transcripts expressed from the human PRNP gene. [Figure 8E] FIG. 8E is a table showing the number of dysregulated off-target genes of human PRNP ZFP-TFs tested in human iPSC-derived neurons, corresponding to the volcano plot in FIG. 8D. [Figure 9]Figure 9A is a table showing the full-length AA sequences (helices, R(-5)Q mutants, intra- and inter-module linkers) of ZFP-TFs of mouse Prnp. Figure 9B is a table showing the full-length AA sequences (helices, R(-5)Q mutants, intra- and inter-module linkers) of ZFP-TFs of human PRNP. The assigned SEQ ID NO for each sequence is shown in parentheses after the sequence. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description of the Invention The present disclosure provides ZFP domains that target a site (i.e., sequence) within or near a mammalian PRNP gene. The ZFP domains described herein may be linked to or fused to another functional molecule or domain. The ZFP domains of the present disclosure may be fused to a transcription factor to repress transcription of the mammalian PRNP gene into RNA. The fusion proteins are referred to as zinc finger protein transcription factors (ZFP-TFs). These ZFP-TFs contain a zinc finger protein (ZFP) domain that specifically binds to a target region (i.e., target site) within or near the PRNP gene and a transcriptional repressor domain that reduces transcription of the gene. Reducing neuronal PrP levels by introducing the ZFP-TF into a patient's brain can result in increased PrP levels. Sc It is expected that the formation and spread of prions will be inhibited (e.g., reduced or stopped), thereby treating (e.g., alleviating symptoms, preventing the onset or worsening of symptoms, and increasing survival rates) of prion diseases.

[0017] Our ZFP-TF approach to PRNP inhibition has several advantages over approaches currently being tested by others. ZFP-TFs can achieve higher levels of PrP suppression than those reported for antisense oligonucleotides (ASOs). Furthermore, ZFP-TFs may only need to be administered once (by introducing a ZFP-TF expression construct, e.g., a recombinant virus (e.g., recombinant AAV) into a patient), whereas ASOs require repeated administration. Furthermore, the ZFP-TF approach only requires the involvement of two alleles of the PRNP gene in the genome of each cell. In contrast, ASOs require the involvement of multiple copies of PRNP mRNA in each cell. Furthermore, using recombinant viruses, e.g., recombinant AAVs, ZFP-TFs can be delivered to the desired brain region.

[0018] Our approach to treating prion disease is expected to be safe: Approximately 1 in 18,000 people are estimated to be heterozygous for a loss-of-function PRNP mutation, with no discernible adverse effects in these individuals.

[0019] I. Targeting of ZFP domains The ZFP domain of the fusion protein of the present invention specifically binds to a target region within or near the mammalian (e.g., human, non-human primate, or mouse) PRNP gene. The DNA-binding ZFP domain of the ZFP-TF directs the fusion protein to the target region of the PRNP gene, presenting the transcriptional repressor domain of the fusion protein to the target region. The repressor domain then represses transcription of the PRNP gene by RNA polymerase. The target region can be any suitable site in the PRNP gene that allows for repression of gene expression. For example, the target region includes or is adjacent to the PRNP transcription start site (TSS) or a PRNP transcriptional regulatory element (e.g., promoter, enhancer, RNA polymerase pause site, etc.). For example, the target region can be approximately 500 to 1,000 bp upstream and / or downstream of the TSS. Come

[0020] In certain embodiments, the genomic target region is at least 8 bp in length. For example, the target region can be 8 bp to 40 bp in length, e.g., 12, 15, 18, 21, 24, 27, 30, 33, or 36 bp in length. The target sequence can be on the sense strand of the gene or the antisense strand of the gene. To ensure targeting accuracy and reduce off-target binding or activity by the ZFP-TF, the sequence of the selected PRNP target region preferably has less than 75% homology (e.g., less than 70%, less than 65%, less than 60%, or less than 50%) to the sequence of other genes. In certain embodiments, the target region of the ZFP-TF of the invention is 15 to 18 bp in length and is located within approximately 500 to 1,000 bp of the TSS. Examples of target regions in the mouse PRNP gene are shown in Figures 1 and 4. An example of a target region in the human PRNP gene is shown in Figure 8A.

[0021] In certain embodiments, the engineered ZFPs of the invention preferably bind to the target site (i.e., binding sequence) shown in a row of the table in Figure 4 and Figure 8A with little or no detectable off-target binding or activity.

[0022] Other criteria for further evaluating targeting moieties include the previous availability of ZFPs that bind to such moieties or related moieties, the ease of designing new ZFPs that bind to a particular targeting moiety, and off-target binding assessment.

[0023] II. ZFP Domain "Zinc finger protein" or "ZFP" refers to a protein with a DNA-binding domain stabilized by zinc. ZFPs bind to DNA in a sequence-specific manner. Each DNA-binding unit of a ZFP is called a zinc "finger." Each finger typically consists of seven amino acid residues and contains a DNA-binding "recognition helix" that determines the specificity of DNA binding. A ZFP domain has at least one finger, and each finger binds to two to four base pairs of DNA, typically three or four base pairs. Each zinc finger typically consists of approximately 30 amino acids and chelated zinc. Engineered ZFPs can have novel binding specificities compared to naturally occurring ZFPs. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, the use of databases containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to a particular triplet or quadruplet sequence (see, e.g., U.S. Patent Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,140,081; 6,200,759; 6,453,242; 6,534,261; 6,979,539; and 8,586,526; and International Patent Publications WO 95 / 19431; WO 96 and WO03 / 016496. The ZFP domains described herein may be linked or fused to another molecule, e.g., a protein.Such ZFP fusions may include domains that enable gene activation (e.g., activation domain), gene repression (e.g., repression domain), ligand binding (e.g., ligand-binding domain), high-throughput screening (e.g., ligand-binding domain), localized hypermutation (e.g., activation-induced cytidine deaminase domain), chromatin modification (e.g., histone deacetylase domain), recombination (e.g., recombinase domain), targeted integration (e.g., integrase domain), DNA modification (e.g., DNA methyltransferase domain), base editing (e.g., base editor domain), or targeted DNA cleavage (e.g., nuclease domain). Examples of engineered ZFP domains are shown in Figures 4 and 8A.

[0024] The ZFP domain of the engineered ZFP fusion proteins of the invention can comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more) zinc finger. A ZFP domain with one finger typically recognizes a target site containing 3 or 4 nucleotides. A ZFP domain with two fingers typically recognizes a target site containing 6 or 8 nucleotides. A ZFP domain with three fingers typically recognizes a target site containing 9 or 12 nucleotides. A ZFP domain with four fingers typically recognizes a target site containing 12-15 nucleotides. A ZFP domain with five fingers typically recognizes a target site containing 15-18 nucleotides. A ZFP domain with six fingers can recognize a target site containing 18-21 nucleotides.

[0025] In certain embodiments, an engineered ZFP of the invention comprises a DNA-binding recognition helix sequence shown in the tables of Figures 4 and 8 A. For example, an engineered ZFP can comprise the sequence of F1, F2, F3, F4, F5, or F6, as shown in the tables of Figures 4 and 8 A.

[0026] In certain embodiments, an engineered ZFP of the invention comprises two adjacent DNA-binding recognition helix sequences shown in a row of the tables in Figures 4 and 8 A. For example, an engineered ZFP can comprise the sequences F1-F2, F2-F3, F3-F4, F4-F5, or F5-F6 shown in a row of the tables in Figures 4 and 8 A.

[0027] In certain embodiments, an engineered ZFP of the invention comprises a DNA-binding recognition helix sequence shown in a row of the table in Figures 4 and 8 A. For example, an engineered ZFP can comprise the sequences of F1, F2, F3, F4, F5, and F6 (e.g., F1-F6) shown in a row of the table in Figures 4 and 8 A.

[0028] The target specificity of the ZFP domain can be improved by mutations to the ZFP backbone sequence, for example, as described in U.S. Patent No. 2018 / 0087072. These mutations include mutations made to residues in the ZFP backbone that can nonspecifically interact with phosphates on the DNA backbone but are not relevant to nucleotide target specificity (see, e.g., Miller et al., Nat Biotechnil. (2019) 37(8):945-52). In some embodiments, these mutations include mutating cationic amino acid residues to neutral or anionic amino acid residues. In some embodiments, these mutations include mutating polar amino acid residues to neutral or nonpolar amino acid residues. In further embodiments, mutations are made at positions (-5), (-9), and / or (-14) relative to the DNA-binding helix. In some embodiments, the zinc finger may contain one or more mutations at positions (-5), (-9), and / or (-14). In further embodiments, one or more zinc fingers in a multi-fingered ZFP domain can contain mutations at positions (-5), (-9), and / or (-14). In certain embodiments, the amino acid at positions (-5), (-9), and / or (-14) (e.g., arginine (R) or lysine (K)) is mutated to alanine (A), leucine (L), serine (S), aspartic acid (N), glutamic acid (E), tyrosine (Y), and / or glutamine (Q). Examples of engineered ZFPs with one, two, or three backbone mutations are shown in the tables of Figures 4, 8A, 9A, and 9B. The symbol "^" in Figures 4 and 8A indicates that the arginine (R) residue four positions upstream of the first amino acid of the indicated recognition helix has been changed to glutamine (Q), and is shown in bold in the tables of Figures 9A and 9B. In each recognition helix sequence, the seven DNA-binding amino acid positions are numbered -1, +1, +2, +3, +4, +5, and +6, so the R to Q substitution position is numbered (-5).

[0029] In certain embodiments, an engineered ZFP of the invention comprises a DNA-binding recognition helix sequence and associated backbone mutations as shown in the tables of Figures 4 and 8 A. In certain embodiments, an engineered ZFP of the invention comprises a DNA-binding recognition helix sequence and associated backbone mutations as shown in a row of the tables of Figures 4 and 8 A.

[0030] In certain embodiments, the engineered ZFPs described herein comprise the recognition helices and backbone portions of the sequences shown in the first row of the tables in Figures 9A and 9B. In certain embodiments, the engineered ZFPs described herein comprise the recognition helices and backbone portions of the sequences shown in the first row of the tables in Figures 9A and 9B as the sequences appear after post-translational modification. For example, the post-translational modification may remove an initiator methionine residue from the sequence shown in the first row of the tables in Figures 9A and 9B.

[0031] In certain embodiments, the ZFP-TFs of the invention comprise one or more zinc finger domains. The domains may be linked together via an extensible, flexible linker, e.g., such that one domain comprises one or more (e.g., four, five, or six) zinc fingers and another domain comprises one or more (e.g., four, five, or six) additional zinc fingers. In certain embodiments, the linker is a standard interfinger linker, such that the finger array comprises a single DNA-binding domain comprising 8, 9, 10, 11, or 12 or more fingers. In other embodiments, the linker is an atypical linker, such as a flexible linker. For example, two ZFP domains may be linked to a transcriptional repressor TF in the structures (N- to C-terminus) ZFP-ZFP-TF, TF-ZFP-ZFP, ZFP-TF-ZFP, or ZFP-TF-ZFP-TF (two ZFP-TF fusion proteins fused via a linker).

[0032] In some embodiments, ZFP-TFs are "two-handed," i.e., they contain two zinc finger clusters (two ZFP domains) separated by an intervening amino acid, and the two ZFP domains bind to two discontinuous target sites. An example of a two-handed zinc finger binding protein is SIP1, in which a cluster of four zinc fingers is located at the amino terminus of the protein and a cluster of three fingers is located at the carboxyl terminus (Remacleetal., EMBO J. (1999) 18(18):5073-84). Each zinc finger cluster in these proteins can bind to a unique target sequence, and the spacing between the two target sequences can include many nucleotides.

[0033] Alternatively, the DNA-binding domain may be derived from a nuclease, such as homing endonucleases and meganucleases, for example, those with known recognition sequences such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII. See, for example, U.S. Patent Nos. 5,420,032 and 6,833,252; Belfort et al., Nucleic Acids Res. (1997) 25:3379-88; Dujon et al., Gene (1989) 82:115-8; Perler et al., Nucleic Acids Res. (1994) 22:1125-7; Jasin, Trends Genet (1996) 12:224-8; Gimble et al., J Mol Biol. (1996) 263:163-80; Argast et al., J Mol Biol. (1998) 280:345-53; and the New England Biolabs catalog. Furthermore, the DNA-binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites. See, e.g., Chevalier et al., Mol Cell (2002) 10:895-905; Epinat et al., Nucleic Acids Res. (2003) 31:2952-62; Ashworth et al., Nature (2006) 441:656-59; Paques et al., Current Gene Therapy (2007) 7:49-66; and U.S. Patent Publication No. 2007 / 0117128.

[0034] III. Zinc finger protein transcription factors The ZFP domain described herein may be fused to a transcription factor. In some embodiments, the transcription factor is a transcription repressor domain, and the ZFP and repressor domain may be linked to each other via a direct peptidyl bond or a peptide linker, or by dimerization (e.g., via a leucine zipper, a STAT protein N-terminal domain, or an FK506-binding protein). As used herein, "fusion protein" refers to a polypeptide having covalently linked domains, as well as a complex of polypeptides linked to each other via non-covalent bonds. The transcription repressor domain can be linked to the ZFP domain at any suitable position, including the C-terminus or N-terminus of the ZFP domain.

[0035] In certain embodiments, two or more ZFP-TFs of the invention are used simultaneously in a patient, wherein the ZFP-TFs bind to different target regions of the PRNP gene to achieve optimal repression of PRNP expression.

[0036] A. Transcriptional repressor domain The ZFP-TF of the present invention comprises an engineered ZFP domain described herein and one or more transcriptional repressor domains that attenuate the transcriptional activity of the PRNP gene. One or more engineered ZFP domains and one or more transcriptional repressor domains may be connected by a flexible linker. Non-limiting examples of transcriptional repressor domains include the KRAB domain of KOX1, KAP-1, MAD, FKHR, EGR-1, ERD, SID, TGF-beta-inducible early gene (TIEG), v-ERB-A, MBD2, MBD3, TRa, histone methyltransferase, histone deacetylase (HDAC), nuclear hormone receptor (e.g., estrogen receptor or thyroid hormone receptor), DNMT family members (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2. See, e.g., 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. Further exemplary repression domains include, but are not limited to, ROM2 and AtHD2A. See, e.g., Chem et al. (1996) Plant Cell 8:305-321; and Wu et al. (2000) Plant J. 22:19-27.

[0037] In some embodiments, the transcriptional repressor domain comprises a sequence from the Krüppel-associated box (KRAB) domain of human zinc finger protein 10 / KOX1 (ZNF10 / KOX1) (e.g., GenBank number NM_015394.4). An exemplary KRAB domain sequence is: DAKSLTAWSR TLVTFKDVFV DFTREEWKLL DTAQQIVYRN VMLENYKNLV SLGYQLTKPD VILRLEKGEE PWLVEREIHQ ETHPDSETAF EIKSSV (SEQ ID NO: 261) is. Variants of this KRAB sequence may also be used so long as they have the same or similar transcriptional repression function.

[0038] In certain embodiments, the engineered ZFP-TFs described herein preferably bind to the target sites shown in a row of the tables in Figures 4 and 8A with little or no detectable off-target binding or activity. Off-target binding may be determined, for example, by measuring the activity of the ZFP-TF at the off-target gene.

[0039] In certain embodiments, an engineered ZFP-TF described herein comprises a DNA-binding recognition helix sequence shown in the tables of Figures 4 and 8A. In certain embodiments, an engineered ZFP-TF described herein comprises two adjacent DNA-binding recognition helix sequences shown in a row of the tables of Figures 4 and 8A. In certain embodiments, an engineered ZFP-TF described herein comprises a DNA-binding recognition helix sequence shown in a row of the tables of Figures 4 and 8A. In certain embodiments, an engineered ZFP-TF described herein comprises the recognition helix and backbone portions of the sequence shown in a row of the tables of Figures 9A and 9B. In certain embodiments, an engineered ZFP-TF described herein comprises the amino acid sequence shown in a row of the tables of Figures 9A and 9B.

[0040] In certain embodiments, an engineered ZFP-TF described herein comprises the recognition helix and backbone portion of the sequence shown in a row of the table in Figures 9A and 9B, as the sequence appears after post-translational modification. In certain embodiments, an engineered ZFP-TF described herein comprises the amino acid sequence shown in a row of the table in Figures 9A and 9B, as the sequence appears after post-translational modification. For example, the post-translational modification may remove an initiator methionine residue from the sequence shown in the table in Figures 9A and 9B.

[0041] B. Peptide Linker The ZFP domain and transcriptional repressor domain of the ZFP-TF of the invention, and / or the zinc fingers within the ZFP domain, may be linked via a peptide linker, e.g., a non-cleavable peptide linker of about 5 to 200 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids). Preferred linkers are flexible amino acid subsequences that are typically synthesized as recombinant fusion proteins. See, e.g., U.S. Patent Nos. 6,479,626; 6,903,185; 7,153,949; 8,772,453; and 9,163,245; and WO 2011 / 139349. The proteins described herein may contain any combination of suitable linkers. Non-limiting examples of linkers are DGGGS (SEQ ID NO:252), TGEKP (SEQ ID NO:253), LRQKDGERP (SEQ ID NO:254), GGRR (SEQ ID NO:255), GGRRGGGS (SEQ ID NO:256), LQRDGERP (SEQ ID NO:257), LRQKDGGGSERP (SEQ ID NO:258), LRQKD(G3S)2ERP (SEQ ID NO:259), and TGSQKP (SEQ ID NO:260). In certain embodiments, TGEKPFA (SEQ ID NO:348) and / or TGSQKPFQ (SEQ ID NO:349) links a zinc finger within the ZFP domain, and / or LRQKDAARGSGG (SEQ ID NO:350) or LRGSGG (SEQ ID NO:351) links the ZFP domain to the transcriptional repressor domain.

[0042] In certain embodiments, the peptide linker is 3-20 amino acid residues in length, with a predominance of G and / or S. A non-limiting example of such a linker is a G4S-type linker (SEQ ID NO: 347), i.e., a linker containing one or more (e.g., two, three, or four) GGGGS (SEQ ID NO: 251) motifs, or variations of said motifs (such as those with insertions, deletions, and substitutions of one, two, or three amino acids from said motif).

[0043] IV. Expression of ZFP-TFs The ZFP-TF of the present disclosure may be introduced into a patient via a nucleic acid molecule encoding it. The nucleic acid molecule may be an RNA or cDNA molecule. The nucleic acid molecule may be introduced into the patient's brain via injection of a composition comprising a lipid:nucleic acid complex (e.g., a liposome). Alternatively, the ZFP-TF may be introduced into a patient via a nucleic acid expression vector comprising a sequence encoding the ZFP-TF. The expression vector may comprise expression control sequences, such as a promoter, enhancer, transcription signal sequence, and transcription termination sequence, that enable expression of the ZFP-TF coding sequence in cells of the nervous system. In certain embodiments, the expression vector remains present in the cell as a stable episome. In other embodiments, the expression vector is integrated into the cell's genome.

[0044] In some embodiments, the promoter on the vector for directing ZFP-TF expression in the brain is a constitutively active promoter or an inducible promoter.Suitable promoters include, but are not limited to, retrovirus RSV LTR promoter (optionally with RSV enhancer), CMV promoter (optionally with CMV enhancer), CMV immediate early promoter, SV40 promoter, dihydrofolate reductase (DHFR) promoter, β-actin promoter, phosphoglycerate kinase (PGK) promoter, EF1α promoter, MoMLV LTR, CK6 promoter, TK promoter, tetracycline-responsive promoter (TRE), HBV promoter, chimeric liver-specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter, CMV enhancer / chicken β-actin / rabbit β-globin promoter (CAG promoter; Niwa et al., Gene (1991) 108 (2): 193-9), and RU-486-responsive promoter. Neuron-specific promoters, such as the synapsin I promoter, the MeCP2 promoter, the CAMKII promoter, the PrP promoter, the GFAP promoter, or engineered or native promoters that restrict expression to neurons and glial cells, can also be used.

[0045] Any method for introducing nucleotide sequences into cells may be used, including, but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, liposomes combined with nuclear localization signals, naturally occurring liposomes (e.g., exosomes), or viral transduction.

[0046] Viral transduction may be used for in vivo delivery of expression vectors.Various viral vectors known in the art, such as vaccine vectors, adenoviral vectors, lentiviral vectors, poxvirus vectors, adeno-associated viral (AAV) vectors, retroviral vectors, and hybrid viral vectors, can be used by those skilled in the art for use in the present disclosure.In some embodiments, the viral vector used herein is a recombinant AAV (rAAV) vector.AAV vectors are particularly suitable for CNS gene delivery because they can infect both dividing and non-dividing cells, exist as stable episomal structures for long-term expression, and have very low immunogenicity (Hadaczek et al., MolTher.(2010)18:1458-61; Zaiss, et al., GeneTher.(2008)15:808-16).Any suitable AAV serotype may be used. For example, the AAV may be AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, or AAVrhlO, or a pseudotype such as AAV2 / 8, AAV2 / 5, AAV2 / 6, or AAV2 / 9 (i.e., AAV derived from multiple serotypes; for example, rAAV contains AAV2 inverted terminal repeats (ITRs) in its genome and AAV8, 5, 6, or 9 capsid). In certain embodiments, the expression vector is an AAV viral vector, and is introduced into target human cells by recombinant AAV virions containing a construct whose genome comprises AAV inverted terminal repeat (ITR) sequences at both ends to enable the production of AAV virions in a production system such as an insect cell / baculovirus production system or a mammalian cell production system. The capsid protein of the AAV may be engineered to reduce immunogenicity or enhance transduction ability in humans or non-human primates.In some embodiments, AAV9 is used.The viral vectors described herein can be produced using methods known in the art.Any suitable permissive cell or packaging cell may be used to produce viral particles.For example, mammalian or insect cells can be used as packaging cell lines.

[0047] V. Medicinal Uses The ZFP-TFs of the present invention can be used to treat patients in need of downregulation of PrP expression. The patient is suffering from or at risk of developing a prion disease. The prion disease to be treated may be familial, sporadic, or acquired, and may be CJD, sCJD, vCJD, GSS, FFI, sFI, kuru, or VSPPr. At-risk patients include those with a genetic predisposition as well as those exposed to prions from mad cow disease or other environmental sources. The present disclosure provides a method for treating a neurodegenerative disease in a subject, such as a human patient, in need of treatment, comprising introducing a therapeutically effective amount (e.g., an amount sufficient to suppress PRNP expression) of a ZFP-TF (e.g., an rAAV vector expressing the same) into the nervous system of the subject. In certain embodiments, the neurodegenerative disease is a prion disease. The term "treating" encompasses alleviating symptoms, preventing the onset of symptoms, delaying disease progression, improving quality of life, and increasing survival. Biomarkers, including but not limited to, prion or neurofilament light chain (NfL) levels in cerebrospinal fluid or plasma, may also be measured to monitor the progress of treatment.

[0048] The present disclosure provides pharmaceutical compositions comprising a viral vector, such as a recombinant rAAV whose recombinant genome includes an expression cassette for a ZFP-TF. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, such as water, saline (e.g., phosphate-buffered saline), dextrose, glycerol, sucrose, lactose, gelatin, dextran, albumin, or pectin. Additionally, the composition may contain auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, or other reagents that enhance the effectiveness of the pharmaceutical composition. The pharmaceutical composition may also comprise a delivery vehicle, such as a liposome, nanocapsule, microparticle, microsphere, lipid particle, or vesicle.

[0049] Cells targeted by the disclosed therapeutic methods include, but are not limited to, neuronal cells (e.g., motor neurons, sensory neurons, dopaminergic neurons, cholinergic neurons, glutamatergic neurons, GABAergic neurons, or serotonergic neurons); glial cells (e.g., oligodendrocytes, astrocytes, pericytes, Schwann cells, or microglial cells); ependymal cells; or neuroepithelial cells. Brain regions targeted by therapeutic methods may be, for example, the cerebral cortex (classical CJD), thalamus (FFI), brainstem (scrapie, BSE, and chronic wasting disease), and cerebellum (kuru). The targeted region may be reached directly by intrastriatal, intrathalamic, intracerebral, intracisternal (ICM) injection, or more commonly, intraparenchymal, intracerebroventricular (ICV), intrathecal, or intravenous injection. Other routes of administration include, but are not limited to, intracerebral, intraventricular, intranasal, or intraocular administration. In some embodiments, viral vectors are administered directly into cerebrospinal fluid (CSF), for example, via intrathecal and / or intracerebral injection, or via intracisternal injection, and then spread throughout CNS tissue. In other embodiments, viral vectors cross the blood-brain barrier and achieve widespread distribution throughout the CNS tissue of a subject after intravenous administration. In other embodiments, viral vectors are delivered directly to the target area via intraparenchymal injection. In some cases, viral vectors can undergo retrograde or anterograde transport to other brain regions after intraparenchymal delivery. In some embodiments, viral vectors have different CNS tissue targeting capabilities (e.g., CNS tissue tropism), which allows for highly efficient, stable, and non-toxic gene transfer.

[0050] For example, the pharmaceutical composition can be administered intraventricularly to the ventricular region of the patient's forebrain, such as the right lateral ventricle, left lateral ventricle, third ventricle, or fourth ventricle. The pharmaceutical composition can be administered intracerebrally, for example, by injection of the composition into or near the striatum, caudate nucleus, putamen, substantia nigra, midbrain, olfactory bulb, cerebrum, medulla, pons, cerebellum, locus coeruleus pons, medulla, brainstem, globus pallidus, hippocampus, cerebral cortex, cerebrum, intracranial cavity, meninges, dura mater, arachnoid mater, or pia mater. Intracerebral administration can, in some cases, include administering the agent to the cerebrospinal fluid (CSF) in the subarachnoid space surrounding the brain.

[0051] In some cases, intracerebral administration involves injection using stereotaxic techniques. Stereotactic techniques are well known in the art and typically involve the use of a computer and a three-dimensional scanning device used together to guide the injection to a specific brain region, such as the ventricular region. A microinjection pump (e.g., manufactured by World Precision Instruments) may also be used. In some cases, a microinjection pump is used to deliver a composition containing a viral vector. In some cases, the infusion rate of the composition ranges from 0.1 μl / min to 100 μl / min. As will be understood by those skilled in the art, the infusion rate depends on various factors, including, for example, the subject's species, the subject's age, the subject's weight / size, the AAV serotype, the required dosage, and the brain region to be targeted. Therefore, other infusion rates may be considered appropriate by those skilled in the art in a particular situation.

[0052] rAAV can be delivered to a subject, for example, by intravenous administration. In some cases, it may be desirable to locally deliver rAAV to brain tissue, spinal cord, cerebrospinal fluid (CSF), neurons, glial cells, meninges, astrocytes, oligodendrocytes, microglia, interstitial spaces, etc. In some cases, recombinant AAV can be directly delivered to the CNS by injection into the ventricular region, as well as to the parenchyma, cortex, cerebellar lobes, thalamus, hippocampus, or other brain regions, or a combination of brain regions. AAV can be delivered with a needle, catheter, or related device using neurosurgical techniques known in the art, such as by stereotactic injection (see, e.g., Stein et al., J Vir. (1999) 73:3424-9; Davidson et al., PNAS. (2000) 97:3428-32; Davidson et al., Nat Genet. (1993) 3:219-223; and Alisky and Davidson, Hum. Gene Ther. (2000) 11:2315-29).

[0053] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure. In the case of conflict, the present specification, including definitions, will control. In general, the nomenclature used in connection with, and techniques related to, neurology, medicine, medicinal chemistry, and cell biology described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly practiced in the art or as described herein. Furthermore, unless the context requires otherwise, singular forms include plural forms and plural forms include the singular. Throughout this specification and the embodiments, the words "have" and "comprise," or variations thereof, such as "has," "having," "comprises," or "comprising," are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of other integers or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although many documents are cited herein, this citation does not acknowledge that any of these documents form part of the common general knowledge in the art. As used herein, the term "approximately" or "about" when applied to one or more target values ​​means a value that is similar to the stated reference value. In certain embodiments, unless otherwise specified or clear from the context, the term refers to a range of values ​​that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or smaller) of the stated reference value.

[0054] In order that this invention may be better understood, the following examples are set forth, which are for illustrative purposes only and are not to be construed as limiting the scope of this invention in any way. The present disclosure relates, for example, to the following: [Section 1] A fusion protein comprising a zinc finger protein (ZFP) domain and a transcriptional repressor domain, wherein the ZFP domain binds to a target region in a mammalian prion protein gene (PRNP gene). [Section 2] Item 1, wherein the target region is within about 1 kb or 500 bp of the transcription start site (TSS) in the PRNP gene. [Section 3] Item 3. The fusion protein of item 1 or 2, wherein the PRNP gene is a human, non-human primate, rodent, or mouse PRNP gene. [Section 4] 4. The fusion protein of any one of paragraphs 1 to 3, wherein the ZFP domain comprises six zinc fingers and optionally inhibits expression of the PRNP gene by at least about 40%, 75%, 90%, 95%, or 99% with no detectable off-target binding or activity. [Section 5] Item 10. The fusion protein of any one of items 1 to 9, wherein the transcriptional repressor domain comprises the amino acid sequence of the KRAB domain of KOX1. [Section 6] Item 6. The fusion protein according to any one of Items 1 to 5, wherein the ZFP domain is linked to the transcriptional repressor domain via a peptide linker. [Section 7] Item 7. The fusion protein of any one of items 1 to 6, wherein the ZFP domain comprises a DNA-binding recognition helix sequence shown in Figures 4 and 8A. [Section 8] Item 8. The fusion protein of any one of items 1 to 7, wherein the ZFP domain comprises a DNA-binding recognition helix sequence shown in a row in the tables of Figures 4 and 8A. [Section 9] Item 9. A nucleic acid construct comprising a coding sequence for the fusion protein according to any one of Items 1 to 8, wherein the coding sequence is operably linked to a transcriptional regulatory element. [Section 10] 10. The nucleic acid construct of paragraph 9, wherein the transcriptional regulatory element is a mammalian promoter that is constitutively active or inducible in brain cells, and optionally the promoter is the human synapsin I promoter. [Section 11] A host cell comprising the nucleic acid construct of item 9 or 10. [Section 12] Item 12. The host cell of Item 11, wherein the host cell is a human cell. [Section 13] 12. The host cell of paragraph 11, wherein the host cell is a brain cell or a pluripotent stem cell, and the stem cell is optionally an embryonic stem cell or an induced pluripotent stem cell (iPSC). [Section 14] A recombinant virus comprising the nucleic acid construct of item 9 or 10. [Section 15] 15. The recombinant virus of clause 14, wherein the recombinant virus is a recombinant adeno-associated virus (AAV) of serotype 6 or 9, as appropriate. [Section 16] A pharmaceutical composition comprising the nucleic acid construct of paragraph 9 or 10, or the recombinant virus of paragraph 14 or 15, and a pharmaceutically acceptable carrier. [Section 17] A method for inhibiting expression of prion protein (PrP) in mammalian brain cells, the method comprising introducing the fusion protein of any one of Items 1 to 6 into the cells by, as appropriate, introducing the nucleic acid construct of Items 7 or 8 or the recombinant virus of Items 14 or 15, thereby inhibiting expression of PrP in the cells. [Section 18] 18. The method of paragraph 17, wherein the mammalian brain cells are human, non-human primate, rodent, or mouse cells. [Section 19] Item 19. The method of item 17 or 18, wherein the mammalian brain cells are neurons, glial cells, ependymal cells, or neuroepithelial cells. [Section 20] 20. The method according to any one of Items 17 to 19, wherein the cells are in the brain of a patient suffering from or at risk of developing a prion disease, and the prion disease is, as appropriate, a familial, sporadic, or acquired prion disease. [Section 21] 21. The method of claim 20, wherein the prion disease is Creutzfeldt-Jakob disease (CJD), sporadic CJD, variant CJD, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), sporadic fatal insomnia (sFI), kuru, or variably protease-sensitive prionopathy (VPSPr). [Section 22] 22. The method according to any one of items 17 to 21, comprising introducing the recombinant virus according to item 14 or 15 into the cell. [Section 23] A method for treating or preventing a neurodegenerative disease in a patient, comprising administering to the patient the recombinant AAV described in paragraph 15. [Section 24] 24. The method of claim 23, wherein the neurodegenerative disease is a prion disease, and optionally the prion disease is a familial, sporadic, or acquired prion disease. [Section 25] 25. The method of paragraph 23 or 24, wherein the AAV is introduced into the patient via intravenous, intrathecal, intracerebroventricular, intracisternal, or intrathalamic injection, or injection into any brain region. [Section 26] 26. The method of claim 24 or 25, wherein the prion disease is Creutzfeldt-Jakob disease (CJD), sporadic CJD, variant CJD, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), sporadic fatal insomnia (sFI), kuru, or variably protease-sensitive prionopathy (VPSPr). [Section 27] A fusion protein according to any one of Items 1 to 8, a nucleic acid construct according to Item 9 or 10, or a recombinant virus according to Item 14 or 15, for use in the method according to any one of Items 17 to 26. [Section 28] Use of the fusion protein of any one of Items 1 to 8, the nucleic acid construct of Items 9 or 10, or the recombinant virus of Items 14 or 15 for the manufacture of a medicament for treating a patient in the method of any one of Items 18 to 25. [Example]

[0055] Example 1: Screening for ZFP-TFs To identify ZFP-TFs that repress the expression of the mouse PRNP gene, we designed and screened a library of 384 ZFP-TFs predicted to bind to 15- to 22-bp sequences in the region of the mouse PRNP gene spanning approximately 500 bp upstream of the transcription start site (TSS) to approximately 500 bp downstream of the TSS (Figures 1 and 2). The target regions of the ZFP-TFs are indicated by arrows in the two figures, and the direction of the arrows indicates the DNA strand (5' to 3') to which the ZFP-TFs bind. 192 of the ZFP-TFs were parent proteins, and the remaining half were mutants of the parent proteins containing three R-to-Q mutations (Miller et al., NatBiotechnol. (2019) 37(8):945-52). In this study, the KRAB domain sequence (SEQ ID NO: 262) was used as a transcriptional repressor and fused to the C-terminus of the ZFP domain.

[0056] Messenger RNA encoding each ZFP-TF was prepared and dispensed into 96-well plates at six dilutions. Mouse Neuro2a cells were transfected with the mRNA using an Amaxa® Nucleofector® instrument (Lonza, Switzerland). After 24 hours, total RNA was extracted from the cells, and the expression of PRNP and two reference genes (ATP5b and EIF4A2) was monitored using real-time RT-qPCR. For this purpose, the cells were lysed and reverse-transcribed using a C2CT kit according to the manufacturer's instructions. The expression level of PRNP was measured using TaqMan quantitative polymerase chain reaction (qPCR). PRNP expression levels were normalized to the geometric mean expression levels of the housekeeping genes EIF4A2 and ATP5B. Mock transfection and transfection with a ZFP-TF known not to target PRNP were used as negative controls.

[0057] Figures 3A-D show normalized PRNP expression 24 hours after administration of the indicated amounts of ZFP-TF in mRNA form (from left to right: 3 ng, 10 ng, 30 ng, 100 ng, 300 ng, or 1000 ng of the indicated ZFP-TF mRNA dose). The ability of ZFP-TF to suppress PRNP expression is also shown by the color gradient in Figure 2 (more potent ZFP-TFs are darker).

[0058] Of the 384 ZFP-TFs, 36 were selected for further study. The mouse PRNP genomic sequences targeted by these 36 ZFP-TFs, as well as the DNA-binding amino acid sequences of the six zinc fingers in the ZFP-TFs, are shown in Figure 4. The full amino acid sequences of the corresponding ZFPs are shown in Figure 9A. The activity of each of these 36 ZFP-TFs in Neuro2a cells is shown in Figure 5. The data in Figure 5 indicate that ZFP-TFs ##81185, 81187, 81189, 81193, 81199, 81201, 81208, 81210, 81228, 81230, 81234, 81240, 81244, 81278, 81282, 81295, 81303, 81309, and 81312 are particularly potent, demonstrating a dose-dependent inhibitory effect on mouse PRNP gene expression.

[0059] Example 2: PRNP-inhibitory activity of selected ZFP-TFs in primary neurons Next, the activity of the selected 36 ZFP-TFs was tested in primary mouse cortical neurons. Primary mouse cortical neurons (MCN; Gibco) were cultured according to the manufacturer's protocol. The coding sequences of ZFP-TFs were cloned into recombinant AAV2 / 6 vectors using the human SYN1 promoter to promote expression. The viruses were produced in HEK293T cells, purified using a CsCl density gradient, and titrated by real-time qPCR according to methods known in the art. The purified viruses were used to culture primary MCNs on DIV2 at 1x10 2 vg / cell, 3x10 2 vg / cell, 1x10 3 vg / cell, 3x10 3vg / cell, 1x10 4 vg / cell, or 3x10 4 After 7 days, total RNA was extracted from neurons, and the expression of PRNP mRNA and three reference genes (ATP5b, EIF4A2, and GAPDH) was monitored using real-time RT-qPCR.

[0060] The data show that all 36 selected ZFP-TFs exhibited potent dose-dependent inhibitory activity on PRNP expression in mouse neurons (FIGS. 6A and 6B).

[0061] Example 3: Off-target activity of ZFP-TFs of mouse PRNP To assess the off-target effects of mouse PrnpZFP-TFs on broad gene expression, we performed microarray experiments using total RNA isolated from primary mouse cortical neurons treated with AAV6 encoding a representative prion ZFP-TF.

[0062] Primary mouse cortical neurons were purchased from Gibco. Cells were plated at 200,000 cells / well on poly-D-lysine-coated 24-well plates and maintained using Gibco Neurobasal Medium (containing GlutaMAX® I supplement, B27 supplement, and penicillin / streptomycin) according to the manufacturer's instructions. Forty-eight hours after plating (at DIV2), the cells were infected with AAV6 at a multiplicity of infection (MOI) of 3E3 VG / cell and harvested 7 days later (DIV9; 50% medium changes were performed every 3–4 days). RNA isolation and microarray analysis were then performed.

[0063] Off-target analysis was performed using the GeneTitan® platform (Clariom S kit) according to the manufacturer's instructions. The assay results were analyzed using TAC software. Genes were considered differentially regulated if the FDR-corrected p-value was 0.05 or less. ZFP-TFs known to have minimal off-target activity and mock transfections were used as negative controls.

[0064] Figure 7A shows the microarray results of 36 representative mouse PRNP ZFP-TFs tested in primary mouse cortical neurons. A wide range of off-target specificity was observed, with some ZFP-TFs exhibiting very little or no off-target activity. Figure 7B shows the number of dysregulated events for each ZFP-TF.

[0065] Example 4: Activity of human PRNP ZFP-TFs in human iPSC-derived neurons Twelve ZFP-TFs designed to target human PRNP were tested in human iPSC-derived GABAergic neurons (Cellular Dynamics International). The human PRNP genomic sequence targeted by these 12 ZFP-TFs, as well as the DNA-binding amino acid sequences of the zinc fingers in the ZFP-TFs, are shown in Figure 8A. The full amino acid sequences of the corresponding ZFPs are shown in Figure 9B. The cells were plated at a density of 40,000 cells per well in 96-well plates coated with poly-L-ornithine and laminin and maintained according to the manufacturer's instructions. 48 hours after plating, the cells were transfected with AAV6 expressing the desired ZFP-TFs at six different MOIs (1E3, 3E3, 1E4, 3E4, 1E5, and 3E5). Transduced cells were maintained for up to 33 days (with 50–75% medium changes every 3–5 days). Cells were harvested 31 days after AAV infection.

[0066] Harvested cells were lysed, and reverse transcription was performed using the C2CT kit according to the manufacturer's instructions. PRNP expression levels were measured using TaqMan quantitative polymerase chain reaction (qPCR). PRNP expression levels were normalized to the geometric mean of the expression levels of the housekeeping genes EIF4A2, ATP5B, and GAPDH. Mock infection was used as a negative control.

[0067] Dose-dependent inhibition was demonstrated using ZFP-TFs targeting human PRNP. While maximal inhibition of over 99% was achieved, ZFP-TFs with lesser degrees of prion inhibition were also identified (e.g., ∼90%, ∼75%, or ∼80% at the highest dose).

[0068] The dose-dependent activity of 12 exemplary ZFP-TFs targeting human PRNP is shown in Figures 8B and 8C. These data demonstrate that the ZFP-TFs exhibit a broad range of prion-inhibitory activity profiles, ranging from approximately 75% to over 99% prion mRNA inhibition at the highest doses tested.

[0069] Example 5: Off-target activity of human PRNPZFP-TF To assess the off-target effects of human PRNPZFP-TFs on broad gene expression, we performed microarray experiments using total RNA isolated from human iPSC-derived neurons treated with AAVs encoding representative human PRNPZFP-TFs.

[0070] Human iPSC-derived neurons were processed as described in Example 4. For microarray analysis, the cells were plated onto poly-L-ornithine and laminin-coated 24-well plates at a density of 260,000 cells per well, transfected with AAV6 expressing the ZFP-TF of interest at 1E5 VG / cell 48 hours after plating, and harvested 19 days after viral transfection. Total RNA was isolated from the harvested cells and used for microarray analysis.

[0071] Off-target analysis was performed using the GeneTitan® platform (Clariom S kit) according to the manufacturer's instructions. Assay results were analyzed using TAC software. Genes were considered differentially regulated if their FDR-corrected p-value was 0.05 or less. ZFP-TFs known to have minimal off-target activity and mock transfections were used as negative controls.

[0072] Figure 8D shows microarray results for 12 human PRNP ZFP-TFs tested in human iPSC-derived neurons. A wide range of off-target specificity was observed, with some ZFP-TFs exhibiting very low or no off-target activity. Figure 8E shows the number of dysregulation events for each ZFP-TF. A wide range of different off-target specificity was observed, with some ZFP-TFs exhibiting very low off-target activity.

Claims

1. A fusion protein comprising a zinc finger protein (ZFP) domain and a transcriptional repressor domain, wherein the ZFP domain binds to a target region in any of SEQ ID NOs: 274-284 in a mammalian prion protein gene (PRNP gene), and the ZFP domain is selected from the group consisting of a nucleotide sequence shown in the following table. Table 1 wherein "^" in the table indicates that the arginine (R) residue at the fourth position upstream of the first amino acid of the indicated helix has been changed to glutamine (Q); and the assigned SEQ ID NO for each sequence is shown in parentheses below the sequence.

2. The fusion protein according to claim 1 , wherein the PRNP gene is a human or non-human primate PRNP gene.

3. The fusion protein according to any one of claims 1 to 2, wherein the transcriptional repressor domain comprises the amino acid sequence of the KRAB domain of KOX1.

4. The fusion protein of any one of claims 1 to 3, wherein the ZFP domain is linked to the transcriptional repressor domain via a peptide linker.

5. A nucleic acid construct comprising a coding sequence for the fusion protein of any one of claims 1 to 4, wherein the coding sequence is operably linked to a transcriptional regulatory element.

6. 6. The nucleic acid construct of claim 5, wherein the transcriptional regulatory element is a mammalian promoter that is constitutively active or inducible in brain cells.

7. A host cell comprising the nucleic acid construct of claim 5 or 6.

8. The host cell of claim 7 , wherein the host cell is a human cell.

9. The host cell of claim 7 , wherein the host cell is a brain cell or a pluripotent stem cell.

10. A recombinant virus comprising the nucleic acid construct of claim 5 or 6.

11. The recombinant virus of claim 10, wherein the recombinant virus is a recombinant adeno-associated virus (AAV).

12. A pharmaceutical composition comprising the nucleic acid construct of claim 5 or 6, or the recombinant virus of claim 10 or 11, and a pharmaceutically acceptable carrier.

13. 12. A pharmaceutical composition for use in a method for inhibiting expression of prion protein (PrP) in mammalian brain cells, the method comprising introducing a nucleic acid construct of claim 5 or 6 or a recombinant virus of claim 10 or 11, thereby introducing a fusion protein of any one of claims 1 to 4 into the cells, thereby inhibiting expression of PrP in the cells.

14. 14. The pharmaceutical composition for use according to claim 13, wherein the mammalian brain cells are human, non-human primate, rodent, or mouse cells.

15. 15. The pharmaceutical composition for use according to claim 13 or 14, wherein the mammalian brain cells are neurons, glial cells, ependymal cells, or neuroepithelial cells.

16. The pharmaceutical composition for use according to any one of claims 13 to 15, wherein the cells are in the brain of a patient suffering from or at risk of developing a prion disease.

17. 17. The pharmaceutical composition for use according to claim 16, wherein the prion disease is Creutzfeldt-Jakob disease (CJD), sporadic CJD, variant CJD, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), sporadic fatal insomnia (sFI), kuru, or variably protease-sensitive prionopathy (VPSPr).

18. The pharmaceutical composition for use according to any one of claims 13 to 17, wherein said method comprises introducing a recombinant virus according to claim 10 or 11 into said cells.

19. A pharmaceutical composition for use in a method for treating or preventing a neurodegenerative disease in a patient, comprising administering to the patient the recombinant AAV of claim 11.

20. 20. The pharmaceutical composition for use according to claim 19, wherein the neurodegenerative disease is a prion disease.

21. 21. The pharmaceutical composition for use according to claim 19 or 20, wherein the AAV is introduced into the patient via intravenous, intrathecal, intracerebroventricular, intracisternal, or intrathalamic injection, or injection into any brain region.

22. 21. The pharmaceutical composition for use according to claim 20, wherein the prion disease is Creutzfeldt-Jakob disease (CJD), sporadic CJD, variant CJD, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), sporadic fatal insomnia (sFI), kuru, or variably protease-sensitive prionopathy (VPSPr).

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