LOW DOSE siRNA TREATMENTS OF HUNTINGTON'S DISEASE

Abstract

A composition for treating Huntington's disease (HD) by reducing endogenous Huntingtin (HTT) levels in a target includes a siRNA including a siRNA sequence selected from SEQ ID NOs:47 to 149, and a carrier peptide comprising a peptide sequence of SEQ ID NO:1. The carrier peptide may target the nicotinic acetylcholine receptor (nAChR) of neuronal cells in order to deliver the siRNA across the blood-brain barrier (BBB). In some embodiments, the siRNA may be conjugated with the carrier peptide. A method of reducing endogenous HTT levels to treat HD in the target may include administering a composition including a siRNA sequence selected from SEQ ID NOs: 47 to 149 to the target.

Description

LOW DOSE siRNA TREATMENTS OF HUNTINGTON’S DISEASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 63 / 592,542, filed October 23, 2023, the entire disclosure of which is incorporated herein by reference.BACKGROUND

[0002] Huntington’s disease (HD) is a progressive and fatal neurodegenerative disease associated with emotional disturbances, uncontrolled movements (chorea), and cognitive decline. Certain destabilizing mutations in the human Huntington gene, which encodes for the Huntingtin (HTT) protein, have been implicated in development of HD.

[0003] While palliative therapies are available to help manage and reduce the severity of certain symptoms of HD, to this date, no effective therapies have been developed to treat the root cause of the disease or to alleviate cognitive decline.

[0004] Small interfering RNA (siRNA) can be used to effectively silence or modulate genes and repress protein expression. As such, siRNA targeted to knockdown (e.g., reduction) or knockout (e.g., substantially complete reduction) of mutated HTT would be of interest as a potential biopharmaceutical treatment for HD. However, therapeutic use of siRNA has been limited by poor bioavailability, non-specific targeting, and immunogenic effects. Design of suitable siRNA sequences and delivery of such siRNA to intracellular targets, particularly those in the central nervous system (CNS), remain challenging.SUMMARY

[0005] One or more aspects of embodiments of the present disclosure are directed toward siRNA compositions and methods for reducing endogenous Huntingtin (HTT) levels in a target, for example, to treat Huntington’s disease (HD). In some embodiments, the siRNA compositions are conjugated with a carrier to facilitate intravascular administration and delivery to targets across the blood brain barrier (BBB).

[0006] One or more embodiments of the present disclosure provide a composition for treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein and / or mRNA levels in a target, the composition including: a siRNA including a siRNA sequence selected from SEQ ID NOs:18 to 149 or a variant thereof; and a carrier peptide including a base sequence of SEQ ID NO:1 or a variant thereof.

[0007] In some embodiments, the siRNA sequence includes a common sub-sequence selected from SEQ ID NOs: 18 to 31 or any sub-set of 2 to 18 residues within SEQ ID NO:46.

[0008] In some embodiments, the siRNA sequence is a 19-mer selected from SEQ ID NOs:47 to 149. In some embodiments, the siRNA sequence is selected from SEQ ID NQs:90, 61 , 68, 47, and 51. In some embodiments, the siRNA binds to Exon 1 of HTT mRNA, and the siRNA sequence is selected from SEQ ID NOs:47 to 52.

[0009] In some embodiments, the siRNA includes one or more chemical modifications selected from 2'-O-methylation of A, G, and / or U; 2'-fluorination of C and / or U; and substitution of phosphorothioate in a phosphodiester backbone unit.

[0010] In some embodiments, the carrier peptide includes a linker sequence of SEQ ID NO: 14, prepended to the base sequence. In some embodiments, the carrier peptide is selected from SEQ ID NOs: 16 and 17.

[0011] In some embodiments, the siRNA is conjugated with the carrier peptide. In some embodiments, a ratio of the siRNA and the carrier peptide is 1 :5 to 1 :20.

[0012] One or more embodiments of the present disclosure provide a method of treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein or mRNA levels in a target, the method including: administering a composition including a siRNA sequence selected from SEQ ID NOs:18 to 149 or a variant thereof.

[0013] In some embodiments, the siRNA sequence includes a common sub-sequence selected from SEQ ID NOs: 18 to 31 or any sub-set of 2 to 18 residues within SEQ ID NO:46.

[0014] In some embodiments, the siRNA sequence includes a 19-mer selected from SEQ ID NOs:47 to 149. In some embodiments, the siRNA sequence is selected from SEQ ID NOs: 90, 61 , 68, 47, and 51. In some embodiments, the siRNA binds to Exon 1 of HTT mRNA, and the siRNA sequence is selected from SEQ ID NOs: 47 to 52.

[0015] In some embodiments, the siRNA includes one or more chemical modifications selected from 2'-O-methylation of A, G, and / or U; 2'-fluorination of C and / or II; and substitution of phosphorothioate in a phosphodiester backbone unit.

[0016] One or more embodiments of the present disclosure provide a method of treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein or HTT mRNA levels, the method including: administering the above-described composition to a target.

[0017] In some embodiments, the composition is prepared by mixing the siRNA or the variant thereof and the carrier peptide or the variant thereof in a ratio of 1 :4 to 1 :20 in asolution that is acceptable for parenteral administration. In some embodiments, the solution is a saline solution including 0.1 wt% to 1 wt% PEG 8000. In some embodiments, the solution is a dextrose solution including 0.01 wt% to 1.5 wt% Polysorbate 80.

[0018] In some embodiments, the administering of the composition occurs in a human subject. In some embodiments, the target is a cell selected from the group consisting of neurons, neuronal cells, brain cells, glial cells, astrocytes, neuronal supporting cells, and cells of the central nervous system.

[0019] In some embodiments, the method further includes conjugating the siRNA to the carrier peptide, prior to administering the composition. In some embodiments, the siRNA is conjugated with the carrier peptide via a linker sequence selected from SEQ ID NOS:14 and 15.

[0020] In some embodiments, the HTT mRNA levels are decreased by 15% or greater, for example 25% or greater, or 15% to 65%.

[0021] One or more embodiments of the present disclosure provide a composition for treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein and / or mRNA levels in a target, the composition including: a 19 to 27-mer siRNA comprising a common sub-sequence selected from SEQ ID NOs:18 to 31 or any sub-set of 2 to 18 residues within SEQ ID NO:46; and a carrier peptide comprising a base peptide sequence of SEQ ID NO:1 or a variant thereof.

[0022] The siRNA is complementary to a HTT mRNA sequence. In some embodiments, the siRNA is a 19 to 21 -mer. In some embodiments, the siRNA is a 19-mer.

[0023] In some embodiments, the siRNA sequence includes one or more chemical modifications selected from 2'-O-methylation of A, G, and / or U; 2'-fluorination of C and / or II; and substitution of phosphorothioate in a phosphodiester backbone unit.

[0024] In some embodiments, the carrier peptide further comprises a linker sequence of SEQ ID NO: 14, prepended to the base sequence. In some embodiments, the carrier peptide is selected from SEQ ID NOs:16 and 17. In some embodiments, the siRNA is conjugated with the carrier peptide via the linker sequence.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a flowchart diagram summarizing the in silico procedure used to design and select siRNA sequences for Huntingtin (HTT) knockdown.

[0026] Figure 2 is a bar chart summarizing the fold-change in measured mRNA levels in SHSY5Y cells treated with Peptide 1 (SEQ ID NO:16):siRNA or Peptide 4 (SEQ IDN0:17):siRNA conjugates (siRNAs 1 -5, SEQ ID NOS: 90, 61 , 68, 47, and 51 ), as normalized against the change in HTT mRNA in a DMEMF12 growth media-only control.

[0027] Figure 3 is a bar chart summarizing the fold-change in measured mRNA levels in SHSY5Y cells treated with Peptide 1 (SEQ ID NO:16):siRNA conjugates (siRNAs 3, 4 (SEQ ID NOs: 68 and 47, respectively) and NTsiRNA), as normalized against synuclein alpha (SNCA) mRNA levels and relative to a dextrose-0.6% polysorbate 80 only control.

[0028] Figure 4 is a bar chart summarizing the fold-change in measured HTT mRNA expression levels in the hippocampus (HP) and striatum (ST) of wild-type C57bl6 mice treated with Peptide 4 (SEQ ID NO:17):siRNA3 (SEQ ID NO:68) or Peptide 4 (SEQ ID NO:17):siRNA4 (SEQ ID NO:47) conjugates, as normalized against synuclein alpha (SNCA) mRNA levels and relative to a saline control.

[0029] Figure 5 is a bar chart summarizing the fold-change in measured HTT mRNA expression levels in the cortex (CTX) and striatum (ST) of BACHD mice expressing human HTT and treated with Peptide 4 (SEQ ID NO:17):siRNA3 (SEQ ID NO:68) or Peptide 4 (SEQ ID NO:17):siRNA4 (SEQ ID NO:47) conjugates, as normalized against SNCA mRNA levels and relative to a saline control.

[0030] Figure 6 is a bar chart summarizing the fold-change in measured HTT mRNA expression levels in the hippocampus (HP) and striatum (ST) of wild-type C57bl6 mice treated with Peptide 4 (SEQ ID NO:17):siRNA3 (SEQ ID NO:68) or Peptide 1 (SEQ ID NO:16):siRNA3 (SEQ ID NO:68) conjugates, as normalized against SNCA mRNA levels and relative to a saline control.

[0031] Figure 7A is a bar chart summarizing the fold-change in measured HTT mRNA expression levels in the striatum (ST) of Q175 mice and treated with varying doses of a Peptide 1 (SEQ ID NO:16):siRNA3 (SEQ ID NO:68) conjugate, as normalized against SNCA mRNA levels and relative to a dextrose / polysorbate 80 control.

[0032] Figure 7B is a bar chart summarizing the non-normalized fold-change in measured HTT mRNA expression levels in the striatum (ST) of Q175 mice and treated with varying doses of a Peptide 1 (SEQ ID NO:16):siRNA3 (SEQ ID NO:68) conjugate relative to a dextrose / polysorbate 80 control.

[0033] Figure 8A is a bar chart summarizing the fold-change in measured HTT mRNA expression levels in the cortex (CTX) of Q175 mice and treated with varying doses of a Peptide 1 (SEQ ID NO:16):siRNA3 (SEQ ID NO:68) conjugate, as normalized against SNCA mRNA levels and relative to a dextrose / polysorbate 80 control.

[0034] Figure 8B is a bar chart summarizing the non-normalized fold-change in measured HTT mRNA expression levels in the cortex (CTX) of Q175 mice and treated with varying doses of a Peptide 1 (SEQ ID NO:16):siRNA3 (SEQ ID NO:68) conjugate relative to a dextrose / polysorbate 80 control.

[0035] Figure 9A is a bar chart summarizing the fold-change in measured HTT mRNA expression levels in the hippocampus (HP) of Q175 mice and treated with varying doses of a Peptide 1 (SEQ ID NO:16):siRNA3 (SEQ ID NO:68) conjugate, as normalized against SNCA mRNA levels and relative to a dextrose / polysorbate 80 control.

[0036] Figure 9B is a bar chart summarizing the non-normalized fold-change in measured HTT mRNA expression levels in the hippocampus (HP) of Q175 mice and treated with varying doses of a Peptide 1 (SEQ ID NO:16):siRNA3 (SEQ ID NO:68) conjugate and relative to a dextrose / polysorbate 80 control.DETAILED DESCRIPTIONSelected definitions

[0037] Abbreviations for nucleic acids and associated structures are used throughout this disclosure and follow the standard IUPAC nomenclature known in the art. For example, as would be understood by those of ordinary skill in the art, Adenine is Ade or A, Cytosine is Cyt or C, Guanine is Gua or G, Thymine is Thy or T, and Uracil is Ura or U.

[0038] Abbreviations for amino acids are used throughout this disclosure and follow the standard nomenclature known in the art. For example, as would be understood by those of ordinary skill in the art, Alanine is Ala or A; Arginine is Arg or R; Asparagine is Asn or N; Aspartic Acid is Asp or D; Cysteine is Cys or C; Glutamic acid is Glu or E; Glutamine is Gin or Q; Glycine is Gly or G; Histidine is His or H; Isoleucine is He or I; Leucine is Leu or L; Lysine is Lys or K; Methionine is Met or M; Phenylalanine is Phe or F; Proline is Pro or P; Serine is Ser or S; Threonine is Thr or T; Tryptophan is Trp or W; Tyrosine is Tyr or Y; and Valine is Vai or V.

[0039] As used herein, the term “carrier peptides” may refer to various peptides, including those described by SEQ ID NOS: 16 and 17, combinations of one of SEQ ID NOS:1 to 7 and 9 to 13 with one of SEQ ID NOS: 14 and 15, and variants thereof, that can be used as targeting or “carrier” molecules to facilitate delivery of an active molecule or effector agent to a target. For example, delivery of an effector agent to a target may be limited when the effector agent is administered alone, but may be increased in the presence of the carrier peptide, for example when the effector agent is conjugated with the carrier peptide. It will be understood that alternative carrier peptides or carrier molecules may be substituted for the specifically disclosed peptide sequences to thereby provide additional embodiments.

[0040] As used herein, the term "effector agent" refers to any molecule that imparts an effect on a target inside the blood brain barrier (BBB), for example, within tissues of the central nervous system (CNS) or brain. The effector agent may include a siRNA sequence, including those siRNA sequences described herein and variants thereof. Insome embodiments, the effector agent may further include, for example: short hairpin or stem loop RNA (shRNA); microRNA, double stranded RNA (dsRNA); strand template RNA (stRNA); oligonucleotides (DNA or RNA); modified oligonucleotides (DNA or RNA); aptamers; analogs and combinations of DNA and RNA; antisense oligomers (ASO); triplex-forming oligonucleotides (TFO); genes; peptides including antibody and antigen fragments; proteins, including antibodies and antigens; small chemical molecules; large chemical molecules; viral particles; liposomes; endosomes; exosomes; nanoparticles; dendrimers such as poly(amidoamine) (PAMAM); positron emission tomography (PET) ligands; eukaryotic cells; prokaryotic cells; microspheres; nanogels; and / or bionanocapsules. The effector agent can be conjugated or linked to a carrier, such as a carrier peptide or carrier peptide variant.

[0041] As used herein, “small interfering RNA,” “short interfering RNA,” “silencing RNA,” and “siRNA” are used interchangeably to refer to a non-coding, double stranded RNA (dsRNA) molecule, typically 19-27 (or e.g., 21 -25) base pairs in length. Each strand is phosphorylated at its 5' end, and is hydroxylated at its 3' end. Once delivered to a suitable target, the siRNA is incorporated into an RNA-lnduced Silencing Complex (RISC) and then unwound to provide a single stranded siRNA capable of binding a complementary mRNA sequence. The RISC subsequently binds to and cleaves the complementary mRNA, which can then no longer be translated into protein. References to siRNAs and / or siRNA variants will be understood to encompass both the double-stranded forms and single-stranded (ssRNA) counterparts (e.g., portions) of the double stranded siRNA, unless otherwise clear from the context. In certain embodiments, for example when referring to pharmaceutical compositions and methods of treatment, these terms may refer to the single-stranded (ssRNA) form that is selectively incorporated into the RISC. Further, the terms explicitly include siRNA molecules that have been chemically modified as described below, for example, to increase their stability and / or bioavailability, and / or to decrease their immunostimulatory effects.

[0042] As used herein, “antisense oligonucleotide (ASO)” and “antisense therapeutic” are used interchangeably to refer to a non-coding, single stranded oligonucleotide sequence, typically 16-27 (or e.g., 18-22) nucleotides in length. The ASO sequence is selected to be complementary to and capable of binding or hybridizing with a target mRNA sequence. Once delivered to a suitable target (e.g., once co-localized with the target mRNA sequence), the hybridization of the ASO with the mRNA may trigger cleavage of the mRNA via one or more cellular mechanisms. For example, an ASO based on single stranded DNA (ssDNA) may activate one or more RNAase H enzymes, while an ASO based on single stranded RNA (ssRNA) may activate the RNA-induced silencing complex (RISC). Once the mRNA is cleaved by either type of enzyme, the mRNA can no longer be translated into protein. References to ASOs and / or ASO variants will be understood to encompass DNA-based as well as RNA-based counterparts or equivalents, unless otherwise clear from the context. Further, the terms explicitly include ASO molecules that have been chemically modified as described below, for example, toincrease their stability and / or bioavailability, and / or to decrease their immunostimulatory effects.

[0043] As used herein, “triplex-forming oligonucleotides (TFO)” and “triplex-forming oligonucleotide therapeutics” refer to a non-coding, single stranded oligonucleotide sequence, typically 10-30 nucleotides in length, or a mimic thereof, such as peptide nucleic acids (PNAs) and / or polyamides. The oligonucleotide sequence or TFO is selected to be capable of binding within the major groove of a target genomic DNA sequence to thereby form a triple helix (triplex) structure. When binding of the TFO outcompetes binding of transcription factors or polymerases to the DNA (e.g., within regulatory sequences or coding sequences, respectively), gene transcription can be controlled or blocked. Additional information about TFOs is disclosed in e.g., Ohkubo et al., “Synthesis and triplex-forming properties of oligonucleotides capable of recognizing corresponding DNA duplexes containing four base pairs,” Nucl. Acids Res., 2015, 43(12), 5675-5686, and Jain et al, “DNA Triple Helices: biological consequences and therapeutic potential,” Biochimie. 2008, 90(8), 1117-1130, the entire content of each of which is incorporated herein by reference.

[0044] As used herein, a "fragment" of a polynucleotide or polypeptide refers to a smaller set (e.g., a contiguous sub-set) of nucleotides or peptides with respect to the referenced polynucleotide or polypeptide. The length of the fragment may range from two to N-1 nucleotides or peptides (where N is the number of residues in the referenced parent), unless further specified. For example, a fragment of a carrier peptide (described below) may refer to a peptide including any sub-sequence or truncation of the reference carrier peptide. In some embodiments, the fragment may retain at least about 80%, 85%, 90%, 95%, or 99% of the same biochemical functions as the reference. The peptide fragment may be identical to the corresponding sub-sequence in the reference carrier peptide, or may further include additional amino acid mutations or chemical derivatizations (e.g., may also be a mutant or a chemical derivative), as described herein.

[0045] As used herein, a “mutant” of a peptide (e.g., a polypeptide) refers to a polypeptide that includes one or more point mutations (amino acid mutations, e.g., residue additions, substitutions, and / or deletions) compared to a reference polypeptide, but still achieves similar biological functions. For example, a "mutant " of a polypeptide or carrier peptide may have a sequence and / or structural homology of at least about 80%, 85%, 90%, 95%, or 99% to the reference polypeptide; and may retain at least about 80%, 85%, 90%, 95%, or 99% of the same biochemical functions as the reference. The term “amino acid mutations” refers to one or more residue additions, substitutions, and / or deletions with respect to a reference polypeptide.

[0046] As used herein, a "chemical derivative" of a peptide (e.g., a polypeptide) is a modification of a referenced peptide to include a chemical modification at one or more positions, for example, to improve stability, mediate intermolecular interactions, facilitate visual localization, etc. The modification may include replacement of standard amino acidswith non-standard residues and / or addition of new functional groups (e.g., methylation at a hydroxyl group). Non-limiting examples of chemical modifications of peptides include the products of techniques such as ubiquitination, methylation, fluorination, fluorescent labeling, phosphorescent labeling, and PEGylation (e.g., derivatization with ubiquitin, methyl, fluorine, a fluorescent dye, a phosphorescent dye, and polyethylene glycol, respectively). The point(s) of modification (e.g., position or starting functional group) is (are) not particularly limited, and for example, may include the N- and C-termini and suitable amino acid side chain groups. The term “chemical derivatizations” refers to one or more of the above-described chemical modifications.

[0047] As used herein, a "chemical derivative" of a polynucleotide is a modification of a referenced polynucleotide to include a chemical modification at one or more positions, for example, to improve stability, mediate intermolecular interactions, facilitate visual localization, etc. The modification may include replacement of reference functional groups (e.g., replacement of a phosphodiester with a phosphorothioate) and / or addition of new functional groups (e.g., methylation at a hydroxyl group). The point(s) of modification is (are) not particularly limited, and for example, may include the phosphates and ribose hydroxides of the backbone, and the aromatic rings of the nucleobases. Examples of such chemical modifications include moieties added to the 2’ position of one or more ribose (sugar) units; substitution or modification of internucleotide phosphodiesters in the backbone with phosphorothioates, phosphorodithioates, triazoles, amides, boranophosphates, etc.; nucleobase modifications to incorporate fluorescent groups or replace standard bases with difluorotoluene, dichlorobenzene, halogenated variants, etc.; and modification of terminus or conjugate groups with aromatic compounds, PEG, triphosphate groups, etc. Selected example modifications are disclosed in Selvam et al., “Therapeutic potential of chemically modified siRNA: Recent trends,” Chem. Biol. Drug. Des. 2017, 90(5): 665-678, and in Deleavey et al., “Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing,” Chemistry & Biology, 2012, 19, 937-954, the entire content of each of which is incorporated herein by reference and is attached to the priority application (U.S. Provisional Application No. 63 / 592,542, filed October 23, 2024 in the name of Ophidion Inc.) as Appendices A and B. In particular, the chemical modifications described in the context of siRNA may be applied to other types of nucleotides.

[0048] A molecule may be described as being a “functional equivalent” of another molecule if both molecules possess a similar biological activity (e.g., fulfill at least about 80%, 85%, 90%, 95%, or 99% of the same biological or biochemical role(s)), and do so at similar levels (e.g., the functional equivalent may possess at least about 80%, 85%, 90%, 95%, or 99% of the biochemical activity of the reference, and in some embodiments greater than 100% of the biochemical activity of the reference). The molecule may possess a similar activity as quantified above and be considered a functional equivalent as that term is used herein, even if, for example, the sequence of residues (e.g., primary structure) is not identical, or different chemical modifications are made to a shared basic sequence. However, it will be understood that a functional equivalent may be a fragment,mutant, or chemical derivative (as those terms are defined herein) of a reference, and may therefore possess certain structural similarities with respect to the reference.

[0049] As used herein, fragments, mutants, chemical derivatives, and functional equivalents (e.g., as defined above) of a carrier peptide may be collectively referred to as “carrier peptide variants,” “carrier-derived peptides,” or “Peptide n-derived peptides,” where “n” is any integer and is used to refer to a disclosed peptide having a specified sequence. Fragments, chemical derivatives, and functional equivalents (e.g., as defined above) of a siRNA polynucleotide may be collectively referred to as “siRNA variants” or “siRNA-derived sequences.” References to a sequence identification number (SEQ ID NO) will be understood as disclosing and encompassing variants thereof, for example, variants having 80% to 99% sequence identity (e.g., homology or similarity) when compared to the named sequence, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity.

[0050] As used herein, "conjugated," "linked," and "complexed" are used interchangeably to describe a state in which two molecules are connectively joined. As used herein, the term "conjugate" or "conjugation" refers to the attachment of two or more entities to form one entity (molecule). For example, embodiments of the present disclosure provide for a composition including a siRNA effector agent conjugated with a carrier peptide. The attachment can be by means of chemical modification, protein fusion, covalent bonds, or non-covalent bonds (e.g., ionic or intramolecular attractions), and in some embodiments may include the use of intermediary linkers, including peptide linkers, chemical (e.g., functional group or small molecule) linkers, or any means known to one skilled in the art. The joining can be permanent or reversible. For example, linkers can be enzymatically cleavable, acid cleavable, photocleavable, and / or heat sensitive (e.g., thermally cleavable). In some embodiments, several linkers can be included according to the desired properties of each linker and each component in the conjugate. Flexible linkers and linkers that increase the solubility of the conjugates are contemplated for use alone, or in combination with other linkers disclosed herein. Methods for conjugation are well known by persons skilled in the art. For example, peptide linkers can be attached and deployed by prepending and / or appending a DNA sequence encoding the linker to one or more proteins in the conjugate (e.g., as a recombinant protein).

[0051] As used herein, "target" refers to any cell that is intended to receive a carrier peptide and / or an effector agent. The target may be entirely within the central nervous system (CNS) tissue, which is isolated from the intervascular system by the BBB. In other embodiments, the target may be or include cells located in tissues outside the BBB.

[0052] The term "target cells" as used herein may also refer to cells expressing the alpha (a) subunit and / or the beta (P) subunit of the nicotinic acetylcholine receptor (nAChR). Carrier peptides that bind to the a-subunit of the nicotinic acetylcholine receptor, including carrier peptides, can thus be used to selectively bind target cells expressing the a subunit of the nicotinic acetylcholine receptor. Examples of cells that express the asubunit of the nicotinic acetylcholine receptor include, for example, the neurons, glial cells, and endothelial cells comprising the BBB. Target cells of the present disclosure also include cells whose endogenous milieu is separated by the BBB, for example, cells in the CNS, including brain cells, spinal cord cells, glial cells and other cells supporting neurons, e.g., astrocytes or "nursing cells". In some embodiments, the target cells may include any cell expressing the alpha subunit of the nicotinic acetylcholine receptor or a homologue thereof, such as but not limited to neuronal cells in a subject (i.e. in vivo), neuronal cells ex vivo, or cultured neuronal cells (i.e. in vitro) such as, for example primary neuronal cultured cells, or immortalized cell lines expressing alpha and / or beta subunits of the nicotinic acetylcholine receptor either naturally or through stable selection of transfected alpha and / or beta nicotinic acetylcholine receptor constructs. In some embodiments, the target cells are neuronal precursor or neuronal progenitor cells, such as neuronal progenitor stem cells that express an alpha subunit of the nicotinic acetylcholine receptor or a homologue thereof. In some embodiments of the present disclosure, the target is present within a subject, for example a mammalian subject, such as a human subject or patient. In alternative embodiments, the target is ex vivo, and in further embodiments, the target is in a biological sample, for example in vitro.

[0053] In some embodiments, the effector agent can be transported to various target cells or tissues. For example, the effector agent can be transported to any nerve cell, e.g., a nerve cell in the central nervous system, olfactory, or visual system. In some embodiments, the effector agent can also be transported to a neurologically related target cell or tissue, e.g., cells or tissues that interact with or are targets of the nervous system.

[0054] All nucleotides and peptides disclosed herein, including siRNA sequences and carrier peptides, may be constructed by using any suitable method available in the art. For example, peptides may be synthesized using a peptide synthesizer (Applied Biosystems Model 433) or can be synthesized recombinantly by methods well known in the art, such as those described in Merrifield, "Solid Phase Synthesis," J. Am. Chem. Soc., 1963, 83:2149-2154. Nucleotides may be synthesized using an oligonucleotide sequencer or by using in vitro transcription, PCR cassette, or expression vector methods well known in the art. The method by which a peptide or nucleotide of the present disclosure is synthesized does not limit the present disclosure.

[0055] In some embodiments, an effector agent or therapeutic agent, for example an siRNA as disclosed herein, can be prepared to be delivered in a "prodrug" form. The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and / or conditions.

[0056] As used herein, the term "gene" refers to a nucleic acid comprising an open reading frame encoding a polypeptide, and may be inclusive of all coding segment sequences (exons) as well as (optionally) non-coding intervening sequences (introns). For example, "gene" may refer to the coding sequence of a gene product, or the codingsequence together with the non-coding regions of the gene product, including the 5'UTR and 3'IITR regions, introns, and promoters or enhancers of the gene product. As used herein, the term "gene product(s)" may encompass the RNA (e.g., mRNA) transcribed from a gene, and / or the polypeptide (e.g., protein) produced by translation of the RNA. A "promoter" is a region of a genomic nucleic acid sequence at which initiation and rate of transcription are controlled. It can contain elements at which regulatory proteins and molecules can bind (such as RNA polymerase and other transcription factors) to initiate the transcription of a specific nucleic acid sequence. The term "enhancer" refers to a cisacting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence, e.g., to increase transcription of a particular gene product. An enhancer can function in either orientation and can be included upstream or downstream of the promoter.

[0057] Although some of the above definitions generally refer to a single-stranded molecule (oligonucleotide), in some embodiments and contexts the terms and usage may also encompass one or more additional strands that are partially, substantially, or fully complementary to the single-stranded molecule. Thus, a nucleic acid can encompass a double-stranded molecule or a double- or multi-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence. As used herein, a single stranded nucleic acid can be denoted by the prefix "ss", a double stranded nucleic acid by the prefix "ds", and a triple stranded nucleic acid by the prefix "ts."

[0058] The terms "composition" and "pharmaceutical composition" are used interchangeably herein to refer to compositions or formulations administered for therapeutic, diagnostic, or prophylactic purposes, and which usually comprise one or more excipients, such as a pharmaceutically acceptable carrier (e.g., solvent or diluent) in the art that is suitable for administration to mammals, and for example, humans or human cells. Cells that are administered a composition as disclosed herein can be part of a living subject, animal, or human, including a transgenic animal for research purposes. The cells can also be cultured, for example as cells as part of an assay for screening potential pharmaceutical compositions. In addition, compositions for topical administration (e.g., to oral and respiratory mucosa) or oral administration can be provided in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, oral rinses, or powders, as known in the art. The compositions also can include stabilizers, preservatives, lubricants, and other adjuvants and excipients. For additional examples of carriers, stabilizers and adjuvants, see Genarro, AR., Remington: The Science and Practice of Pharmacy with Facts and Comparisons, 21 st Ed., the entire content of which is incorporated herein by reference. Those having ordinary skill in the art are capable of choosing suitable formulations depending on the administration route, as described herein.

[0059] The term "pharmaceutically acceptable carrier" refers to any pharmaceutically acceptable means to mix and / or deliver an effector agent or pharmaceutical composition to a subject. For example, the term may refer to any pharmaceutically acceptablematerial, formulation, or vehicle (such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material) involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. The carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation as well as being compatible with administration to a subject, for example a human. The carrier can be in the form of a solid, semi-solid, or liquid diluent, cream, or capsule. Such pharmaceutical preparations are a further object of some embodiments of the present disclosure. The amount of active compound (effector agent) may be between 0.001-95% by weight of the preparation, for example, between 0.01 -1 % by weight in preparations for parenteral use and for example, between 1 and 50% by weight in preparations for oral administration.

[0060] The compositions as disclosed herein can be administered by any convenient route, including oral, sublingual, parenteral, enteral, mucosal, topical, subcutaneous, intravascular, intravenous, intraarterial, intramuscular, intraperitoneal, transdermal, rectal, vaginal, intranasal, intraocular, intraspinal, or intracerebroventricular. In some embodiments, the compositions as disclosed herein are not topically administered. In some embodiments, the delivery is by oral administration of the composition formulation. In some embodiments, the delivery is by intranasal administration of the composition, especially for use in therapy of the brain and related organs (e.g., meninges and spinal cord). Along these lines, intraocular administration is also possible. In some embodiments, the delivery means is by intravenous (IV) administration of the composition, which is especially advantageous when a longer-lasting IV formulation is desired. Suitable formulations can be found in Remington's Pharmaceutical Sciences, 16th and 18th Eds., Mack Publishing, Easton, Pa. (1980 and 1990), and Introduction to Pharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia (1985), the entire content of each of which is incorporated herein by reference.

[0061] The terms "parenteral administration" and "administered parenterally" as used herein refer to modes of administration other than enteral and topical administration. In some embodiments, the term typically refers to injection, but may include, without limitation, intravenous, intraarterial, intramuscular, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. The phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" as used herein refer to administration of an effector agent, drug, or other material other than directly into the central nervous system, for example, by subcutaneous administration, such that it enters the subject or patient’s system and is subject to metabolism and other like processes.

[0062] In the preparation of pharmaceutical formulations according to or containing embodiments of the present disclosure for oral administration, the effector agent can be mixed with solid, powdered ingredients, such as lactose, saccharose, sorbitol, mannitol,starch, amylopectin, cellulose derivatives, gelatin, or another suitable ingredient as a bulking agent to prepare the desired number of dosage units. The composition may also be mixed with disintegrating agents and / or lubricating agents (such as magnesium stearate, calcium stearate, sodium stearyl fumarate, and / or polyethylene glycol waxes) to aid in ingestion and / or bioavailability. The mixture may then be processed into granules or pressed into tablets.

[0063] The compositions as disclosed herein can be administered in prophylactically or therapeutically effective amounts. A “prophylactically or therapeutically effective amount” refers to an amount necessary to at least partially attain a desired effect, e.g., to delay the onset of, inhibit the progression of, or halt altogether the onset or progression of the particular disease or disorder being treated (e.g., Huntington’s disease). Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, and individual patient parameters including age, physical condition, size, weight, co-morbidities, and concurrent medications or treatment protocols. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose can be administered for medical reasons, psychological reasons or for virtually any other reason.

[0064] In some embodiments, the compositions disclosed herein may be administered to a patient or human subject in an amount of 0.1 mg per kg to 100 mg per kg (mpk) of subject body weight, for example, 0.1 mpk to 50 mpk, 0.2 mph to 20 mpk, 0.3 mpk to 10 mpk, or 0.5 mpk to 5 mpk. The compositions may be administered using any suitable dose volume, for example, a dose volume of 100 pL to 1000 mL, 200 pL to 750 mL, 500 pL to 500 mL, 1 mL to 200 mL, or 5 mL to 100 mL. For example, the compositions may be administered in an amount of 0.1 to 10 mpk and in a dose volume of 100 pL to 5 mL.

[0065] However, in some embodiments, the compositions disclosed herein may be particularly effective in lower doses. Without being bound by any particular theory, and without limiting the scope of the present disclosure, it is believed that since the compositions and methods disclosed herein rely on receptors, high doses of the compositions according to embodiments of the present disclosure may - in some instances - lead to receptor down regulation and limited efficacy. Indeed, it has been surprisingly found that the conjugates and compositions according to the present disclosure can be more effective in lower doses, i.e., doses lower than 0.1 mg per kg (body weight), or lower than 4,000 pg per kg (body weight).

[0066] For example, in some embodiments, the compositions disclosed herein may be administered to an animal subject (e.g., a mouse) in an amount of 1 pg per kg to 4,000 pg per kg (or 0.001 mg per kg to 4 mg per kg) of subject body weight, for example, 1 pg per kg to 3,000 pg per kg, 1 pg per kg to 2,000 pg per kg, 1 pg per kg to 1 ,000 pg per kg, 2 pg per kg to 4,000 pg per kg, 2 pg per kg to 3,000 pg per kg, 2 pg per kg to 2,000 pg per kg,2 g per kg to 1 ,000 pg per kg, 10 pg per kg to 4,000 pg per kg, 10 pg per kg to 3,000 pg per kg, 10 pg per kg to 2,000 pg per kg, 10 pg per kg to 1 ,000 pg per kg, 20 pg per kg to 4,000 pg per kg, 20 pg per kg to 3,000 pg per kg, 20 pg per kg to 2,000 pg per kg, 20 pg per kg to 1 ,000 pg per kg, 40 pg per kg to 4,000 pg per kg, 40 pg per kg to 3,000 pg per kg, 40 pg per kg to 2,000 pg per kg, 40 pg per kg to 1 ,000 pg per kg, 100 pg per kg to 4,000 pg per kg, 100 pg per kg to 3,000 pg per kg, 100 pg per kg to 2,000 pg per kg, 100 pg per kg to 1 ,000 pg per kg, 200 pg per kg to 4,000 pg per kg, 200 pg per kg to 3,000 pg per kg, 200 pg per kg to 2,000 pg per kg, 200 pg per kg to 1 ,000 pg per kg, 350 pg per kg to 4,000 pg per kg, 350 pg per kg to 3,000 pg per kg, 350 pg per kg to 2,000 pg per kg, 350 pg per kg to 1 ,000 pg per kg, 1 pg per kg to 350 pg per kg, 2 pg per kg to 350 pg per kg, 10 pg per kg to 350 pg per kg, 20 pg per kg to 350 pg per kg, 40 pg per kg to 350 pg per kg, 100 pg per kg to 350 pg per kg, 200 pg per kg to 350 pg per kg, 1 pg per kg to 200 pg per kg, 2 pg per kg to 200 pg per kg, 10 pg per kg to 200 pg per kg, 20 pg per kg to 200 pg per kg, 40 pg per kg to 200 pg per kg, 1 pg per kg to 50 pg per kg, 2 pg per kg to 50 pg per kg, 10 pg per kg to 50 pg per kg, 20 pg per kg to 50 pg per kg, 40 pg per kg to 50 pg per kg, 1 pg per kg to 40 pg per kg, 2 pg per kg to 40 pg per kg, 10 pg per kg to 40 pg per kg, 20 pg per kg to 40 pg per kg, 1 pg per kg to 20 pg per kg, 2 pg per kg to 20 pg per kg, 10 pg per kg to 20 pg per kg, 1 pg per kg to 10 pg per kg, 2 pg per kg to 10 pg per kg. For example, in some embodiments, the compositions disclosed herein may be administered to an animal subject (e.g., a mouse) in an amount of 1 pg per kg, 2 pg per kg, 10 pg per kg, 20 pg per kg, 40 pg per kg, 100 pg per kg, 200 pg per kg, 350 pg per kg, 1 ,000 pg per kg, or 2000 pg per kg. It is understood that any and all ranges between the end points and specific amounts described above are also within this disclosure, including any and all sub-ranges subsumed in the ranges above, and other ranges that may span between or across the disclosed ranges, sub-ranges or specific amounts.

[0067] Those of ordinary skill in the art would be capable of discerning the human equivalent dose from the animal (e.g., mouse) doses described above. For example, as would be understood by those of ordinary skill in the art, the same doses (per kg of body weight) described above may be used in human patients when scaling by body weight, or doses 1 .7-fold higher may be used in human patients when scaling by brain weight. In some embodiments, for example, when scaling by brain weight, the compositions disclosed herein may be administered to a human subject or patient in an amount of 1 .7 pg per kg to 6,800 pg per kg of subject body weight, for example, 1 .7 pg per kg to 5, 100 pg per kg, 1 .7 pg per kg to 3,400 pg per kg, 1 .7 pg per kg to 1 ,700 pg per kg, 3.4 pg per kg to 6,800 pg per kg, 3.4 pg per kg to 5,100 pg per kg, 3.4 pg per kg to 3,400 pg per kg, 3.4 pg per kg to 1 ,700 pg per kg, 17 pg per kg to 6,800 pg per kg, 17 pg per kg to 5,100 pg per kg, 17 pg per kg to 3,400 pg per kg, 17 pg per kg to 1 ,700 pg per kg, 34 pg per kg to 6,800 pg per kg, 34 pg per kg to 5,100 pg per kg, 34 pg per kg to 3,400 pg per kg, 34 pg per kg to 1 ,700 pg per kg, 68 pg per kg to 6,800 pg per kg, 68 pg per kg to 5,100 pg per kg, 68 pg per kg to 3,400 pg per kg, 68 pg per kg to 1 ,700 pg per kg, 170 pg per kg to 6,800 pg per kg, 170 pg per kg to 6,800 pg per kg, 170 pg per kg to 3,400 pg per kg, 170 pg per kg to 1 ,700 pg per kg, 340 pg per kg to 6,800 pg per kg, 340 pg per kg to 5, 100 pg per kg, 340 pg per kg to 3,400 pg per kg, 340 pg per kg to 1 ,700 pg per kg, 595 pg per kgto 6,800 g per kg, 595 pg per kg to 6,800 pg per kg, 595 pg per kg to 6,800 pg per kg, 595 pg per kg to 1 ,700 pg per kg, 1 .7 pg per kg to 595 pg per kg, 3.4 pg per kg to 595 pg per kg, 17 pg per kg to 595 pg per kg, 34 pg per kg to 595 pg per kg, 68 pg per kg to 595 pg per kg, 170 pg per kg to 595 pg per kg, 340 pg per kg to 595 pg per kg, 1 .7 pg per kg to 340 pg per kg, 3.4 pg per kg to 340 pg per kg, 17 pg per kg to 340 pg per kg, 34 pg per kg to 340 pg per kg, 68 pg per kg to 340 pg per kg, 1 .7 pg per kg to 85 pg per kg, 3.4 pg per kg to 85 pg per kg, 3.4 pg per kg to 85 pg per kg, 34 pg per kg to 85 pg per kg, 68 pg per kg to 85 pg per kg, 1 .7 pg per kg to 68 pg per kg, 3.4 pg per kg to 68 pg per kg, 17 pg per kg to 68 pg per kg, 34 pg per kg to 68 pg per kg, 1 .7 pg per kg to 34 pg per kg, 3.4 pg per kg to 34 pg per kg, 17 pg per kg to 34 pg per kg, 1 .7 pg per kg to 17 pg per kg, 3.4 pg per kg to 17 pg per kg. For example, in some embodiments, when scaling by brain weight, the compositions disclosed herein may be administered to a human subject or patient in an amount of 1 .7 pg per kg, 3.4 pg per kg, 17 pg per kg, 34 pg per kg, 68 pg per kg, 85 pg per kg, 170 pg per kg, 340 pg per kg, 595 pg per kg, 1 ,700 pg per kg, or 3,400 pg per kg.. It is understood that any and all ranges between the end points and specific amounts described above are also within this disclosure, including any and all sub-ranges subsumed in the ranges above, and other ranges that may span between or across the disclosed ranges, sub-ranges, or specific amounts.

[0068] As used herein, the terms "administering," "introducing," and "providing" are used interchangeably, and according to embodiments of the present disclosure, may refer to the placement of the pharmaceutical composition including a described effector agent or siRNA sequence into an in vivo subject or in vitro culture by a method or route that results in at least partial localization of the agents at a desired site (e.g., target cells and / or molecules). The agents of the present disclosure can be administered by any appropriate route that results in an effective treatment in the subject. siRNA compositions

[0069] Huntingtin (HTT) is a protein encoded by the HTT gene in humans (NCBI Ref. NP_002102) and mice (NP_034544). Although the exact function of HTT has not yet been determined, HTT is known to be highly expressed in neuronal cells and involved in axonal transport. Further, HTT is essential for normal, successful embryonic development. The 5’ end of a normal (wild-type) HTT gene typically includes 5 to 35 CAG repeats. However, mutations in the HTT gene that result in 36 or more CAG repeats (e.g., resulting in transcription of 36 or more N-terminus Glu residues) can cause the resulting mutated HTT protein (mHTT) to be unstable and subject to enzyme cleavage, and / or have altered activity and function. Higher numbers of repeats in mHTT and higher expression of mHTT are associated with earlier onset of Huntington’s disease (HD), as characterized by neuronal damage in the striatum (ST), hippocampus (HP), and cerebral cortex (CTX). The mRNA for HTT (NCBI Ref. NM_002111 ) includes 67 exons, with the CAG repeat region being located in Exon 1 . Embodiments of the present disclosure provide for compositions including siRNA capable of binding to one or more of the 67 HTT mRNA exons, thereby causing knockdown of the HTT protein (or e.g., mHTT protein). The knockdown ordecrease in mHTT protein may therapeutically treat or alleviate the symptoms of Huntington’s disease (HD).

[0070] In some embodiments, a composition for treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) levels includes a siRNA as an effector agent, and a carrier peptide for delivery to a target. Each component is described in more detail below.

[0071] The siRNA may be synthesized and initially provided in a double-stranded form. As referred to herein, the siRNA includes a “sense strand,” which is incorporated into the RISC and binds to HTT mRNA, and an “antisense strand,” which is complementary to the sense strand.

[0072] One or both strands of the siRNA may additionally include a 2-mer 3’ overhang (e.g., a 2-mer sequence at the 3’ end of the strand that is not paired with the complementary strand). In some embodiments, the antisense strand may include a 3’ overhang while the sense strand does not. The 3’ overhang may be selected from any 2- mer nucleobase sequence, such as UU, dUdll, dTdT, or a sequence that would be complementary to the existing sequence on the mRNA target (as discussed below) and in some embodiments may be UU.

[0073] In some embodiments, the siRNA included in the siRNA compositions may be 19 to 27 nucleobases in length (e.g., may be a 19-mer to 27-mer), exclusive of any 2-mer 3’ overhangs. In some embodiments, for example, the siRNA may be a 19-mer to 25-mer, or a 19-mer, 20-mer, or 21 -mer. In some embodiments, the siRNA may be a 19-mer.

[0074] Certain HTT mRNA sub-sequences that are as long as or longer than the siRNA according to embodiments of the present disclosure (e.g., 19 bases or longer) may serve as suitable mRNA target regions for siRNA-induced binding and transcriptional arrest. Such target regions may be located within various exons of the HTT mRNA. In some embodiments, the target region within the HTT mRNA may be located within exon 1 , 2, 3, 6, 6-7, 8, 8-9, 12 to 13, 17, 27, 30, 35, 36, 36-37, 42, 42-43, 54, 56, or 67, where the notation “i-j” indicates that the target region spans adjacent portions of the two exons, numbered i and j. Stated another way, the siRNA embodiments may be capable of binding to one of the above-listed target regions.

[0075] In some embodiments, multiple siRNAs according to embodiments of the present disclosure may be capable of binding within the same target region at different starting base positions. Such siRNAs (e.g., the sense strands of the siRNAs) may thus be related to each other by a series of frame shifts along an RNA sequence that is complementary to the HTT mRNA target region. The related siRNAs may share a common sub-sequence of between 1 to 18 nucleobases in length, for example, 2 to 16 or 4 to 14 nucleobases in length, where the common sub-sequence is included at a different starting base position (position number) in each member of the group of related siRNAs.Some of the siRNAs according to embodiments of the present disclosure may thus include a common sub-sequence as listed in Table 1 below, which includes sequences of the sense and anti-sense strands (SEQ ID NOS: 18 to 31 and 32 to 45, respectively), and also lists the starting position and exon of the corresponding sequence in the HTT mRNA.Table 1

[0076] In some embodiments, the common sub-sequence of the siRNA sense strands may be, for example, 4 to 14 nucleobases in length, and may be selected from SEQ ID NOs: 18, 19, 25, and 29. In some embodiments, the common sub-sequence of the siRNAs may be, for example, 6 to 11 nucleobases in length, and may be selected from SEQ ID NOs:25 and 29.

[0077] In some embodiments, the target region within the HTT mRNA may be located within exon 67, and may be a 37-residue window centered around the g residue at position 10151 of the HTT mRNA. Accordingly, a common sub-sequence of the siRNA sense strand capable of binding to this target region in exon 67 may encompass any oligomer having a length of 18 residues or shorter (e.g., 2 to 18, 3 to 15, 4 to 12, or 5 to 10 residues) from within the sequence uuaaaauuaaucucuuuacugauauaauuaaauuuua (SEQ ID NO:46).

[0078] In some embodiments, the siRNA included in the siRNA compositions may be specifically designed or selected to have a reduced risk of cross-reactivity (e.g., with non- HTT mRNA) and immunotoxicity. For example, the siRNA may meet the following sequence-related criteria:

[0079] 1 ) The siRNA includes a strand (e.g., a sense strand) that is complementary to an HTT mRNA exon sequence;

[0080] 2) The siRNA is not complementary to any HTT mRNA sub-sequence having one or more nucleotide polymorphisms with an occurrence frequency of greater than 1 %;

[0081] 3) The siRNA is not complementary to the mRNA of any human genes (e.g., any sequences in the target transcriptome) other than HTT. For example, the siRNA does not appreciably bind to other mRNA sequences due to the presence of at least 2 mismatches;

[0082] 4) The siRNA does not have a sequence that lends itself to formation of a strong secondary structure (such as a loop or pseudoknot); and

[0083] 5) The siRNA does not include any known toxic motifs (e.g., motifs that trigger a hepatotoxic physiological response), such as tgc, tcc, and gc.

[0084] Stated another way, the siRNA compositions according to embodiments of the present disclosure may exclude sequences that: 1 ) are not complementary to an HTT mRNA exon sequence; 2) are complementary to any HTT mRNA sequence having one or more nucleotide polymorphisms with an occurrence frequency of greater than 1 %; 3) are complementary to the mRNA of any transcriptome sequence other than HTT; 4) have a strong secondary structure; and / or 5) include any of the above-described toxic motifs.

[0085] In some embodiments, the siRNA may be selected from particular 19-mer siRNA sequences and variants thereof that meet the preceding combined criteria. In some embodiments, for example, the siRNA may be selected from the siRNA sequences listed in Table 2 below, which includes sequences of the sense and anti-sense strands (SEQ ID NOS: 47 to 149 and 150 to 252, respectively), and also lists the starting position and exon of the corresponding sequence in the HTT mRNA. In some embodiments, the siRNA sequences may be selected from the Table 2 sequences and variants thereof, including sequences having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.Table 2

[0086] In some embodiments, the siRNA may include a sense strand sequence selected from SEQ ID NOs:90, 61 , 68, 47, and 51 , and variants thereof (including sequences having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.) The sequences may be alternatively referred to herein by the names in Table 3:Table 3

[0087] As noted above, an excessive number of copies in the CAG repeat region within exon 1 of the HTT mRNA is associated with the development of HD symptoms. As such, it has been hypothesized that targeting exon 1 of HTT may result in more effective knockdown and / or treatment. Accordingly, in some embodiments, the siRNA may specifically bind to Exon 1 of HTT mRNA. For example, the siRNA may include a sense strand sequence selected from SEQ ID NOs:47 to 52 and variants thereof (including sequences having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.)Chemical modifications of siRNA sequences

[0088] It is well known that unmodified siRNAs (e.g., siRNA sequences including only standard bases) have short half-lives in vivo due to rapid degradation, which limits their utility as therapeutics. Accordingly, in some embodiments, the siRNA may be chemically modified at one or more bases to provide improved in vivo stability and therapeutic efficacy. Examples of such chemical modifications include those described above in connection with the term “chemical derivatives”, but not are limited thereto.

[0089] In some embodiments, the siRNA (siRNA variant) may include one or more chemical modifications selected from 2'-O-methylation of A, G, and / or U; 2'-fluorination of C and / or U; and substitution of a phosphodiester backbone unit with phosphorothioate. In some embodiments, the siRNA may have a sense strand that is modified to include one or more 2'-O-methyl modified RNA bases. In some embodiments, the siRNA may have an anti-sense strand that is modified to include one or more 2'-fluoro modified RNA bases and an overhang with phosphorothioate linkages.

[0090] In some embodiments, for example, the siRNA may include a sense strand that is modified to include a 2'-O-methyl group at bases 1 to 4, 7 to 10, 12, 13, 15, 16, 18, and 19. The siRNA may include an anti-sense strand, which may be paired with or complementary to the sense strand, that is modified to include a fluoro group at bases 3, 6, 9, 14, and 15. The anti-sense strand may also be modified to include a *U*U overhang at the 3’-end of the antisense strand, in which the phosphate linkages are replaced with phosphorothioate linkages (e.g., between the 19-mer sequence and the overhang, and between the UU bases of the overhang).

[0091] For example, the siRNA variant may be represented by the following generic sequences showing the positions and types of chemical modifications to each strand:

[0092] Sense strand: 5'-P-mNmNmN mNNN mNmNmN mNNmN mN-3'

[0093] Antisense strand: 5'-P-NN(2'-F-N) NN(2'-F-N) NN(2'-F-N) NNN N(2'-F-N)(2'-F- N) NNN N*U*U-3’

[0094] In the above sequences, mN indicates a 2'-O-methyl RNA base, 2'-F-N indicates a 2'-fluoro modified RNA base, II indicates uracil, and * indicates aphosphorothioate linkage. It is understood that the underlying or original base sequence may be the same as any siRNA sequence according to embodiments of the present disclosure, for example, as represented by any of SEQ ID NOS:47 to 149 (sense) and 150 to 252 (anti-sense).

[0095] For example, the siRNA may be a variant of siRNA6 (SEQ ID NO:69), and may have the following sequence and chemical modifications:

[0096] Sense Strand: 5'-P-mCmCmU mllAA mUmUmU mCAmll mUGmU mCAmU mU-3'

[0097] Antisense Strand: 5'-P-AA(2'-F-U) GA(2'-F-C) AA(2'-F-U) GAA A(2'-F-U)(2'-F-U) AAG G*U*U-3’,

[0098] where the antisense strand is modified to include a 2-mer *U*U overhang at the 3’-end.

[0099] In the above sequences, A, G, and U are the standard RNA bases; mC, mil, mA are 2'-O-methyl modified RNA bases; (2'-F-U) and (2'-F-C) are 2'-fluoro modified RNA bases; and * indicates replacement of a phosphate linkage with a phosphorothioate linkage.

[0100] In some embodiments, the siRNA may be any of siRNAI to 5 (SEQ ID NOs: 90, 61 , 68, 47, and 51 ) including substantially the same modifications as described for siRNA6 (e.g., with respect to position within the 19-mer sequence) as detailed above. For example, the Example data presented herein were collected using siRNA variants that were chemically modified in this manner.

[0101] It will be understood that the descriptions herein of siRNA compositions encompass embodiments including siRNA variants as defined above. For example, additional embodiments may include siRNA fragments, extensions (e.g., longer siRNA sequences complementary to the HTT mRNA target regions), and / or functional equivalents having at least about 80%, 85%, 90%, 95%, or 99% sequence and / or structural homology to siRNA sequences described herein, as well as chemical derivatives of all of the above.Carrier Peptide

[0102] In certain embodiments, the composition further includes a carrier peptide (e.g., carrier-derived peptide) to facilitate delivery of the siRNA or another effector agent to a suitable target (e.g., a cell or tissue in which HTT is expressed). The carrier peptide may target (e.g., bind to) a receptor included on a particular cell or tissue of interest, and may subsequently facilitate passage of the siRNA payload into that cell or tissue.

[0103] The carrier peptide may include or be formed of two oligomers: a base sequence; and a linker sequence. The linker sequence may facilitate conjugation of the carrier peptide to the siRNA or effector agent (e.g., may be the point of contact between the effector agent and the carrier peptide), and the base sequence may facilitate delivery of the conjugate to the specific target. The C-terminus of the base sequence may be linked to the N-terminus of the linker sequence (e.g., the linker sequence is prepended to the base sequence) to form the carrier peptide.Base sequence

[0104] Lynxl is a protein that binds to neuronal nicotinic receptors (NNRs), also referred to as nicotinic acetylcholine receptors (nAChRs). Detailed information about lynxl is disclosed in Ibanez-Tallon et al., 2002, Neuron, 33:893-903, the entire content of which is incorporated herein by reference. nAChRs are frequently expressed in target cells of the blood brain barrier (BBB), and the Ioop2 region of lynxl is the putative binding domain for nAChRs. As such, lynxl -Ioop2-derived base sequences (e.g., base sequences derived from the Ioop2 domain of human lynxl , in lieu of the full lynxl protein) can be used as carriers for targeting and delivery of a therapeutic effector agent across the BBB via the nAChRs.

[0105] Cellular uptake and transport of the effector agent across the BBB via the interaction of lynxl -Ioop2-derived base sequences with nAChRs can be determined by, for example, cellular imaging or confocal microscopy of relevant tissue sections. Lynxl - Ioop2-derived peptides are described in more detail in e.g., U.S. 8,629,114, issued on January 14, 2014; U.S. 9,522,193, issued on December 20, 2016; U.S. 9,913,915, issued on March 13, 2018; U.S. 10,328,156, issued on June 25, 2019; U.S. 10,772,966, issued on September 15, 2020, and U.S. 17 / 020,613, filed on September 14, 2020, the entire content of each of which is incorporated herein by reference. The terms “lynxl -Ioop2- derived peptide” and “base sequence” may be interchangeably used herein.

[0106] In some embodiments, for example, the composition may include a carrier peptide including a base sequence having the general sequence of X1-X2-X3-X4-X5-X6-X7- X8-X9-X10-R-X12-K-X14-X15-X16, (SEQ ID NO:1 ), in which for SEQ ID NO:1 :

[0107] Xi is M or T;

[0108] X2 is T or I;

[0109] Xs is T or W;

[0110] X4is R or C;

[0111] Xs is T or D;

[0112] Xe is Y, I, or G;

[0113] Xy is F or Y;

[0114] X8is T or C;

[0115] X9is P, N, or S;

[0116] X10 is Y, T, or S;

[0117] X12 is M or G;

[0118] X is V or R;

[0119] X15 is R, S, A, or I; and

[0120] X is K, S, or D.

[0121] In some embodiments, the carrier peptide may include a base sequence that is similar to or conserved with a sequence in an analogue lynxl protein of another (e.g., non-human) species. In some embodiments, for example, the base sequence may be represented by any of MTTRTYFTPYRMKVRK (SEQ ID NO:2), MTTRTYYTPTRMKVSK (SEQ ID NO:3), MTWCDYFTPSRGKVRKS (SEQ ID NO:4), or MTTRTYFTPYRGKVRK (SEQ ID NO:5). In some embodiments, the base sequence may be selected from SEQ ID NOS:2 to 5 and sequences having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.

[0122] In some embodiments, the carrier peptide may include a lynxl -Ioop2 -derived base sequence with the sequence MTTRTYATPYRMKVRKS (SEQ ID NO:6), which is similar to SEQ ID NO:2 except that the phenylalanine (F) at position 7 is substituted with alanine (A). In some embodiments, the carrier peptide may include a lynxl -Ioop2-derived base sequence with the sequence MTTRTYFTPYAMADRKS (SEQ ID NO:7), which is similar to SEQ ID NO:2 except that the RMKV residues at positions 11-14 are respectively substituted with AMAD. In some embodiments, the base sequence may be selected from SEQ ID NOS:6 and 7 and sequences having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.

[0123] In some embodiments, additional amino acids may be added (appended, prepended, or inserted) to the lynxl -Ioop2-derived base sequence. For example, the sequence of MPENPRPGTP (SEQ ID NO:8) may be added to a suitable position within the 16-mer peptide of SEQ ID NO:1. In one embodiment, MPENPRPGTP (SEQ ID NO: 8) is added between residues X3 and X4 of SEQ ID NO:1 as defined above, resulting in X1X2X3MPENPRPGTPX4X5X6X7X8X9X10RX12KX14X15X16 (SEQ ID NO:9). In some embodiments, the carrier peptide may include a lynxl -Ioop2-derived base sequence with the sequence MTTMPENPRPGTPRTYFTPYRMKVRKS (SEQ ID NO: 10), or a sequence having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.

[0124] In some embodiments, the carrier peptide may include the lynx'! -Ioop2-derived base sequence of SEQ ID NO:4.

[0125] In some embodiments, the lynx'! -Ioop2-derived base sequence may be a 12- mer peptide (e.g., peptide fragment) having a sequence derived from SEQ ID NO:1 , and for example may be X3-X4-X5-X6-X7-X8-X9-X10-R11-X12-K13-X14 (SEQ ID NO:11 ), in which for SEQ ID NO:11:

[0126] XsisTorW;

[0127] X4is R or C;

[0128] Xs is T or D;

[0129] Xe is any amino acid;

[0130] XyisForY;

[0131] Xs is any amino acid;

[0132] X9 is any amino acid;

[0133] X10 is any amino acid;

[0134] X12 is M or G; and

[0135] X isVorR.

[0136] In some embodiments, a SEQ ID NO: 1 -derived 12-mer base sequence has the sequence X3-X4-X5-X6-X7-X8-X9-X10-R-X12-K-X14 (SEQ ID NO: 12), in which for SEQ ID NO:12:

[0137] X3isTorW;

[0138] X4is R or C;

[0139] Xs is T or D;

[0140] X6is Y, G or I;

[0141] X7isForY;

[0142] X8is T or C;

[0143] Xg is P, N, or S;

[0144] Xw is Y, T, or S;

[0145] X12 is M or G; and

[0146] X is V.

[0147] In some embodiments, a SEQ ID NO: 1 -derived 12-mer base sequence has the sequence X3-X4-X5-X6-X7-X8-X9-X10-R11-X12-K13-X14 (SEQ ID NO: 13), in which for SEQ ID NO:13:

[0148] Xs is T or W;

[0149] X4is R or C;

[0150] Xs is T or D;

[0151] Xe is Y, G or i;

[0152] Xy is F or Y;

[0153] Xs is T;

[0154] Xg is P;

[0155] Xw is Y or T;

[0156] X12 is M or G; and

[0157] Xu is V.

[0158] In some embodiments, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids may be made to a Iynx1 -loop2-derived base sequence of the present disclosure (e.g., any of SEQ ID NOs: 1 to 7 or 9 to 13). Such substitutions, insertions, and / or deletions may typically be in the range of about 1 to 5 amino acids. The choice of amino acids for conservative substitutions may be selected according to the location of the amino acid to be substituted in the peptide. For example, a conservative substitution for an amino acid on the exterior of the peptide, which is exposed to solvents and / or potential intermolecular interactions, may differ from a conservative substitution for an amino acid in the interior of the peptide, which is not exposed to solvents and intermolecular interactions. Such context-sensitive selection of conservative amino acid substitutions are well known in the art, for example as disclosed in Dordo et al, J. Mol. Biol., 1999, 217, 721 -739; Taylor et al, J. Theor. Biol., 119 (1986);205-218; and S. French and B. Robson, J. Mol. Evol., 19 (1983)171 , the entire content of each of which is incorporated herein by reference. For example, conservative amino acid substitutions suitable for amino acids on the exterior of a protein or peptide(i.e. amino acids exposed to a solvent), may include the following substitutions: substitution of Y with F; T with S, K, or A; P with A; E with D or Q; N with D or G; R with K; G with N or A; T with S, K, or A; D with N or E, I with L or V, F with Y or L; S with T or A, R with K, G with N or A, K with R; A with S, K, P, G, T, or V; W with Y; and M with L. Accordingly, in some embodiments, the carrier peptide may include a lynx'! -Ioop2-derived base sequence variant with a sequence selected from SEQ ID NOs: 1 to 7 or 9 to 13 that is modified to include one or more conservative substitutions.

[0159] However, embodiments of the present disclosure are not limited thereto, and for example, compositions according to embodiments of the present disclosure may incorporate lynxl -Ioop2 base sequence variants, other base sequences having no sequence relationship to the lynxl -Ioop2 base sequence, or other components and methods of transporting the siRNA across the BBB.Linker sequence

[0160] The linker sequence may include any suitable peptide sequence that is capable of mediating or facilitating an inter- or intramolecular association between the carrier peptide and the effector agent. Stated another way, the linker sequence of the carrier peptide may participate in one or more conjugate-forming interactions with the effector agent. Non-limiting examples of such interactions include electrostatic attraction, hydrogen bonding, Van der Waals interactions, covalent or ionic bond formation, and combinations thereof. Although embodiments of the present disclosure provide compositions in which the carrier peptide is mixed and conjugated with siRNA as the effector agent, it will be understood that in alternative embodiments, the carrier peptide may be suitably conjugated with other effector agents, such as drugs, antibodies, and oligonucleotides.

[0161] In some embodiments, the carrier peptide may include a linker sequence including 1 to 4 C-terminal glycine residues.

[0162] In some embodiments, the carrier peptide may include a linker sequence including 1 to 4 C-terminal glycine residues (for example, 1 , 2, 3, or 4 Gly residues), followed by a 3 to 12 residue sub-sequence capable of facilitating an interaction with an effector agent. For example, because siRNA, ASO, and TFO are negatively charged, the carrier peptide residue sub-sequence may include or consist of positively charged residues so that an electrostatic attraction can be formed with the negatively charged effector agent (e.g., siRNA, ASO, or TFO).

[0163] Stated another way, in some embodiments, the effector agent (e.g., siRNA, ASO, or TFO) may be conjugated with the carrier peptide via the linker sequence due an electrostatic attraction between the positively charged linker sequence and the negatively charged effector agent.

[0164] In some embodiments, for example, at least 3 of the 3 to 12 residues may be selected from arginine and lysine, and the remaining residues may be any standard amino acid. In some embodiments, 4 to 10, or 6 to 8 residues may be selected from arginine and lysine, and the remaining residues may be any standard amino acid.

[0165] In some embodiments, the sub-sequence that is capable of facilitating an electrostatic interaction between the linker sequence and a negatively charged effector agent may include 3 to 11 amino acids, for example, 5 to 10 amino acids, or 6 to 9 amino acids as described above (e.g., such that at least 3 are selected from Arg and Lys).

[0166] For example, the linker sequence may be represented by GGGG-X5-X6-X7-X8- X9-X10-X11-X12-X13-X14-X15-X16 (SEQ ID NO: 14), where the glycine residues at positions 2 to 4 are optional (e.g., may be present or absent), and the amino acids of Xs to X16 are selected from the standard amino acids or are absent, provided that at least three of these residues are present and are selected from Arg and Lys. For example, the linker sequence may be represented by (G)n-Xs-X6-X7-X8-X9-Xio-Xu-Xi2-Xi3-Xi4-Xi5-Xi6, where n is an integer of 1 to 4 and the amino acids of Xs to X16 are the same as above.

[0167] In some embodiments, the 3 to 12 residue sub-sequence may only include positively charged amino acids, and for example, all of the 3 to 12 amino acids may be independently selected from arginine and lysine. For example, in the linker sequence of SEQ ID NO: 14, the amino acids of Xs to X16 are selected from Arg and Lys or are absent, provided that at least of these three residues are present. Stated in other words, the linker sequence may be represented by (G)n-(Xs)m, where n is an integer of 1 to 4, each instance of Xs is independently selected from arginine and lysine, and m is an integer of 3 to 12.

[0168] In some embodiments, the carrier peptide may include a linker sequence including 4 C-terminal glycine residues, followed by a 7 to 9 residue sub-sequence capable of facilitating an electrostatic interaction between the linker sequence and the siRNA. In some embodiments, the 7 to 9 amino acids may include or consist of arginine or lysine residues.

[0169] For example, the linker sequence may be represented by GGGG-Xs-Xe-XyXs- X9-X10-X11-X12-X13 (SEQ ID NO: 15), where the amino acids of X5 to X are selected from Arg and Lys or are absent, provided that at least seven of these residues are present. For example, the linker sequence may be represented by (G)n-(X5)m, where n is an integer of 1 to 4, each instance of X5 is independently selected from arginine and lysine, and m is an integer of 7 to 9.

[0170] In some embodiments, the carrier peptide may include a linker sequence including 4 C-terminal glycine residues, followed by 7 or 9 arginine residues. In some embodiments, the carrier peptide (which includes the base sequence and the linker sequence) may have a sequence of MTWCDYFTPSRGKVRKSGGGGRRRRRRRRR(SEQ ID NO: 16). In some embodiments, the carrier peptide may have a sequence of MTWCDYFTPSRGKVRKSGGGGRRRRRRR (SEQ ID NO: 17). In some embodiments, the carrier peptide may be selected from SEQ ID NOS: 16 and 17 and sequences having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.

[0171] Each of the amino acids in the linker sequence may have any suitable stereochemistry. When the linker sequences according to embodiments of the present disclosure includes one or more arginine and / or lysine residues, the arginine and / or lysine residues may each independently be L- or D-residues.

[0172] In some embodiments, the linker sequence may include, consist essentially of, or consist of a poly-L-arginine peptide having 7, 8, or 9 arginine residues. In some embodiments, the linker sequence may include, consist essentially of, or consist of a poly- D-arginine peptide having 7, 8, or 9 arginine residues.

[0173] It will be understood that the descriptions herein of carrier peptides encompass embodiments including carrier peptide variants as defined above. For example, additional embodiments may include carrier peptide fragments, mutants, and / or functional equivalents having at least about 80%, 85%, 90%, 95%, or 99% sequence and / or structural homology to peptide sequences described herein, as well as chemical derivatives of all of the above.Treatment Methods

[0174] A method of treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein and / or mRNA levels in a target (e.g., a target cell and / or a patient), according to embodiments of the present disclosure, includes: administering a composition including any of the above-described siRNA sequences, siRNA variants, or chemical derivatives thereof to the target. The siRNA may be a sequence of SEQ ID NOs:47 to 149, as described above. The target may be in vivo or in vitro.

[0175] In some embodiments, the administering the siRNA composition may include administering a composition comprising the siRNA conjugated to a carrier peptide. In some embodiments, the administering of the siRNA composition (whether alone or conjugated to a carrier peptide) may occur in a mouse or human subject (e.g., a patient with Huntington’s disease).

[0176] In some embodiments, the target may be a cell selected from the group consisting of neurons, neuronal cells, brain cells, glial cells, astrocytes, neuronal supporting cells, and cells of the central nervous system (CNS). In some embodiments, the target (e.g., cell) includes or expresses a receptor that is targeted by the carrier peptide, for example, a nicotinic acetylcholine receptor. In some embodiments, the target may be within or across the BBB. As such, according to embodiments of the present disclosure, the administering the siRNA composition to the target includes delivery of thesiRNA across the BBB. Any suitable method or manner of delivering the siRNA across the BBB may be used without limitation. In some embodiments, delivery across the BBB may be accomplished by conjugating the siRNA to one of the carrier peptides disclosed herein.

[0177] The relative amounts of effector agent (e.g., siRNA) and carrier peptide in the siRNA composition may be selected to maximize or increase the amount of siRNA that is conjugated to carrier peptide and therefore available for delivery to the target. The concentration of the conjugate is related to the equilibrium constant for association (Ka) between the two molecules. Accordingly, the relative concentrations of the siRNA and the carrier peptide may be modified or selected to account for various intermolecular forces (including electrostatic attraction) affecting the Ka. In some embodiments, a molar ratio of the siRNA and the carrier peptide may be 1 :4 to 1 :20, for example, 1 :7 to 1 :18, or 1 : 10 to 1 :15. In some embodiments, the ratio of the siRNA and the carrier peptide may be 1 : 10 or 1 :13.

[0178] In some embodiments of the method, the composition is a pharmaceutical composition. When the composition is prepared as a pharmaceutical solution, the siRNA and the carrier peptide may be dissolved or suspended in any suitable aqueous carrier or solution that is acceptable for parenteral administration (such as a saline and / or dextrose solution). In some embodiments, for example, the solution may be a 0.5 wt% to 5 wt% saline solution (e.g., a 0.5 wt% to 3 wt%, or 0.9 wt% to 2 wt% saline solution) or a 1 wt% to 10 wt% dextrose solution (e.g., a 2 wt% to 8 wt%, or 3 wt% to 5 wt% dextrose solution). In some embodiments, the solution may include additional stabilizing agents in the art, such as PEG8000 (e.g., at a concentration of 0.01 wt% to 1 wt%, for example, 0.05 wt% to 0.5 wt%, 0.1 wt% to 0.4 wt%, or 0.2 wt% to 0.3 wt%) and / or Polysorbate 80 (e.g., at a concentration of 0.01 wt% to 1 .5 wt%, for example, 0.05 wt% to 1 .2 wt%, 0.1 wt% to 1 wt, 0.15 wt% to 0.8 wt%, 0.2 wt% to 0.7 wt%, or 0.3 wt% to 0.6 wt%).

[0179] In some embodiments, the siRNA may be included in the pharmaceutical solution in an amount of 0.01 % to 1 % by weight, for example, 0.02% to 0.5%, 0.03% to 0.25%, 0.04% to 0.1 %, or 0.05% to 0.08% by weight. For example, a pharmaceutical solution may include about 50 pg of siRNA (about 200 pg of conjugate) in 100 pL of solution.

[0180] As discussed above, in some embodiments, the method may further include conjugating the siRNA to the carrier peptide, prior to administering the composition. The chemical or biochemical steps used to achieve such a conjugation depend on the particular linker or interaction between the two molecules, and those having ordinary skill in the art are capable of selecting suitable methods, parameters, etc., to achieve the conjugation.

[0181] Administration of the composition to the target may result in an observable decrease in HTT mRNA levels and / or HTT protein expression. The decrease in HTT mRNA levels in targeted cells and tissues (e.g., particular regions of the brain, includingthose that are more strongly affected by HD) can be measured by extracting RNA from a suitable sample of the target (e.g., from cells including a receptor that can be targeted by the carrier peptide, such as a nAChR receptor), generating cDNA from a set or predetermined volume of the RNA extract, and subjecting the cDNA to PCR. The relative decrease (e.g., knock-down) of HTT mRNA with respect to an untreated control can be quantified by comparing the change in HTT mRNA levels in the presence and absence of the siRNA:carrier peptide composition with an unrelated mRNA that is expected to remain at a constant level even in the presence of the siRNA:carrier peptide composition. For example, the unrelated mRNA standard may be the synuclein alpha (SNCA) protein, which is also expressed in neuronal cells.

[0182] In some embodiments, the method of treating HD according to embodiments of the present disclosure may result in a statistically significant decrease in (e.g., knockdown of) HTT mRNA levels (e.g., with a P-value of less than 0.05, and in some embodiments less than 0.01 ). For example, the methods according to embodiments of the present disclosure may result in a decrease in HTT mRNA levels of greater than 15%, for example, a decrease of 15% to 65%, 20% to 60%, 25% to 55%, 30% to 50%, or 35% to 45%.

[0183] In some embodiments, for example, when the target is an in vitro neuronal cell culture, HTT mRNA levels may be decreased by 15% to 40%, for example, 20% to 35%, or 25 to 30% relative to untreated (control) levels.

[0184] In some embodiments, for example, when the target is in vivo in a mouse (e.g., wild-type mouse), HTT mRNA levels may be decreased by 40% to 65% in the hippocampus, for example, 45% to 60%, or 50% to 55%, and by 15% to 40% in the striatum, for example, 20% to 35% or 25% to 30% relative to untreated (control) levels.

[0185] In some embodiments, for example, when the target is in vivo in a transgenic mouse, HTT mRNA levels may be decreased by 30% to 65% in the cortex, for example, 35% to 60%, 40% to 55%, or 45% to 50%, and by 20% to 60% in the striatum, for example, 25% to 55%, 30% to 50%, or 35% to 45% relative to untreated (control) levels.

[0186] The following Examples are presented for illustrative purposes only, and do not limit the scope or content of the present application.Example 1. Selection of lead siRNAs

[0187] siRNA sequences with the potential to bind to HTT mRNA and block its translation to protein were identified using in silico methods as follows. Figure 1 is a flowchart diagram summarizing the selection procedure. As shown in box 10 of the flowchart, 13,456 complementary 20-mer oligonucleotides to human HTT mRNA were identified, and then filtered to include only the 570 sequences that would be conserved between humans, monkeys, and mice.

[0188] As shown in boxes 12 and 14, potentially risky candidates were eliminated, including 5 siRNAs matched to mRNA sequences having nucleotide polymorphisms with an occurrence frequency of greater than 1 %, and 49 siRNAs having 0 or 1 mismatches (e.g., 95% to 100% complementarity) to the mRNAs of human genes other than HTT.

[0189] As shown in box 16, the minimal free energy (MFE) of each oligonucleotide was calculated. Lower MFEs are correlated with higher chances of forming a strong secondary structure (e.g., a hairpin loop, a pseudoknot, etc.), which is undesirable in a siRNA. Oligomers predicted to form secondary structures with a MFE less than -4 kcal / mol were eliminated, resulting in 472 remaining candidates.

[0190] As shown in box 18, candidates with known toxic motifs (such as tgc, tcc, and gc) were eliminated, resulting in 97 remaining siRNA candidates represented by SEQ ID NOS:47 to 149 and 150 to 252. Such toxic motifs are further described in e.g., Burdick et al., “Sequence motifs associated with hepatotoxicity of locked nucleic acid - modified antisense oligonucleotides,” Nucleic Acids Res., 2014, 42(8), pg. 4882-4891 , the entire content of which is incorporated herein by reference.

[0191] Some studies have suggested that the CAG repeat region (which encodes polyglutamines) in Exon 1 may be the source of HTT toxicity, and that HD treatment may require specifically blocking mRNA function in Exon 1. Because the list of 97 candidates did not include any siRNA sequences corresponding to any portion of Exon 1 , the oligomer selection criteria was relaxed to list 19-mers and to allow oligomers for which the target mRNA was not conserved between human and mouse. 6 siRNAs corresponding to Exon 1 (SEQ ID NOS:47 to 52 and 150 to 155) were subsequently added to the candidate list.Example 2. in vitro knockdown of HTT mRNA by selected siRNAs

[0192] Selected HTT siRNA candidates were initially evaluated for HTT knockdown efficacy and specificity in vitro, using Iynx1-loop2-derived carrier peptides to mediate delivery into human neuronal cells.

[0193] The key genes involved in Iynx1-loop2-mediated delivery via the nAChRs include the nicotinic receptor alpha7 subunit (CHRNA7), nicotinic receptor alpha4 subunit (CHRNA4), nicotinic receptor beta2 subunit (CHRNB2), and NACHO. Because differentiated SHSY5Y human cells express all such genes of interest, they were selected to test for knockdown of HTT mRNA. The HTT mRNA knockdown efficiency was assessed by comparison to synuclein alpha (SNCA), which is a neuronally expressed gene that is regulated independently of HTT.

[0194] Methods: SHSY5Y cells were grown according to standard protocols as specified by the vendor (ATCC). When cells reached 80% confluency, the cells were passaged and 160K cells were plated in 0.5 mL of growth media in a 24-well plate. 24 hours later, the growth media was replaced with the differentiation media (day 1 ofdifferentiation protocol). Two days later, 30% of the differentiation media was replaced with fresh differentiation media (day 3 of differentiation protocol). siRNA1 -5 (SEQ ID NOS:90, 61 , 68, 47, and 51 ) were tested with Peptide 1 (SEQ ID NO: 16) or Peptide 4 (SEQ ID NO: 17) as follows.

[0195] On day 4 of the differentiation protocol, Peptide 1 (SEQ ID NO:16):siRNA conjugates or Peptide 4 (SEQ ID NO:17):siRNA conjugates were mixed in DMEMF12 media and incubated for 15 min at room temperature (e.g., 25 °C). At the end of the incubation period, 0.1 mL of the differentiation media was replaced with 0.1 mL of the peptide:siRNA conjugates. The final concentrations in the cell growth media were 10 pM-1 |uM for Peptide 1 (SEQ ID NO:16):siRNA conjugates and 10 pM-0.77 uM for Peptide 4 (SEQ ID NO: 17):siRNA conjugates (10:1 and 13:1 ratio respectively).

[0196] 24 hours after exposure to the conjugates, the cells were washed once with PBS, and RNA was extracted using an RNeasy Mini Kit (Qiagen, # 74106) with an on column DNase I treatment (Qiagen, RNase-Free DNase Set, # 79256). cDNA was generated from 0.5 ug of RNA using the KIT CDNA SUPERMIX QSCRIPT (Quanta, VWR, # 101414-108). Each qPCR reaction contained 20 ng of cDNA. The KIT QPCR FASTMIX II ROX (Quanta, VWR, # 97065-998) was used for the qPCR reaction, and triplicates were run on the Applied Biosystems™ 7500 Real-Time PCR System. Knockdown of appropriate HTT target mRNA was assessed by comparison to SNCA using suitable primers for RT-qPCR: Taqman assay Hs00918174_m1 (HTT1 ) for Peptide 1 (SEQ ID NO:16) with the 5 siRNAs, Peptide 4 (SEQ ID NO:17):siRNA1 (SEQ ID NQ:90), and Peptide 4 (SEQ ID NO:17):siRNA4 (SEQ ID NO:47); Taqman assay Hs00918128_m1 (HTT2) for Peptide 4 (SEQ ID NO:17):siRNA3 (SEQ ID NO:68); and Taqman assay Hs00240907_m1 (SNCA) for all samples. The qPCR values of the HTT and SNCA gene were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Taqman assay Hs02786624_g1 ) and peptidylprolyl isomerase (PPIA, Taqman assay Hs04194521_s1 ). A DMEMF12-only sample served as a control.

[0197] Results: Differentiated SHSY5Y cells showed a statistically significant decrease of HTT mRNA expression upon exposure to siRNA3 (SEQ ID NO:68) or siRNA4 (SEQ ID NO:47) when conjugated with either of Peptide 1 (SEQ ID NO: 16) or Peptide 4 (SEQ ID NO: 17). No significant change in expression of SNCA was observed in those same samples.

[0198] Table 4 summarizes the change in measured mRNA levels as normalized against the change in HTT1 mRNA in the DMEMF 12-only control; values in bold highlight statistically significant knockdowns. Figure 2 presents the same data in bar chart form; the error bars represent the standard deviation. In particular, SHSY5Y exposure to Peptide 1 (SEQ ID NO:16):siRNA3 (SEQ ID NO:68) was correlated with a 23% (P < .01 ) reduction of HTT mRNA. Exposure to Peptide 1 (SEQ ID NO:16):siRNA4 (SEQ ID NO:47) was correlated with a 16% (P < .05) reduction of HTT mRNA. Exposure to Peptide 4 (SEQ ID NO:17):siRNA3 (SEQ ID NO:68) was correlated with a 37% (P < .05) reduction of HTTmRNA. Exposure to Peptide 4 (SEQ ID NO:17):siRNA4 (SEQ ID NO:47) was correlated with a 29% (P < .05) reduction of HTT mRNA. Exposure to Peptide 1 (SEQ ID NO: 16) alone (e.g., in the absence of any siRNA) did not show any statistically significant effect on HTT mRNA levels.Table 4In Table 4, “Peptide 1” corresponds to (SEQ ID NO: 16); Peptide 4” corresponds to (SEQ ID NO: 17); and “siRNAI ,” “siRNA2,” “siRNA 3,” “siRNA4” and “siRNA5” correspond to (SEQ ID NQS:90, 61 , 68, 47 and 51 ).

[0199] Conclusions: Statistically significant and specific knockdown of HTT mRNA by siRNA3 (SEQ ID NO. 68) or siRNA4 (SEQ ID NO. 47), when conjugated with either Peptide 1 (SEQ ID NO: 16) or Peptide 4 (SEQ ID NO: 17), was observed in vitro in differentiated SHSY5Y cell lines. Based on the data from this study, siRNA3 and siRNA4 were further tested in vivo for their efficacy in specific brain regions, discussed below.

[0200] To assess differences in knockdown, including according to differences in concentration of the siRNA, selected siRNAs were evaluated for HTT knockdown efficacy and specificity in vitro, using different ratios of the Iynx1-loop2-derived carrier peptides to the siRNAs. The protocol was generally as described above, except that the final concentrations of the peptide:siRNA mixtures in the cell growth media were varied between a 10:1 ratio (10 pM peptide: 1 pM siRNA), an 8:1 ratio (8 pM peptide: 1 pM siRNA), and a 6:1 ratio (6 pM peptide: 1 pM siRNA). Also, the final peptide:siRNA mixtures (having different peptide:siRNA ratios) were prepared in a solution of dextrose with 0.6% polysorbate 80. And knockdown of appropriate HTT target mRNA was assessed by comparison to SNCA using the HTT1 and SNCA primers noted above in addition to another primer: Taqman assay Hs00918178_m1 (HTT3) for HTT. Knockdown was also assessed against dextrose only control and a non-targeting siRNA (NTsiRNA) as a negative control (i.e., the siSTABLE non-targeting siRNA (catalog number D-001700-01 ) from Dharmacon, Colorado).

[0201] Result: Differentiated SHSY5Y cells showed a statistically significant decrease of HTT mRNA expression upon exposure to all tested siRNAs (including those with siRNA3 (SEQ ID NO:68) and siRNA4 (SEQ ID NO:47) when conjugated to Peptide 1 (SEQ ID NO:16).

[0202] The cells showed a decrease in HTT mRNA expression upon exposure to siRNA 3 and siRNA4 (SEQ ID NOS: 68 and 47, respectively) when conjugated to Peptide 1 (SEQ ID NO:16).

[0203] Table 5 summarizes the change in measured mRNA levels normalized to SNCA; values in bold highlight statistically significant knockdowns. Figure 3 presents the same data in bar chart form; the error bars represent the standard deviation. As shown in Table 5 and Figure 3, all of the peptide:siRNA conjugates at all ratios exhibited significant knockdown, with the siRNA4 (SEQ ID NO:47) conjugates showing better knockdown than the siRNA3 (SEQ ID NO:68) conjugates. And the siRNA3 (SEQ ID NO:68) conjugates exhibited similar knockdown at the 10:1 ratio across three batches. Also, the siRNA4 (SEQ ID NO:47) conjugates at the 10:1 and 8:1 ratios exhibited significantly improved knockdown compared to the 6:1 ratio. Exposure to Dextrose-0.6% alone (e.g., in theabsence of any siRNA or peptide) did not show any statistically significant effect on HTT mRNA levels.Table 5Example 3. In vivo knock-down of mouse HTT mRNA by siRNA with Peptide 4 (SEQ ID NON 7)

[0204] Methods: Animals were allowed to acclimate at an approved vendor (Charles River Laboratories International, Inc., (CRL), Shrewsbury, MA) for at least 2 days prior to the start of the experiment. Wild-type C57bl6 mice were 2-5 months of age, fed Teklad global rodent chow, and were housed in groups of 2 to 5 per cage. siRNAs were synthesized by Dharmacon (Lafayette, CO). Carrier peptides were synthesized by Lifetein (Hillsborough, NJ).

[0205] siRNA3 (SEQ ID NO:68) or siRNA4 (SEQ ID NO:47) was administrated to each animal using the following procedure. Briefly, 50 pg of a test siRNA and Peptide 4 (SEQ ID NO: 17) peptide were mixed in vital dye (Evans Blue) / saline solution to produce a 13:1 molar ratio of Peptide 4 (SEQ ID NO:17) to siRNA, and incubated for 15 minutes at room temperature. The peptide:siRNA conjugates were then delivered as a 100 pL injection per mouse via the lateral tail vein. An identical volume of saline solution was injected in control animals.

[0206] Knockdown of HTT mRNA in the hippocampus (HP) and striatum (ST) was assessed by RT-qPCR. Seventy-two hours post-dose, animals were sacrificed and whole brains were removed without perfusion. Left-side brain tissues (HP and ST) were stored in RNAIater solution prior to RNA extraction. RNA was extracted from the HP and ST brain regions using an RNeasy Mini Kit (Qiagen, # 74106) with an on column DNAse I treatment (Qiagen, # 79256). cDNA was generated from 1 pg of RNA using the KIT CDNA SUPERMIX QSCRIPT (Quanta, VWR, # 101414-108). Each qPCR reaction contained 30- 50 ng of cDNA. The KIT QPCR FASTMIX II ROX (Quanta, VWR, # 97065-998) was used for the qPCR reaction; triplicates were run on the Applied Biosystems™ 7500 Real-Time PCR System. Knockdown of appropriate HTT target mRNA was assessed by comparison to SNCA using suitable primers for RT-qPCR: Taqman assay Mm01213763 (HTT) in the ST, Taqman assay Mm01213782_m1 (HTT) in the HP, and Taqman assayMm01 188700_m1 (SNCA) in the ST and HP. The qPCR values of HTT and SNCA were normalized to GAPDH and PPI as in Example 1 .

[0207] Result: siRNA3 (SEQ ID NO:68) and siRNA4 (SEQ ID NO:47) conjugated with Peptide 4 (SEQ ID NO: 17) each showed a statistically significant decrease in HTT mRNA expression in the HP and ST of wild-type mice. Table 6 summarizes the change in HTTmRNA levels as normalized against SNCA and relative to the saline control; values in bold highlight statistically significant knockdowns. Figure 4 presents the same data in bar chart form; the error bars represent the standard deviation. In particular, a 57% (P < .01 ) reduction of HTT mRNA was observed in the HP of mice injected with Peptide 4 (SEQ ID NO:17):siRNA3 (SEQ ID NO:68), and a 24% (P < .01 ) was observed in the ST. Meanwhile, a 57% (P < .01 ) reduction of HTT mRNA was observed in the HP of mice injected with Peptide 4 (SEQ ID NO:17):siRNA4 (SEQ ID NO:47), and a 34% (P < .01 ) reduction was observed in the ST.Table 6In Table 6, “Peptide 4” corresponds to (SEQ ID NO: 17); and “siRNA3” and “siRNA4” respectively correspond to (SEQ ID NOS:68 and 47).

[0208] Conclusions: Statistically significant and specific knockdown of HTT mRNA was observed in vivo in wild-type mice treated with siRNA3 (SEQ ID NO:68) or siRNA4 (SEQ ID NO:47), conjugated with Peptide 4 (SEQ ID NO: 17). Based on the data from this study, siRNA3 (SEQ ID NO:68) and siRNA4 (SEQ ID NO:47) were further tested in a humanized HTT mouse model.Example 4. In vivo knock-down of human HTT mRNA in transgenic mice

[0209] Methods: Male and female BACHD (FVB / N-Tg(HTT*97Q)IXwy / J) mice were obtained from The Jackson Laboratory (Bar Harbon, ME) and were acclimated for several days before the start of the experiment. Fasting was not required for this study. The siRNA and carrier peptides were obtained as in Example 3.

[0210] siRNA3 (SEQ ID NO:68) or siRNA4 (SEQ ID NO:47) was administrated to each animal using the following procedure. Briefly, 50 pg of a test siRNA and Peptide 4 (SEQ ID NO:17) were mixed in saline solution to produce a 13:1 molar ratio of peptide to siRNA, and incubated for 15 minutes at room temperature. The peptide:siRNA conjugates were then delivered as a 100 pL injection per mouse via the lateral tail vein. An identical volume of saline solution was injected in control animals.

[0211] Knockdown of HTT mRNA in the HP and ST was assessed by RT-qPCR. After two weeks for the ST and four weeks for the cortex (CTX), animals were sacrificed and whole brains were removed without perfusion. Left-side brain tissues (HP and ST) were stored in RNAIater solution prior to RNA extraction. RNA was extracted from the HP and ST brain regions as in Example 3. cDNA was generated from 1 pg of RNA and qPCR reaction triplicates were run as in Example 3. Knockdown of appropriate HTT target mRNA was assessed by comparison to SNCA using suitable primers for RT-qPCR: Taqman assay Hs00918134_m1 (HTT) and Taqman assay Mm01188700_m1 (SNCA). The qPCR values of HTT and SNCA were normalized to GAPDH and PPI as in Example 1.

[0212] Result: siRNA3 (SEQ ID NO:68) and siRNA4 (SEQ ID NO:47) conjugated with Peptide 4 (SEQ ID NO: 17) each showed a statistically significant decrease in HTT mRNA expression in the cortex and ST of BACHD mice.

[0213] Table 7 summarizes the change in measured mRNA levels as normalized against SNCA and relative to the saline control; values in bold highlight statistically significant knockdowns. Figure 5 presents the same data in bar chart form; the error bars represent the standard deviation. In particular, a 39% (P < .05) reduction of HTT mRNA was observed in the CTX of mice injected with Peptide 4 (SEQ ID NO:17):siRNA3 (SEQ ID NO:68), and a 37% (P < .05) reduction was observed in the ST. A 52% (P < .05), reduction of HTT mRNA was observed in the CTX of mice injected with Peptide 4 (SEQ ID NO:17):siRNA4 (SEQ ID NO:47), and a 49% (P < .01 ), reduction was observed in the ST.Table 7In Table 7, “Peptide 4” corresponds to (SEQ ID NO: 17); and “siRNA3” and “siRNA4” respectively correspond to (SEQ ID NOS:68 and 47).

[0214] Conclusions: Statistically significant and specific knockdown of human HTT RNA was observed in vivo by siRNA3 and siRNA4, confirming their candidacy for treatment of human Huntington Disease patients.Example 5: In vivo knock-down of mouse HTT mRNA by siRNA with Peptide 1 (SEQ ID NO: 16) and Peptide 4 (SEQ ID NO: 17)

[0215] Methods: The same methods were used as in the experiment of Example 3, except that Peptide 1 (SEQ ID NO: 16) peptide was used in some samples in place of Peptide 4 (SEQ ID NO: 17), and siRNA3 (SEQ ID NO:68) was used in all samples.

[0216] Result: Like Peptide 4 (SEQ ID NO:17), Peptide 1 (SEQ ID NO:16) is also an effective peptide to mediate transport of siRNA into the brain. Both Peptide 1 (SEQ ID NO:16) and Peptide 4 (SEQ ID NO:17), when conjugated with siRNA3 (SEQ ID NO:68), provided a specific and statistically significant decrease of HTT mRNA expression in the HP and ST of wild-type mice. Table 8 summarizes the change in HTT mRNA levels as normalized against SNCA and relative to the saline control; values in bold highlight statistically significant knockdowns. Figure 6 presents the same data in bar chart form; the error bars represent the standard deviation. A 57% (P < .01 ) reduction of HTT mRNA in the HP and a 24% (P <0.1 ), reduction in the ST was observed in mice injected with Peptide 4 (SEQ ID NO:17):siRNA3 (SEQ ID NO:68). Similarly, a 56% (P < .01 ) reduction of HTT mRNA in the HP and a 34% (P <0.1 ) reduction in the ST was observed in mice injected with Peptide 1 (SEQ ID NO:16):siRNA3 (SEQ ID NO:68).Table 8

[0217] Conclusions: The results show that both Peptide 1 (SEQ ID NO:16) and Peptide 4 (SEQ ID NO: 17) are potent carrier peptides that deliver siRNA in the brain and into neurons, resulting in significant knock-down of the target gene’s mRNA by the siRNAs according to embodiments of the present disclosure.Example 6. In vivo knock-down of human HTT mRNA in Q175 mice

[0218] Methods: Male and female Q175 mice at PsychoGenics (Paramus, New Jersey) were acclimated for several days before the start of the experiment. Fasting was not required for this study. The siRNA and carrier peptides were obtained as in Example 3.

[0219] siRNA3 (SEQ ID NO:68) was administrated to each animal using the following procedure. Briefly, 50 pg of a test siRNA and Peptide 1 (SEQ ID NO: 16) were mixed in saline solution to produce a 10:1 molar ratio of peptide to siRNA, and incubated for 15 minutes at room temperature. The peptide:siRNA conjugates were then delivered with the indicated doses of siRNA3 (i.e. , 50 pg, 5 pg, 2 injections of 0.5 pg for a total of 1 pg, 0.5 pg, and 0.05 pg) as a 100 pL injection per mouse via the lateral tail vein. An identical volume of a dextrose / POLYSORBATE 80 solution was injected in control animals.

[0220] Knockdown of HTT mRNA in the cortex (CTX), hippocampus (HP) and striatum (ST) was assessed by RT-qPCR. After two weeks for the ST and four weeks for the CTX, animals were sacrificed and whole brains were removed without perfusion. Left-side brain tissues (HP and ST) were stored in RNAIater solution prior to RNA extraction. RNA was extracted from the CTX, HP and ST brain regions as in Example 3. cDNA was generated from 1 pg of RNA and qPCR reaction triplicates were run as in Example 3. Knockdown of appropriate HTT target mRNA was assessed by comparison to SNCA using suitable primers for RT-qPCR: Taqman assay Mm01213763_m1 (mHTT2), Taqman assay Mm01213782_m1 (mHTT4), and Taqman assay Mm01188700_m1 (SNCA).

[0221] Result: siRNA3 (SEQ ID NO:68) conjugated with Peptide 1 (SEQ ID NO: 16) showed statistically significant decreases in HTT mRNA expression in the cortex, hippocampus and ST at all tested doses. However, surprisingly, the conjugates administered at the 5 pg dose, the 1 pg dose (2x 0.5 pg doses administered an hour apart), the 0.5 pg dose, and the 0.05 pg doses exhibited comparable or superior knockdown as compared to the higher 50 pg dose. And the 5 pg and 1 pg doses show significantly superior knock-down compared with the 50 pg dose in the striatum and hippocampus. These results show the surprising efficacy of the conjugates disclosed herein at significantly reduced doses.

[0222] Tables 9-11 summarize the change in measured mRNA levels in the striatum, cortex and hippocampus, respectively, as normalized against SNCA and relative to the dextrose / polysorbate 80 control; values in bold highlight statistically significant HTT mRNA knockdowns after delivery of the siRNA3 dose indicated in the first column. Figure 5 presents the same data in bar chart form; the error bars represent the standard deviation. Figures 7A, 8A and 9A present the same data in bar chart form; the error bars represent the standard deviation. Tables 12-14 summarize the non-normalized data. Figures 7B, 8B and 9B present the same data in bar chart form; the error bars represent the standard deviation.Table 9 - Knock-down in Striatum (Normalized)Table 10 - Knock-Down in Cortex (Normalized)Table 11 - Knock-down in Hippocampus (Normalized)Table 12 - Knock-Down in Striatum (Non-normalized)Table 13 - Knock-Down in Cortex (Non-normalized)Table 14 - Knock-Down in Hippocampus (Non-normalized)

[0223] Conclusions: Statistically significant and specific knockdown of human HTT RNA was observed in vivo by siRNA3 at low doses, surprisingly showing efficacious low dose treatments of human Huntington Disease patients.

[0224] As used herein, unless otherwise expressly specified, all numbers such as those expressing values, ranges, amounts or percentages may be read as if prefaced by the word "about," even if the term does not expressly appear. As used herein, the terms "substantially", "about", and similar terms are used as terms of approximation and not as terms of degree, and are intended to account for the inherent deviations in measured or calculated values that would be recognized by those of ordinary skill in the art. Also, any numerical range recited herein is intended to include all sub-ranges of the same numerical precision subsumed within the recited range. For example, a range of "1 .0 to 10.0" is intended to include all subranges between (and including) the recited minimum value of 1.0 and the recited maximum value of 10.0, that is, having a minimum value equal to or greater than 1.0 and a maximum value equal to or less than 10.0, such as, for example, 2.4 to 7.6. Any maximum numerical limitation recited herein is intended to include all lower numerical limitations subsumed therein and any minimum numerical limitation recited in this specification is intended to include all higher numerical limitations subsumed therein. Accordingly, Applicant reserves the right to amend this specification, including the claims, to expressly recite any sub-range subsumed within the ranges expressly recited herein. As used herein, the terms "combination thereof" and "combinations thereof" may refer to a chemical combination (e.g., an alloy or chemical compound), a mixture, or a laminated structure of components.

[0225] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present disclosure. Plural encompasses singular and vice versa. As used herein, the singular forms "a" and "an" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises", "comprising", "includes", and "including," when used in this specification, specify the presence of the stated features, integers, acts, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, acts, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Expressions such as "at least one of", when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0226] While the subject matter of the present disclosure has been described in connection with certain embodiments, it is to be understood that the subject matter of the present disclosure is not limited to the disclosed embodiments, but, on the contrary, the present disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims, and equivalents thereof.

Claims

WHAT IS CLAIMED IS:1 . A composition for treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein and / or mRNA levels in a target, the composition comprising: a siRNA comprising a siRNA sequence selected from SEQ ID NOs:18 to 149 or a variant thereof; and a carrier peptide comprising a base sequence of SEQ ID NO:1 or a variant thereof.

2. The composition of claim 1 , wherein the siRNA sequence comprises a common sub-sequence selected from SEQ ID NOs:18 to 31 or any sub-set of 2 to 18 residues within SEQ ID NO:46.

3. The composition of claim 1 , wherein the siRNA sequence is a 19-mer selected from SEQ ID NOs:47 to 149.

4. The composition of claim 1 , wherein the siRNA sequence is selected from SEQ ID NQs:90, 61 , 68, 47, and 51.

5. The composition of claim 1 , wherein the siRNA binds to Exon 1 of HTT mRNA, and the siRNA sequence is selected from SEQ ID NOs:47 to 52.

6. The composition of claim 1 , wherein the siRNA comprises one or more chemical modifications selected from 2'-O-methylation of A, G, and / or U; 2'-fluorination of C and / or U; and substitution of phosphorothioate in a phosphodiester backbone unit.

7. The composition of any one of claims 1 -6, wherein the carrier peptide further comprises a linker sequence of SEQ ID NO: 14 prepended to the base sequence.

8. The composition of any one of claims 1 -6, wherein the carrier peptide is selected from SEQ ID NOs:16 and 17.

9. The composition of any one of claims 1-6, wherein the siRNA is conjugated with the carrier peptide.

10. The composition of any one of claims 1 -6, wherein a ratio of the siRNA and the carrier peptide is 1 :5 to 1 :20.

11. A method of treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein and / or mRNA levels in a target, the method comprising: administering a composition comprising a siRNA sequence selected from SEQ ID NOs: 18 to 149 or a variant thereof.

12. The method of claim 11 , wherein the siRNA sequence comprises a common sub-sequence selected from SEQ ID NOs: 18 to 31 or any sub-set of 2 to 18 residues within SEQ ID NO:46.

13. The method of claim 11 , wherein the siRNA sequence comprises a 19-mer selected from SEQ ID NOs:47 to 149.

14. The method of claim 11 , wherein the siRNA sequence is selected from SEQ ID NOs: 90, 61 , 68, 47, and 51.

15. The method of claim 11 , wherein the siRNA sequence binds to Exon 1 of HTT mRNA, and the siRNA sequence is selected from SEQ ID NOs: 47 to 52.

16. The method of any one of claims 11 -15, wherein the siRNA sequence comprises one or more chemical modifications selected from 2'-O-methylation of A, G, and / or U; 2'-fluorination of C and / or II; and substitution of phosphorothioate in a phosphodiester backbone unit.

17. A method of treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein and / or HTT mRNA levels in a target, the method comprising: administering the composition of any one of claims 1 -6 to the target.

18. The method of claim 17, wherein the composition is prepared by mixing the siRNA or the variant thereof and the carrier peptide or the variant thereof in a ratio of 1 :4 to 1 :20 in a solution that is acceptable for parenteral administration.

19. The method of claim 18, wherein the solution is a saline solution comprising 0.1 wt% to 1 wt% PEG 8000.

20. The method of claim 18, wherein the solution is a dextrose solution comprising 0.01 wt% to 1 .5 wt% Polysorbate 80.

21. The method of claim 17, wherein the administering of the composition occurs in a human subject.

22. The method of claim 17, wherein the target is a cell selected from the group consisting of neurons, neuronal cells, brain cells, glial cells, astrocytes, neuronal supporting cells, and cells of the central nervous system (CNS).

23. The method of claim 17, further comprising conjugating the siRNA to the carrier peptide, prior to administering the composition.

24. The method of claim 23, wherein the carrier peptide further comprises a linker sequence of SEQ ID NO: 14, and the siRNA is conjugated with the carrier peptide via the linker sequence.

25. The method of claim 17, wherein the HTT mRNA levels are decreased by 15% to 65%.

26. A composition for treating Huntington’s disease (HD) by reducing endogenous Huntingtin (HTT) protein and / or mRNA levels in a target, the composition comprising: a 19 to 27-mer siRNA comprising a common sub-sequence selected from SEQ ID NOs: 18 to 31 or any sub-set of 2 to 18 residues within SEQ ID NO:46; and a carrier peptide comprising a base peptide sequence of SEQ ID NO:1 or a variant thereof.

27. The composition of claim 26, wherein the 19 to 27-mer siRNA is complementary to a HTT mRNA sequence.

28. The composition of claim 26, wherein the siRNA is a 19 to 21 -mer.

29. The composition of claim 26, wherein the siRNA is a 19-mer.

30. The composition of any one of claims 26 to 29, wherein the siRNA sequence comprises one or more chemical modifications selected from 2'-O-methylation of A, G, and / or U; 2'-fluorination of C and / or U; and substitution of phosphorothioate in a phosphodiester backbone unit.

31. The composition of any one of claims 26 to 29, wherein the carrier peptide further comprises a linker sequence of SEQ ID NO: 14, prepended to the base sequence.

32. The composition of any one of claims 26 to 29, wherein the carrier peptide is selected from SEQ ID NOs:16 and 17.

33. The composition of claim 31 , wherein the siRNA is conjugated with the carrier peptide via the linker sequence.

34. The method of any of claims 11-25, wherein the administration comprises administering the composition in a dose of siRNA lower than 0.1 mg per kg, or lower than 4,000 pg per kg.

35. The method of any of claims 11-25 and 34, wherein the administration comprises administering the composition in a dose of siRNA of 1 pg per kg to 4,000 pg per kg.

36. The method of any of claims 11 -25, 34 and 35, wherein the administration comprises administering the composition in a dose of siRNA of 1 pg per kg to 4,000 pg per kg, 1 pg per kg to 3,000 pg per kg, 1 pg per kg to 2,000 pg per kg, 1 pg per kg to 1 ,000 pg per kg, 2 pg per kg to 4,000 pg per kg, 2 pg per kg to 3,000 pg per kg, 2 pg per kg to 2,000 pg per kg, 2 pg per kg to 1 ,000 pg per kg, 10 pg per kg to 4,000 pg per kg, 10 pg per kg to 3,000 pg per kg, 10 pg per kg to 2,000 pg per kg, 10 pg per kg to 1 ,000 pg per kg, 20 pgper kg to 4,000 pg per kg, 20 pg per kg to 3,000 pg per kg, 20 pg per kg to 2,000 pg per kg, 20 pg per kg to 1 ,000 pg per kg, 40 pg per kg to 4,000 pg per kg, 40 pg per kg to 3,000 pg per kg, 40 pg per kg to 2,000 pg per kg, 40 pg per kg to 1 ,000 pg per kg, 100 pg per kg to 4,000 pg per kg, 100 pg per kg to 3,000 pg per kg, 100 pg per kg to 2,000 pg per kg, 100 pg per kg to 1 ,000 pg per kg, 200 pg per kg to 4,000 pg per kg, 200 pg per kg to 3,000 pg per kg, 200 pg per kg to 2,000 pg per kg, 200 pg per kg to 1 ,000 pg per kg, 350 pg per kg to 4,000 pg per kg, 350 pg per kg to 3,000 pg per kg, 350 pg per kg to 2,000 pg per kg, 350 pg per kg to 1 ,000 pg per kg, 1 pg per kg to 350 pg per kg, 2 pg per kg to 350 pg per kg, 10 pg per kg to 350 pg per kg, 20 pg per kg to 350 pg per kg, 40 pg per kg to 350 pg per kg, 100 pg per kg to 350 pg per kg, 200 pg per kg to 350 pg per kg, 1 pg per kg to 200 pg per kg, 2 pg per kg to 200 pg per kg, 10 pg per kg to 200 pg per kg, 20 pg per kg to 200 pg per kg, 40 pg per kg to 200 pg per kg, 1 pg per kg to 50 pg per kg, 2 pg per kg to 50 pg per kg, 10 pg per kg to 50 pg per kg, 20 pg per kg to 50 pg per kg, 40 pg per kg to 50 pg per kg, 1 pg per kg to 40 pg per kg, 2 pg per kg to 40 pg per kg, 10 pg per kg to 40 pg per kg, 20 pg per kg to 40 pg per kg, 1 pg per kg to 20 pg per kg, 2 pg per kg to 20 pg per kg, 10 pg per kg to 20 pg per kg, 1 pg per kg to 10 pg per kg, 2 pg per kg to 10 pg per kg.

37. The method of any of claims 11-25 and 34-36, wherein the administration of the composition comprises administering the composition in a dose of siRNA of 1 pg per kg, 2 pg per kg, 10 pg per kg, 20 pg per kg, 40 pg per kg, 100 pg per kg, 200 pg per kg, 350 pg per kg, 1 ,000 pg per kg, or 2000 pg per kg.

38. The method of any of claims 11 -25, wherein the administration of the composition comprises administering the composition in a dose of siRNA of 1.7 pg per kg to 6,800 pg per kg.

39. The method of any of claims 11-25 and 38, wherein the administration of the composition comprises administering the composition in a dose of siRNA of 1.7 pg per kg to 6,800 pg per kg, 1.7 pg per kg to 5,100 pg per kg, 1.7 pg per kg to 3,400 pg per kg, 1.7 pg per kg to 1 ,700 pg per kg, 3.4 pg per kg to 6,800 pg per kg, 3.4 pg per kg to 5,100 pg per kg, 3.4 pg per kg to 3,400 pg per kg, 3.4 pg per kg to 1 ,700 pg per kg, 17 pg per kg to 6,800 pg per kg, 17 pg per kg to 5,100 pg per kg, 17 pg per kg to 3,400 pg per kg, 17 pg per kg to 1 ,700 pg per kg, 34 pg per kg to 6,800 pg per kg, 34 pg per kg to 5, 100 pg per kg,34 pg per kg to 3,400 pg per kg, 34 pg per kg to 1 ,700 pg per kg, 68 pg per kg to 6,800 pg per kg, 68 pg per kg to 5,100 pg per kg, 68 pg per kg to 3,400 pg per kg, 68 pg per kg to 1 ,700 pg per kg, 170 pg per kg to 6,800 pg per kg, 170 pg per kg to 6,800 pg per kg, 170 pg per kg to 3,400 pg per kg, 170 pg per kg to 1 ,700 pg per kg, 340 pg per kg to 6,800 pg per kg, 340 pg per kg to 5,100 pg per kg, 340 pg per kg to 3,400 pg per kg, 340 pg per kg to 1 ,700 pg per kg, 595 pg per kg to 6,800 pg per kg, 595 pg per kg to 6,800 pg per kg, 595 pg per kg to 6,800 pg per kg, 595 pg per kg to 1 ,700 pg per kg, 1 .7 pg per kg to 595 pg per kg, 3.4 pg per kg to 595 pg per kg, 17 pg per kg to 595 pg per kg, 34 pg per kg to 595 pg per kg, 68 pg per kg to 595 pg per kg, 170 pg per kg to 595 pg per kg, 340 pg per kg to 595 pg per kg, 1 .7 pg per kg to 340 pg per kg, 3.4 pg per kg to 340 pg per kg, 17 pg per kg to 340 pg per kg, 34 pg per kg to 340 pg per kg, 68 pg per kg to 340 pg per kg, 1.7 pg per kg to 85 pg per kg, 3.4 pg per kg to 85 pg per kg, 3.4 pg per kg to 85 pg per kg, 34 pg per kg to 85 pg per kg, 68 pg per kg to 85 pg per kg, 1 .7 pg per kg to 68 pg per kg, 3.4 pg per kg to 68 pg per kg, 17 pg per kg to 68 pg per kg, 34 pg per kg to 68 pg per kg, 1 .7 pg per kg to 34 pg per kg, 3.4 pg per kg to 34 pg per kg, 17 pg per kg to 34 pg per kg, 1 .7 pg per kg to 17 pg per kg, 3.4 pg per kg to 17 pg per kg.

40. The method of any of claims 11-25, 38 and 39, wherein the administration of the composition comprises administering the composition in a dose of siRNA of 1 .7 pg per kg, 3.4 pg per kg, 17 pg per kg, 34 pg per kg, 68 pg per kg, 85 pg per kg, 170 pg per kg, 340 pg per kg, 595 pg per kg, 1 ,700 pg per kg, or 3,400 pg per kg.

41. The method of any of claims 11 -40, wherein the administering the compositions comprising administering the composition to a human or animal subject diagnosed with, or suffering one or more symptoms of Huntington’s disease.

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