Compositions and methods for delivery of low dose effector agents

Abstract

A composition for transporting a therapeutic effector agent across the blood brain barrier and for treating, alleviating or reducing the risk of developing symptoms of cognitive decline and other symptoms of neurodegeneration includes a therapeutic effector agent conjugated to a carrier peptide comprising SEQ ID NO:1. The composition can be administered in low doses to great therapeutic effect. Methods of administering the composition include systemic and intranasal delivery routes.

Description

COMPOSITIONS AND METHODS FOR DELIVERY OF LOW DOSE EFFECTOR AGENTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 63 / 449,826, filed March 3, 2023, the entire content of which is incorporated herein by reference.INTRODUCTION

[0002] Neurodegenerative disease and disorders, and their symptoms, can be particularly difficult to treat. For example, while cognitive decline associated with these diseases and disorders is particularly detrimental to quality of life, and is increasingly prevalent in today’s society, very few effective therapies exist to alleviate this devastating symptom or to treat its underlying neurodegenerative conditions.

[0003] One of the main challenges to the effective treatment of neurodegenerative disorders or diseases - and thus the alleviation or treatment of cognitive decline - is the delivery of the therapeutics. Currently, delivery of such therapeutics is accomplished by intracranial or intrathecal administration, but this is an invasive method that also requires overcoming stability issues in the extracellular and intracellular environments, and the formulation of a method for in vivo delivery to specific target cells. Indeed, one of the greatest hurdles for the few existing therapeutics for the treatment of neurodegenerative diseases and disorders is the safe and efficient intracranial or intrathecal delivery of the therapeutic agent to the relevant target in the brain.

[0004] Additionally, existing therapeutics are typically delivered at relatively high doses, increasing the risk of side effects, and other negative drawbacks associated with the therapeutics. Administration of higher doses of the therapeutics also increases the cost of the therapy.

[0005] Accordingly, safe, efficient, and effective delivery of therapeutics at lower doses or dosages would be a welcome improvement, and a much-needed advancement in the treatment of neurodegenerative disorders and diseases, and their highly detrimental symptoms.SUMMARY

[0006] According to embodiments of the present disclosure, a conjugate includes a therapeutic effector agent, and a carrier peptide comprising SEQ ID NO:1. The molar ratio of the therapeutic effector agent to the carrier peptide is 1 : greater than or equal to

[0007] In some embodiments, the therapeutic effector agent may include an oligomer or oligonucleotide. And in some embodiments, the therapeutic effector agent may include siRNA or an anti-sense oligonucleotide.

[0008] According to some embodiments, the carrier peptide may include SEQ ID NO:4.

[0009] In some embodiments of the present disclosure, a conjugate includes a therapeutic effector agent, and a carrier peptide comprising SEQ ID NO:1 , and a molar ratio of the therapeutic effector agent to the carrier peptide is 1 : lower than or equal to 10.

[0010] According to some embodiments, the therapeutic effector agent may include an oligomer or oligonucleotide. And in some embodiments, the therapeutic effector agent may include siRNA or an anti-sense oligonucleotide.

[0011] In some embodiments, the carrier peptide may include SEQ ID NO:4.

[0012] According to embodiments of the present disclosure, a pharmaceutical composition for administration to a subject includes any of the conjugates, and one or more pharmaceutically acceptable excipients.

[0013] In some embodiments, a concentration of the therapeutic effector agent is lower than 4,000 pg per kg of body weight of the subject. And in some embodiments, a concentration of the therapeutic effector agent is lower than 4,000 pg per kg of body weight of the subject. In some embodiments, a concentration of the therapeutic effector agent is lower than 600 pg per kg of body weight of the subject. And in some embodiments, a concentration of the therapeutic effector agent is lower than 100 pg per kg of body weight of the subject.

[0014] According to some embodiments, the carrier peptide of the conjugate comprises SEQ ID NO:4.

[0015] In some embodiments, the therapeutic effector agent comprises an oligomer or oligonucleotide. And in some embodiments, the therapeutic effector agent comprises siRNA or an anti-sense oligonucleotide.

[0016] According to some embodiments, a pharmaceutical composition for administration to a subject includes a conjugate comprising a therapeutic effector agent conjugated to a carrier peptide comprising SEQ ID NO:1 , and one or more pharmaceutically acceptable excipients. A concentration of the therapeutic effector agent is lower than 4,000 pg per kg of body weight of the subject.

[0017] In some embodiments, the concentration of the therapeutic effector agent may be lower than 600 pg per kg of body weight of the subject.

[0018] In some embodiments, the carrier peptide comprises SEQ ID NO:4.

[0019] According to embodiments of the present disclosure, a method of administering any of the conjugates or any of the pharmaceutical compositions to a subject includes administering an amount of the conjugate or pharmaceutical composition sufficient to deliver an amount of the therapeutic effector agent lower than 4,000 pg per kg of body weight of the subject.

[0020] In some embodiments, the amount of the conjugate or pharmaceutical composition may be sufficient to deliver an amount of the effector agent lower than 600 pg per kg of body weight of the subject. And in some embodiments, the amount of the conjugate or pharmaceutical composition may be sufficient to deliver an amount of the effector agent lower than 100 pg per kg of body weight of the subject.

[0021] In some embodiments, the carrier peptide comprises SEQ ID NO:4.

[0022] According to some embodiments, the therapeutic effector agent of the conjugate may include an oligomer or oligonucleotide. In some embodiments, the therapeutic effector agent of the conjugate may include siRNA or an anti-sense oligonucleotide.

[0023] In some embodiments, the subject may have been diagnosed with, suffer from, or be at risk of developing a neurodegenerative disorder or disease.

[0024] According to some embodiments, the subject may be experiencing or may have experienced symptoms of cognitive decline.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a bar chart summarizing the change in measured mRNA levels in SHSY5Y cells treated with Peptide 1 (SEQ ID NO:16):ASO or Peptide 4 (SEQ ID NO:17):ASO conjugates (ASOs 1 to 5, SEQ ID NOS: 18, 19, 20, 21 , and 22), as normalized against the change in HTT1 mRNA in a DMEMF12 growth media-only control.

[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):ASO conjugates (ASOs 6, 3 and 2, SEQ ID NOs: 23, 20 and 19), as normalized against synuclein alpha (SNCA) mRNA levels and relative to a dextrose-0.6% only control.

[0027] Figure 3 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):ASO3 (SEQ ID NO:20) complexes, as normalized against synuclein alpha (SNCA) mRNA levels and relative to a saline control.

[0028] Figure 4A is a graph of Open Field Total Rearing showing total rearing bouts in the three groups of mice studied in Example 3. N values represent the number of animalsat the time of open field assessment. Statistics showing the total rearing bouts for the three groups of mice studied in Example 3.

[0029] Figure 4B is a graph of Open Field Total Distance Traveled, showing the total distance for the three groups of mice studied in Example 3. N values represent the number of animals at the time of open field assessment. Statistics showing the total distance traveled for the three groups of mice studied in Example 3.

[0030] Figure 5A is a graph comparing the soluble expanded mHTT levels in the cortex (CTX) according to Assay 6 (2B7 / MW1-ST) of the vehicle control, and ASO6 mice groups studied in Example 3.

[0031] Figure 5B is a graph comparing the aggregated mHTT levels in the cortex (CTX) according to Assay 45 (MW8 / 4C9-ST) of the vehicle control, and ASO6 mice groups studied in Example 3.

[0032] Figure 5C is a graph comparing the expanded mHTT levels in the striatum (ST) according to Assay 6 (2B7 / MW1-ST) of the vehicle control, and ASO6 mice groups studied in Example 3.

[0033] Figure 5D is a graph comparing the aggregated mHTT levels in the striatum (ST) according to Assay 45 (MW8 / 4C9-ST) of the vehicle control, and ASO6 mice groups studied in Example 3.

[0034] Figure 6A is a graph of Open Field Total Rearing showing total rearing bouts in the three groups of mice studied in Example 4. N values represent the number of animals at the time of open field assessment. Statistics showing the total rearing bouts for the three groups of mice studied in Example 4.

[0035] Figure 6B is a graph of Open Field Statistics Total Distance Traveled showing total distance traveled in the three groups of mice studied in Example 4. N values represent the number of animals at the time of open field assessment. Statistics showing the total distance traveled for the three groups of mice studied in Example 4.

[0036] Figure 7A is a graph comparing the soluble expanded mHTT levels in the cortex (CTX) according to Assay 6 (2B7 / MW1-ST) of the vehicle control, and ASO6 mice groups studied in Example 4.

[0037] Figure 7B is a graph comparing the aggregated mHTT levels in the cortex (CTX) according to Assay 45 (MW8 / 4C9-ST) of the vehicle control, and ASO6 mice groups studied in Example 4.

[0038] Figure 7C is a graph comparing the soluble expanded mHTT levels in the striatum (ST) according to Assay 6 (2B7 / MW1 -ST) of the vehicle control, and ASO6 mice groups studied in Example 4.

[0039] Figure 7D is a graph comparing the aggregated mHTT levels in the striatum (ST) according to Assay 45 (MW8 / 4C9-ST) of the vehicle control, and ASO6 mice groups studied in Example 4.

[0040] Figure 8 is a side-by-side comparison of the Total Rearing Frequency graph of Figure 4A and Total Distance Traveled graph of Figure 4B with the Total Rearing Frequency graph of Figure 6A and the Total Distance Traveled graph of Figure 6B, providing a comparison of these functional measures between the 25 pg dose of Example 3 and the 2.5 pg dose of Example 4.

[0041] Figure 9 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 ID NO:17):siRNA conjugates (siRNAs 1-5, SEQ ID NOS:24, 25, 26, 27, and 28), as normalized against the change in HTT mRNA in a DMEMF12 growth media-only control.

[0042] Figure 10 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:26, 27, respectively) and NTsiRNA), as normalized against synuclein alpha (SNCA) mRNA levels and relative to a dextrose-0.6% polysorbate 80 only control.

[0043] Figure 11 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:26) or Peptide 4 (SEQ ID NO:17):siRNA4 (SEQ ID NO:27) conjugates, as normalized against synuclein alpha (SNCA) mRNA levels and relative to a saline control.

[0044] Figure 12 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:26) or Peptide 4 (SEQ ID NO:17):siRNA4 (SEQ ID NO:27) conjugates, as normalized against SNCA mRNA levels and relative to a saline control.

[0045] Figure 13 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:26) or Peptide 1 (SEQ ID NO:16):siRNA3 (SEQ ID NO:26) conjugates, as normalized against SNCA mRNA levels and relative to a saline control.

[0046] Figure 14A 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:26) conjugate, as normalized against SNCA mRNA levels and relative to a dextrose / polysorbate 80 control.

[0047] Figure 14B is a bar chart summarizing the non-normalized fold-change in measured HTT mRNA expression levels in the striatum (ST) of Q175 mice and treatedwith varying doses of a Peptide 1 (SEQ ID NO:16):siRNA3 (SEQ ID NO:26) conjugate relative to a dextrose / polysorbate 80 control.

[0048] Figure 15A 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:26) conjugate, as normalized against SNCA mRNA levels and relative to a dextrose / polysorbate 80 control.

[0049] Figure 15B 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:26) conjugate relative to a dextrose / polysorbate 80 control.

[0050] Figure 16A 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:26) conjugate, as normalized against SNCA mRNA levels and relative to a dextrose / polysorbate 80 control.

[0051] Figure 16B 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:26) conjugate and relative to a dextrose / polysorbate 80 control.DETAILED DESCRIPTION

[0052] According to embodiments of the present disclosure, safe, efficient, and effective treatments for neurodegenerative disorders and diseases include the systemic or intranasal administration of a therapeutic conjugate to a patient suffering from a neurodegenerative disorder or disease or otherwise expressing symptoms of cognitive decline or impairment. The therapeutic conjugate according to embodiments of the present disclosure includes a carrier peptide, and a therapeutic effector agent.

[0053] 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.

[0054] Abbreviations for amino acids are also used throughout this disclosure and similarly 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.

[0055] 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 in the brain. 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 or complexed with the carrier peptide to form the conjugates according to embodiments of the present disclosure.

[0056] 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 or spinal column. In some embodiments, as disclosed herein, the target may be target cells or extracellular molecules. Non-limiting examples of effector agents that can be conjugated or linked to Iynx1-loop2-derived peptides include: small interfering RNA (siRNA); divalent siRNA (see, e.q., Julia F Alterman, et al., 2019, “A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system,” Nat Biotechnol 2019 Aug; 37(8):884-894, the entire content of which is incorporated herein by reference); 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 or antisense oligonucleotides (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. In some embodiments, however, the effector agent may be or include an oligomer or oligonucleotide. Nonlimiting examples of such oligomer or oligonucleotide effector agents include: siRNA; divalent siRNA; short hairpin or stem loop RNA (shRNA); microRNA, double stranded RNA (dsRNA); strand template RNA (stRNA); an oligonucleotide (DNA or RNA); an anti-sense oligonucleotide (ASO); a modified oligonucleotide (DNA or RNA); aptamers; analogs and combinations of DNA and RNA; antisense oligomers or antisense oligonucleotides (ASO); or triplexforming oligonucleotides (TFO). In some embodiments, for example, the therapeutic effector agent may be or include an siRNA or antisense oligonucleotide. The effector agent can be conjugated or linked to a carrier, such as a carrier peptide or carrier peptide variant, as discussed further herein.

[0057] 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 triggercleavage 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, to increase their stability and / or bioavailability, and / or to decrease their immunostimulatory effects.

[0058] 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.

[0059] 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% to about 99%, for example, 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.

[0060] 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% to about 99%, for example, about 80%, 85%, 90%, 95%, or 99% to the reference polypeptide; andmay retain at least about 80% to about 99%, for example, 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.

[0061] 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 acids with 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.

[0062] 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. Non-limiting 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. In particular, the chemical modifications described in the context of siRNA may be applied to other types of nucleotides. Additionally, nonlimiting examples of suitable chemical derivatives include GalNAc-siRNA conjugates, which are approved by the United States Food and Drug Administration. See, e.g., Aaron D. Springer and Steven F. Dowdy, 2018, “GalNAc-siRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics,” Nucleic Acid Ther. 2018 Jun 1 ; 28(3): 109-118; and Alexandre J. Debacker, Jon Voutila, Matthew Catley, David Blakey, and Nagy Habib, 2020, “Delivery of Oligonucleotides to the Liver with GalNAc:From Research to Registered Therapeutic Drug,” Mol Ther. 2020 Aug 5; 28(8): 1759- 1771 , the entire contents of all of which are incorporated herein by reference.

[0063] 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% to about 99%, for example, 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% to about 99%, for example, 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.

[0064] 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 polynucleotide (e.g., an ASO polynucleotide or siRNA polynucleotide) may be collectively referred to as, for example, “ASO variants” or “ASO-derived sequences,” or “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 about 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.

[0065] 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 therapeutic 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 arewell 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).

[0066] 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 intravascular system by the BBB. In other embodiments, the target may be or include cells located in tissues outside the BBB.

[0067] 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 oc-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. Non-limiting examples of cells that express the a subunit 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.

[0068] 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.

[0069] All nucleotides and peptides disclosed herein, including nucleotide sequences (e.g., ASO and siRNA) 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, orexpression 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.

[0070] In some embodiments, the therapeutic effector agent 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.

[0071] 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 coding sequence together with the non-coding regions of the gene product, including the 5'UTR and 3'UTR 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.

[0072] Although some of the above definitions generally refer to a single-stranded molecule (oligonucleotides), 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."

[0073] 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. The compositions also can include stabilizers, preservatives, lubricants, and other adjuvants and excipients. For additional examples ofcarriers, stabilizers and adjuvants, see Genarro, AR., Remington: The Science and Practice of Pharmacy with Facts and Comparisons, 21st 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.

[0074] 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 acceptable material, 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 . In some embodiments, however, the amount of the effector agent may be significantly reduced without affecting efficacy or therapeutic effect. Indeed, in some embodiments, such a reduced amount of the effector agent may yield improved efficacy or therapeutic effect compared to higher or standard doses or dosages.

[0075] The compositions disclosed herein can be administered by any convenient route, including sublingual, parenteral, enteral, mucosal, topical, subcutaneous, intravascular, intravenous, intraarterial, intramuscular, intraperitoneal, transdermal, rectal, vaginal, intranasal, intraocular, intraspinal, or intracerebroventricular. In some embodiments, for example, the compositions as disclosed herein may be administered via a systemic administration route or an intranasal route. And in some embodiments, the compositions as disclosed herein are not topically administered or administered by intracranial delivery. As the compositions according to some embodiments are delivered by systemic or intranasal administration, these compositions present a safe, effective and efficient way to cross the BBB to deliver the therapeutic conjugates to targets across or within the BBB.

[0076] For example, 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.

[0077] The terms "parenteral administration" and "administered parenterally" as used herein to refer to modes of administration other than enteral and topical administration. In some embodiments, the term 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 (e.g., circulatory system) and is subject to metabolism and other like processes.

[0078] 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 symptom, disease or disorder being treated (e.g., a neurodegenerative disease or disorder). 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. However, as discussed further herein, according to embodiments of the present disclosure, and contrary to conventional wisdom, a reduced or low dose is used - i.e. , a dose rather lower than that which is considered safe and effective according to sound medical judgment.

[0079] 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 therapeutic effector agent in 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 / or compositions according to the present disclosure can enable more effective treatment with lower doses of the therapeutic effector agent, i.e., doses of the effector agent lower than 1 ,000 pg per kg (body weight), 600 pg per kg (body weight) or lower, lower than 4,000 pg per kg (body weight), lower than 1 ,000 pg per kg (body weight), or 100 pg per kg (body weight) or lower.

[0080] For example, in some embodiments, the compositions and / or conjugates disclosed herein may be administered to an animal subject (e.g., a mouse) in an amount sufficient to deliver the effector agent 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, 1 pg per kg to 600 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, 2 pg per kg to 600 pg per kg, 10 pg perkg 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, 10 pg per kg to 600 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, 20 pg per kg to 600 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, 40 pg per kg to 600 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, 100 pg per kg to 600 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, 200 pg per kg to 600 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, 350 pg per kg to 600 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 100 pg per kg, 2 pg per kg to 100 pg per kg, 10 pg per kg to 100 pg per kg, 20 pg per kg to 100 pg per kg, 40 pg per kg to 100 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 and / or conjugates disclosed herein may be administered to an animal subject (e.g., a mouse) in an amount sufficient to deliver the effector agent 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, 600 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.

[0081] 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 and / or conjugates disclosed herein may be administered to a human subject or patient in an amount sufficient to deliver the effector agent 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, 1 .7 pg per kg to 1 ,000 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, 3.4 pg per kg to 1 ,000 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, 17 pg per kg to 1 ,000 pg per kg, 34 pg per kg to 6,800 pg perkg, 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, 34 pg per kg to 1 ,000 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, 68 pg per kg to 1 ,000 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, 170 pg per kg to 1 ,000 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, 340 pg per kg to 1 ,000 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, 595 pg per kg to 1 ,000 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 and / or conjugates disclosed herein may be administered to a human subject or patient in an amount sufficient to deliver the effector agent 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 ,000 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.

[0082] As would be understood by those of ordinary skill in the art, the amount of the conjugate or complex (or composition) including the carrier peptide and the therapeutic effector agent needed to yield these ranges of the dose of the therapeutic effector agent will vary depending on the ratio (or relative amounts) of the carrier peptide to the therapeutic effector agent. By way of example only and without limitation, conjugates or complexes (or compositions) having ratios of effector agent to carrier peptide of 1 :4 and 1 :12, respectively, would require different total amounts of the composition as a whole to achieve the same effector agent dose. Those of ordinary skill in the art are capable of calculating the amount of the conjugate, complex or composition needed to achieve a specified effector agent dose based on the ratio of the carrier peptide to effector agent in the conjugate, complex or composition.

[0083] 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 ASO composition 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 / ormolecules). The agents of the present disclosure can be administered by any appropriate route that results in an effective treatment in the subject.Therapeutic Effector Agents

[0084] As discussed generally above, the effector agent may include 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. In some embodiments, as disclosed herein, the target may be target cells or extracellular molecules. Non-limiting examples of effector agents that can be conjugated or linked to carrier peptides disclosed herein include: siRNA; divalent siRNA; 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 or antisense oligonucleotides (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.

[0085] The effector agent can be conjugated or linked to a carrier, such as a carrier peptide or carrier peptide variant, as discussed further herein. Some non-limiting examples of effector agents and methods by which the effector agents can be conjugated, linked or complexed to a lynxl -derived peptide of the present invention, include: siRNA as disclosed herein and in Kumar et al., Nature 448: 39-43, 2007; Pulford et al., PLoS One 5:e11085, 2010; and Rohn et al., J. Drug Target, 20: 381 -388, 2012, the entire contents of all of which are incorporated herein by reference; hsRNA or microRNA as described in Hwang do et al., Biomaterials, 32: 4968-4975, 2011 , the entire contents of which are incorporated herein by reference; oligonucleotides (DNA or RNA) as described in Pardridge, Jpn J. Pharmacol, 87:97-103, 2001 , the entire contents of which are incorporated herein by reference; modified oligonucleotides (e.g., DNA or RNA) as described in Pardridge, 2011 , supra', genes as described in Pardridge, 2011 , supra, and Gong et al., Biomaterials, 33:3456-3463, 2012, the entire contents of which are incorporated herein by reference; peptides and PET ligands as described in Pardridge, 2011 , supra, proteins as described in Pardridge, 2011 , supra, and Xiang et al., J Drug Target, 19:632-636, 2011 , the entire contents of which are incorporated herein by reference; small chemical molecules as described in Zhan et al., Mol P / iarm, 7: 1940-1947 , 2010, the entire contents of which are incorporated herein by reference; large chemical molecules; viral particles; liposomes as described in Pulford et al., 2010, supra; endosomes: exosomes as described in Alvarez et al., Nat. Biotechnol., 29:341 -345, 2011 , the entire contents of which are incorporated herein by reference; nanoparticles as described in Chen et al., J Drug Target, 19:228-234, 2011 and Liu et al., Biomaterials, 30:4195-4202, 2009, the entire contents of both of which are incorporated herein by reference; dendrimers (e.g. PAMAM) as described in Liu et al., supra', eukaryotic cells; prokaryotic cells; and microspheres, nanogels, and bionanocapsules as described in Patelet al., CA / S Drugs, 23:35-58, 2009, the entire contents of which are incorporated herein by reference.

[0086] In some embodiments, however, the effector agent may be or include an oligomer or oligonucleotide. Nonlimiting examples of such oligomer or oligonucleotide effector agents include: siRNA; divalent siRNA; short hairpin or stem loop RNA (shRNA); microRNA, double stranded RNA (dsRNA); strand template RNA (stRNA); an oligonucleotide (DNA or RNA); an anti-sense oligonucleotide (ASO); a modified oligonucleotide (DNA or RNA); aptamers; analogs and combinations of DNA and RNA; antisense oligomers or antisense oligonucleotides (ASO); or triplex-forming oligonucleotides (TFO). For example, in some embodiments, the therapeutic effector agent may be or include an siRNA or antisense oligonucleotide.

[0087] The length of the oligomer or oligonucleotide effector agent is not particularly limited, and may be any suitable length depending on the intended use of the conjugate and / or composition (e.g., depending on the disease, disorder, or symptoms intended to be treated or alleviated by the therapy or treatment). However, in some embodiments, the oligomer or oligonucleotide (e.g., siRNA or ASO) may be 16 to 27 nucleobases in length (e.g., may be a 16-mer to 27-mer) exclusive of any 2-mer 3’ overhangs. In some embodiments, for example the oligomer or oligonucleotide may be an 18-mer to 22-mer, or a 19-mer, 20-mer, or 21 -mer. In some embodiments, the oligomer or oligonucleotide may be a 19-mer or 20-mer.

[0088] For example, in some embodiments in which the oligomer or oligonucleotide contains siRNA, the siRNA effector agent 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. Some non-limiting examples of suitable siRNA effector agents, for example for treating Huntington’s disease, are described in U.S. Provisional Application No. 63 / 592,542, titled LOW DOSE siRNA TREATMENTS OF HUNTINGTON’S DISEASE, filed on October 23, 2023 in the name of OPHIDION INC., the entire content of which is incorporated herein by reference.

[0089] And in some embodiments in which the oligomer or oligonucleotide contains ASO, the ASO effector agent may be 16 to 27 nucleobases in length (e.g., may be a 16- mer to 27-mer). In some embodiments, the ASO effector agent may be an 18-mer to 22- mer, or a 19-mer, 20-mer, or 21 -mer. In some embodiments, the ASO may be a 20-mer. Some non-limiting examples of suitable siRNA effector agents, for example for treating Huntington’s disease, are described in U.S. Provisional Application No. 63 / 418,945, titled LOW DOSE ANTI-SENSE OLIGONUCLEOTIDE (ASO) TREATMENTS OF HUNTINGTON’S DISEASE, filed on October 24, 2022 in the name of OPHIDION INC., and in corresponding PCT International Application No. PCT / US2023 / 017275, titled COMPOSITIONS AND METHODS FOR ANTI-SENSE OLIGONUCLEOTIDE (ASO) TREATMENT OF HUNTINGTON’S DISEASE, filed on April 3, 2023, the entire contents of all of which are incorporated herein by reference.

[0090] Surprisingly and unexpectedly, conjugation of the carrier peptides described herein with oligonucleotide effector agents enable effective administration of the therapeutic conjugates by systemic - rather than intracranial - administration routes. Additionally, conjugation of the carrier peptides with oligonucleotide effector agents has also surprisingly and unexpectedly been found to enable a significant reduction in the dose or dosage (as described above) of the therapeutic conjugate needed to achieve beneficial or therapeutic effect. Indeed, in some embodiments, administration of a reduced dose or dosage of the therapeutic conjugates yields better therapeutic efficacy than a standard or elevated dose or dosage.Carrier Peptides

[0091] According to embodiments of the present disclosure, the carrier peptide (e.g., carrier-derived peptide) facilitates delivery of the therapeutic effector agent to a suitable target (e.g., a target cell or tissue). 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 therapeutic effector agent payload into that cell or tissue.

[0092] 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 therapeutic effector agent (e.g., may be the point of contact between the therapeutic 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 sequences

[0093] 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.

[0094] 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 suitable as base sequences according to the present disclosure 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 “Iynx1-loop2-derived peptide” and “base sequence” may be interchangeably used herein.

[0095] 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 :

[0096] Xi is M or T;

[0097] X2 is T or I;

[0098] X3is T or W;

[0099] X4is R or C;

[0100] Xs is T or D;

[0101] X6is Y, I, or G;

[0102] Xy is F or Y;

[0103] X8is T or C;

[0104] Xg is P, N, or S;

[0105] X10 is Y, T, or S;

[0106] Xi2is M or G;

[0107] Xu is V or R;

[0108] X15 is R, S, A, or I; and

[0109] X16 is K, S, or D.

[0110] 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.

[0111] In some embodiments, the carrier peptide may include a lynxl -Ioop2-derived base sequence with the sequence MTTRTYATPYRMKVRKS (SEQ ID NO:6), which issimilar to SEQ ID N0:2 except that the phenylalanine (F) at position 7 is substituted with alanine (A). In some embodiments, the carrier peptide may include a lynx'! -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.

[0112] In some embodiments, additional amino acids may be added (appended, prepended, or inserted) to the Iynx1-loop2-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 Iynx1-loop2 -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.

[0113] In some embodiments, the carrier peptide may include the Iynx1 -loop2-derived base sequence of SEQ ID NO:4.

[0114] In some embodiments, the Iynx1 -loop2-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 :

[0115] Xs is T or W;

[0116] X4is R or C;

[0117] Xs is T or D;

[0118] Xe is any amino acid;

[0119] X7is F or Y;

[0120] Xs is any amino acid;

[0121] X9 is any amino acid;

[0122] X10 is any amino acid;

[0123] X12 is M or G; and

[0124] Xu is V or R.

[0125] 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:

[0126] XsisTorW;

[0127] X4is R or C;

[0128] Xs is T or D;

[0129] X6is Y, Gori;

[0130] X7isForY;

[0131] X8is T or C;

[0132] XgisP, N, orS;

[0133] X10 is Y, T, orS;

[0134] X12 is M orG; and

[0135] XuisV.

[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-R11-X12-K13-X14 (SEQ ID NO: 13), in which for SEQ ID NO:13:

[0137] XsisTorW;

[0138] X4is R or C;

[0139] Xs is T or D;

[0140] X6is Y, G or I;

[0141] X7isForY;

[0142] X8is T;

[0143] X9is P;

[0144] Xw isYorT;

[0145] X12 is M or G; and

[0146] XuisV.

[0147] 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 any 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 Iynx1-loop2-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.

[0148] Additionally, in some embodiments, the carrier peptide may include a lynxl - 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 additional amino acids, for example, one or more additional amino acids added to either end of the sequence. For example, in some embodiments, the carrier peptide may include a lynxl -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 up to 5 additional amino acids, for example, up to 5 additional amino acids added to either end of the sequence. However, embodiments of the present disclosure are not limited thereto, and for example, compositions according to embodiments of the present disclosure may incorporate other lynxl -Ioop2 base sequence variants. In some embodiments, for example, such variant sequences have 80% to 99% sequence identity to one of SEQ ID NOs: 1 -7 or 9 to 13, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.Linker sequences

[0149] 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 therapeutic effector agent. Stated another way, the linker sequence of the carrier peptide may participate in one or more conjugate-forming interactions with the therapeutic effector agent. Non-limiting examples of such interactions include electrostatic attraction, hydrogen bonding, Van der Waals interactions, covalent or ionic bondformation, and combinations thereof. In some embodiments of the present disclosure, compositions include a carrier peptide mixed and conjugated with certain oligonucleotides as the therapeutic effector agents, as discussed herein.

[0150] In some embodiments, the carrier peptide may include a linker sequence including 1 to 4 C-terminal glycine residues.

[0151] 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 certain oligonucleotides - such as 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., an oligonucleotide such as siRNA, ASO, or TFO).

[0152] Stated another way, in some embodiments, the effector agent (e.g., an oligonucleotide such as siRNA, ASO, or TFO) may be conjugated with the carrier peptide via the linker sequence due to an electrostatic attraction between the positively charged linker sequence and the negatively charged effector agent.

[0153] 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.

[0154] 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).

[0155] For example, the linker sequence may be represented by GGGG-Xs-Xe-XyXs- 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.

[0156] 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 three of these 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, eachinstance of Xs is independently selected from arginine and lysine, and m is an integer of 3 to 12.

[0157] 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 ASO. In some embodiments, the 7 to 9 amino acids may include or consist of arginine and / or lysine residues. And in some embodiments, the 7 to 9 amino acids may include or consist of all arginine residues, or all lysine residues.

[0158] For example, the linker sequence may be represented by GGGG-X5-X6-X7-X8- 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.

[0159] 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 variant sequences having 80% to 99% sequence identity thereto, e.g., at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto, etc.

[0160] Each of the amino acids in the linker sequence may have any suitable stereochemistry. When the linker sequence 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.

[0161] 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.

[0162] 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% to about 99%, for example, about 80%, 85%, 90%, 95%, or 99%, sequence and / or structural homology to the carrier peptide sequences described herein, as well as chemical derivatives of all of the above.Conjugates, Complexes and Compositions

[0163] As discussed generally above, according to embodiments of the present disclosure, therapeutic conjugates, complexes and / or compositions according to embodiments of the present disclosure include the carrier peptide and the therapeutic effector agent, which may be conjugated to each other. The relative amounts of the effector agent (e.g., oligomer or oligonucleotide) and the carrier peptide in the conjugate, complex and / or composition may be selected to maximize or increase the amount of the effector agent that is conjugated to carrier peptide and therefore available for delivery to the target.

[0164] Alternatively, as also discussed generally above, it has been surprisingly and unexpectedly found that conjugation of the effector agent to the carrier peptides according to embodiments of the present disclosure enables reductions in the amount of the effector agent needed to effect meaningful therapeutic effect. As such, while in some embodiments, the amount of effector agent conjugated to the carrier peptide may be maximized in order to maximize the amount of effector agent delivered to the target, according to some alternative embodiments of the present disclosure, lesser or reduced amounts of the effector agent may be conjugated to the carrier peptide. And in some embodiments, these conjugates or compositions having lesser or reduced amounts of the effector agent may provide similar (comparable) or even improved or increased therapeutic effect compared to their higher effector agent concentration counterparts.

[0165] In addition, the relative concentrations of the conjugates are related to the equilibrium constant for association (Ka) between the two molecules. Accordingly, the relative concentrations of the effector agent 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 effector agent to the carrier peptide may be 1 :12 to 1 :1 , 1 :10 to 1 :1 , 1 :8 to 1 :1 , 1 :6 to 1 :1 , 1 :4 to 1 :1 , or 1 :2 to 1 :1.

[0166] In some embodiments, however, the molar ratio of the effector agent and the carrier peptide may be 1 :4 to 1 :30, for example, 1 :5 to 1 :25,1 :5 to 1 :20, 1 :7 to 1 :25, 1 :7 to 1 :22, 1 :7 to 1 :18, 1 :10 to 1 :20, 1 :10 to 1 :15, or 1 :14 to 1 :18. In some embodiments, for example, the ratio of the effector agent and the carrier peptide may be 1 : 10 to 1 : 13 or 1 :15 to 1 :19. And in some example embodiments, the ratio of the effector agent and the carrier peptide may be 1 : greater than or equal to 10, for example, 1 : greater than 10 to 1 :30, 1 : greater than 10 to 1 :25, 1 : greater than 10 to 1 :22, 1 : greater than 10 to 1 :20, 1 : greater than 10 to 1 : 18, 1 : greater than 10 to 1 : 15. And in some alternative embodiments, the ratio of the effector agent and the carrier peptide may be 1 : lower than or equal to 10, for example, 1 :4 to 1 : lower than 10, 1 :2 to 1 : lower than 10, or 1 :7 to 1 : lower than 10.

[0167] For example, in some embodiments in which the effector agent is an siRNA effector agent, 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 to 1 :13.

[0168] And in some embodiments in which the effector agent is an ASO effector agent, for example, a molar ratio of the ASO effector agent and the carrier peptide may be 1 :5 to 1 :25, for example, 1 :7 to 1 :22, 1 :10 to 1 :20, or 1 :14 to 1 :18. In some embodiments, for example, the ratio of the ASO effector agent and the carrier peptide may be 1 :15 or 1 :19.

[0169] According to some embodiments, a pharmaceutical composition may include the conjugates and / or compositions of the effector agent and carrier peptide, and one or more pharmaceutically acceptable excipients, such as (but not limited to) pharmaceutically acceptable carriers. When the composition is prepared as a pharmaceutical solution, the effector agent 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 known 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%).

[0170] In some embodiments, the effector agent may be included in the pharmaceutical solution in an amount of 0.005% to 1 % by weight, for example, 0.015% 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 2.5 to 5.0 pg of effector agent (about 15 to 30 pg of conjugate) in 100 pL of solution. For example, in some embodiments in which the effector agent contains siRNA, the pharmaceutical solution may include about 5.0 pg of the siRNA effector agent (about 30 pg of conjugate) in 100 pL of solution. And in some embodiments in which the effector agent contains ASO, for example, the pharmaceutical solution may include about 2.5 pg of the ASO effector agent (about 15 pg of conjugate) in 100 pL of solution.

[0171] As discussed above, in some embodiments, the conjugates and / or compositions according to embodiments of the present disclose include effector agent conjugated to the carrier peptides. 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.Doses and Dosages

[0172] Throughout this disclosure, the terms “dose” and “dosage” are used in their art recognized senses. For example, while the term “dose” refers to a specified or measured amount of a therapeutic agent to be taken or given at any one given time, the term “dosage” refers to both the size of the dose to be taken as well as the timing regimen fortaking multiple doses. Accordingly, for example, X amount of a therapeutic agent is a “dose,” while X amount of a therapeutic agent to be taken twice daily for two weeks is a “dosage.”

[0173] According to some embodiments of the present disclosure, a dosage of the conjugate and / or composition may include any number of doses administered according to a dosage plan prescribed by a physician, without limitation. For example, in some embodiments, the dosage plan may include a single dose of the conjugate and / or composition administered only once. In some embodiments, however, the dosage plan may include multiple doses of the conjugate and / or composition administered according to a time interval prescribed by a physician. For example, in some embodiments, the dosage plan may include periodic doses, with subsequent doses administered a prescribed interval from the first dose. The prescribed interval is not particularly limited, and may be any interval prescribed by a physician. In some embodiments, however, the prescribed interval may be 1 hour to 6 months, for example, 1 hour to 3 months, 1 hour to 1 month, 1 hour to 1 week, or 1 hour to 1 day.

[0174] Each dose of the conjugate, complex and / or composition may comprise a pharmaceutical composition including the conjugate, complex and / or composition and one or more pharmaceutically acceptable excipients, such as (but not limited to) pharmaceutically acceptable carriers. The amount of the conjugate, complex and / or composition in each dose may vary depending on the prescription of the physician. But in some embodiments, each dose includes a low dose or reduced dose of the effector agent in the conjugate, complex and / or composition compared to a conventional dose unit. Such doses are described in more detail above. By way of illustration, in some embodiments, as discussed above, each dose may include a pharmaceutical composition including the conjugate, complex and / or composition in an amount sufficient to deliver an amount of the effector agent of lower than 0.1 mg per kg of body weight of the intended subject (or recipient), for example, lower than 4,000 pg per kg body weight of the intended subject, or lower than 1 ,000 pg per kg body weight of the intended subject.Administration and Treatment Methods

[0175] According to some embodiments of the present disclosure, a method of treating a neurodegenerative disorder or disease, or of alleviating, retarding or preventing (or reducing the likelihood of experiencing) symptoms of cognitive decline includes administering the conjugate and / or composition discussed herein to a target. The target may be in vivo or in vitro.

[0176] In some embodiments, the administering the conjugate and / or composition may include administering the conjugate and / or composition comprising the conjugate to a subject in need thereof. As used herein, the term “subject in need thereof” refers to a subject (animal or human) that has been diagnosed with, is suffering from, or is at risk of developing a neurodegenerative disorder or disease, or to a subject (animal or human)that is experiencing adverse cognitive symptoms, but which may not have been diagnosed with a neurodegenerative disease or disorder.

[0177] 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 conjugate and / or composition to the target includes delivery of the conjugate and / or composition across the BBB, which is enabled by the carrier peptide.

[0178] As discussed generally above, according to embodiments of the present disclosure, the conjugates and / or compositions disclosed herein can be administered by any convenient route, including sublingual, parenteral, enteral, mucosal, topical, subcutaneous, intravascular, intravenous, intraarterial, intramuscular, intraperitoneal, transdermal, rectal, vaginal, intranasal, intraocular, intraspinal, or intracerebroventricular. In some embodiments, for example, the conjugates and / or compositions as disclosed herein may be administered via a systemic administration route or an intranasal route. And in some embodiments, the conjugates and / or compositions as disclosed herein are not topically administered nor administered by intracranial delivery. As the compositions according to some embodiments are delivered by systemic or intranasal administration, these compositions present a safe, effective and efficient way to cross the BBB to deliver the therapeutic conjugates to targets across or within the BBB.

[0179] For example, 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.EXAMPLES

[0180] The following Examples are presented for illustrative purposes only, and do not limit the scope or content of the present application.

[0181] Table 1 below lists the sequences for the ASOs referenced in the following Examples.

[0182] Table 1 - Sequences fExample 1. In vitro knockdown of Huntingtin (HTT) mRNA by ASO conjugates

[0183] HTT ASO conjugates were initially evaluated for HTT knockdown efficacy and specificity in vitro, using lynx'! -Ioop2-derived carrier peptides to mediate delivery into human neuronal cells.

[0184] The key genes involved in lynx'! -Ioop2-mediated delivery via the nACHRs include the nicotinic receptor alpha? 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.

[0185] 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 differentiation media (day 1 of differentiation protocol). Two days later, 30% of the differentiation media was replaced with fresh differentiation media (day 3 of differentiation protocol). ASO1 to ASO5 (SEQ ID NOs: 18-22, as shown in the below Table 1 ) were tested with Peptide 1 (SEQ ID NO: 16) or Peptide 4 (SEQ ID NO: 17) as follows.

[0186] On day 4 of the differentiation protocol, Peptide 1 (SEQ ID NO:16):ASO or Peptide 4 (SEQ ID NO: 17): ASO complexes 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:ASOcomplexes. The final concentrations in the cell growth media were 15 |_iM peptide: 1 pM ASO (15:1 ratio).

[0187] 24 hours after exposure to the complexes, 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 pg 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 HTT; and Taqman assay Hs00240907_m1 for alpha-synuclein (SNCA). 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 ).

[0188] Result: Differentiated SHSY5Y cells showed a statistically significant decrease of HTT mRNA expression upon exposure to ASO3 (SEQ ID NQ:20) when complexed 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.

[0189] The cells showed a decrease in HTT mRNA expression upon exposure to ASO1 , ASO2, ASO3, ASO4, and ASO5 (SEQ ID NOS: 18-22) when complexed with either of Peptide 1 (SEQ ID NO: 16) or Peptide 4 (SEQ ID NO: 17), but also showed a simultaneous decrease in SNCA mRNA expression, indicating that these ASOs had comparatively lower specificity for HTT.

[0190] Table 2 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 1 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):ASO3 (SEQ ID NQ:20) was correlated with an 86% (P < .01 ) reduction of HTT target mRNA. Exposure to Peptide 4 (SEQ ID NO:17):ASO3 (SEQ ID NQ:20) was correlated with a 76% (P < .01 ) reduction of HTT target mRNA. Exposure to DMEMF12 (e.g., in the absence of any ASO or peptide) did not show any statistically significant effect on HTT mRNA levels.Table 2In Table 4, “Peptide 1” corresponds to SEQ ID NO. 16; Peptide 4” corresponds to SEQ ID NO. 17; and “ASO1” to “ASO5” correspond to SEQ ID NOS. 18, 19, 20, 21 and 22, respectively.

[0191] Conclusions: Statistically significant and specific knockdown of HTT mRNA by ASO3 (SEQ ID NO. 20), when complexed 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, ASO3 (SEQ ID NQ:20) was further tested in vivo for their efficacy in specific brain regions, discussed below.

[0192] To assess differences in knockdown according to differences in concentration of the ASO, selected ASOs were evaluated for HTT knockdown efficacy and specificity in vitro, using different ratios of the lynxl -Ioop2-derived carrier peptides to the ASOs. The protocol was generally as described above, except that the final concentrations of the peptide:ASO mixtures in the cell growth media were varied between a 15:1 ratio (15 pM peptide: 1 pM ASO), a 10:1 ratio (10 pM peptide: 1 pM ASO), and a 4:1 ratio (4 pM peptide: 1 pM ASO). Also, the final peptide:ASO mixtures (having different peptide: ASO 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.

[0193] Result: Differentiated SHSY5Y cells showed a statistically significant decrease of HTT mRNA expression upon exposure to all tested ASOs (including those with ASO3 (SEQ ID NO:20), ASO6 (SEQ ID NO:23) and ASO2 (SEQ ID NO: 19)) when conjugated to Peptide 1 (SEQ ID NO:16).

[0194] The cells showed a decrease in HTT mRNA expression upon exposure to ASO3, ASO6 and ASO2 (SEQ ID NOS: 20, 23 and 19, respectively) when conjugated to Peptide 1 (SEQ ID NO:16).

[0195] Table 3 summarizes the change in measured mRNA levels normalized to SNCA; values in bold highlight statistically significant knockdowns. Figure 2 presents the same data in bar chart form; the error bars represent the standard deviation. As shown in Table 3 and Figure 2, all of the peptide:ASO conjugates at all ratios exhibited significant knockdown, with the ASO6 (SEQ ID NO:23) conjugates showing better knockdown than the ASO3 (SEQ ID NQ:20) and ASO2 (SEQ ID NO:19) conjugates. And the ASO6 (SEQ ID NO:23) conjugates exhibited similar knockdown at all tested ratios (i.e., 15:1 , 10:1 and 4:1 ). But while all ASO3 (SEQ ID NQ:20) conjugates exhibited significant knockdown, the 15:1 and 10:1 ratios exhibited better knockdown than the 4:1 ratio. Similarly, while all ASO2 (SEQ ID NO: 19) conjugates exhibited significant knockdown, the 15:1 ratio exhibited better knockdown than the 4:1 ratio. Exposure to Dextrose-0.6% alone (e.g., in the absence of any ASO or peptide) did not show any statistically significant effect on HTT mRNA levels.Table 3Example 2. In vivo knock-down of mouse HTT mRNA by AS03 (SEQ ID N0:20) with Peptide 4 (SEQ ID NO: 17)

[0196] 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. Male 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. ASOs were synthesized by Integrated DNA Technologies (IDT, Coralville, IA). Carrier peptides were synthesized by Lifetein (Hillsborough, NJ).

[0197] ASO3 (SEQ ID NO: 20) was administrated to each animal using the following procedure. Briefly, 25 ag of ASO3 (SEQ ID NO:20) and Peptide 4 (SEQ ID NO: 17) peptide were mixed in vital dye (Evans Blue) / saline solution to produce a 15:1 or 19:1 molar ratio of Peptide 4 to ASO3, and incubated for 15 minutes at room temperature. The Peptide 4:ASO3 complexes 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.

[0198] 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 Lig 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 HTT target mRNA was assessed by comparison to SNCA using suitable primers for RT-qPCR: Taqman assay Mm01213763 for HTT, and Taqman assay Mm01188700_m1 for SNCA. The qPCR values of the HTT and SNCA gene were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Taqman assay Mm99999915_g1 ) and peptidylprolyl isomerase (PPIA, Taqman assay Mm02342430_g1 ).

[0199] Result: ASO3 (SEQ ID NQ:20) complexed with Peptide 4 (SEQ ID NO: 17) showed a statistically significant decrease in HTT mRNA expression in the HP and ST of wild-type mice. Table 4 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 3 presents the same data in bar chart form; the error bars represent the standard deviation. In particular, a 48% (P < .01 ) reduction of HTT mRNA was observed in the HP of mice injected with Peptide 4 (SEQ ID NO:17):ASO3 (SEQ ID NQ:20), and a 26% (P < .01 ) reduction was observed in the ST.Table 4In Table 4, “Peptide 4” corresponds to SEQ ID NO. 17; and “ASO3” corresponds to SEQ ID NO: 20.

[0200] Conclusions: Statistically significant and specific knockdown of HTT mRNA was observed in vivo in wild-type mice treated with ASO3 (SEQ ID NO:20) complexed with Peptide 4 (SEQ ID NO: 17).Example 3. Chronic Dosage of 25 g in Humanized zQ175 Huntington’s mice

[0201] Methods: Male and female zQ175 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 ASOs and carrier peptides were obtained as in Example 2.

[0202] Three groups of 7-8 mice each were tested in this study, including: 1 ) ASO6 conjugate treated mice; 2) vehicle control mice; and 3) Wild-Type (WT) mice. Each mouse was 12 weeks old at the initiation of the therapy, and was administered one dose every two weeks for a total of 6 months. Each dose included an ASO6 (SEQ ID NO:23) conjugate administrated to each animal using the following procedure. Briefly, 25 pg of ASO6 (SEQ ID NO:23) and Peptide 1 (SEQ ID NO: 16) were mixed in a dextrose-0.6% polysorbate 80 solution to produce a 10:1 molar ratio of peptide to ASO, and incubated for 15 minutes at room temperature. The peptide:ASO conjugates were then delivered with a dose of 25 pg of ASO6 (SEQ ID NO:23) as a 100 pL injection per mouse via the lateral tail vein. An identical volume of a dextrose-0.6% polysorbate 80 solution was injected in vehicle control animals.

[0203] Total rearing frequency, total distance traveled, and post-treatment mutant HTT protein levels in the cortex (CTX) and striatum (ST) were assessed. Soluble expanded mutant HTT levels in both the CTX and the ST were assessed via Assay 6 (2B7 / MW1 - ST), and aggregated mutant HTT levels in both the CTX and ST were assessed via Assay 45 (MW8 / 4C9-ST). The results of these assessments are shown in Figures 4A (total rearing frequency), 4B (total distance traveled), 5A (soluble expanded mutant HTT levels in the CTX), 5B (aggregated mutant HTT levels in the CTX), 5C (soluble expanded mutant HTT levels in the ST) and 5D (aggregated mutant HTT levels in the ST). Tables 5 and 6, below, show the total values (and standard deviations (SD)) for the distance traveled and rearing frequency. Tables 7-10, below, show the values for the soluble expanded and aggregated mHTT levels (average ± standard deviation (SD)) in the CTX (Tables 7 and 8) and ST (Tables 9 and 10).Table 5: Distance Traveled (25 pg study)Table 6: Rearing Frequency (25 pg study)Table 7: Expanded mHTT in CTX in 25 pg study (Assay 6: 2B7 / MW1-ST)Table 8: Aggregated mHTT in CTX in 25 pg study (Assay 45: MW8 / 4C9-ST)Table 9: Soluble expanded mHTT in ST in 25 pg study (Assay 6: 2B7 / MW1-ST)Table 10: Aggregated mHTT in ST in 25 pg study (Assay 45: MW8 / 4C9-ST)

[0204] Results: As shown in FIG. 4A, the Total Rearing Frequency in the ASO6 treated group was similar to that of the WT group (reversal of phenotype). . There were significant differences in total rearing between the treatment groups (F (3, 27) = 8.797, p = 0.0003) with the untreated WT group (p < 0.01 ) and the group treated with ASO6 (p < 0.05) rearing significantly more compared to the group treated with the vehicle control. It is important to note that the ASO6 treated group is statistically different than the vehicle group, and the ASO6 group is statistically the same as the wild-type group, suggesting the ASO6 treatment prevents neurodegeneration that leads to reduced behavior.

[0205] This improvement in function correlates well with the reduction of the soluble protein level in the cortex (12%) and no change in the aggregated protein in the cortex, soluble or aggregated protein in the striatum. See FIGs. 5A through 5D.Example 4. Chronic Dosage of 2.5 g in Humanized zQ175 Huntington’s mice

[0206] Methods: Male and female zQ175 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 ASOs and carrier peptides were obtained as in Example 2.

[0207] Three groups of 7-8 mice each were tested in this study, including: 1 ) AS06 conjugate treated mice; 2) ASO4 conjugate treated mice; and 3) vehicle control mice. Each mouse was 15 weeks old at the initiation of the therapy, and was administered one dose every two weeks for a total of 3 months. Each dose included an ASO6 (SEQ ID NO:23) or ASO4 (SEQ ID NO:21 ) conjugate administrated to each animal using the following procedure. Briefly, 2.5 pg of ASO6 (SEQ ID NO:41 ) or ASO4 (SEQ ID NO: 100) and Peptide 1 (SEQ ID NO: 16) were mixed in a dextrose-0.6% polysorbate 80 solution to produce a 10:1 molar ratio of peptide to ASO, and incubated for 15 minutes at room temperature. The peptide:ASO conjugates were then delivered with a dose of 2.5 pg of ASO6 (SEQ ID NO:23) or ASO4 (SEQ ID NO:21 ) as a 100 pL injection per mouse via the lateral tail vein. An identical volume of a dextrose-0.6% polysorbate 80 solution was injected in vehicle control animals.

[0208] Total rearing frequency, total distance traveled, and post-treatment expanded and aggregated mutant HTT levels in the cortex (CTX) and striatum (ST) were assessed. Expanded mutant HTT levels in both the CTX and the ST were assessed via Assay 6 (2B7 / MW1-ST), and aggregated mutant HTT levels in both the CTX and ST were assessed via Assay 45 (MW8 / 4C9-ST). The results of these assessments are shown in Figures 6A (total rearing frequency), 6B (total distance traveled), 7A (soluble expanded mutant HTT levels in the CTX), 7B (aggregated mutant HTT levels in the CTX), 7C (soluble expanded mutant HTT levels in the ST) and 7D (aggregated mutant HTT levels in the ST). Tables 11 and 12, below, show the total values (and standard deviations (SD)) for the distance traveled and rearing frequency. Tables 13-16, below, show the values for the expanded and aggregated mHTT levels (average ± standard deviation (SD)) in the CTX (Tables 13 and 14) and ST (Tables 15 and 16).Table 11 : Distance Traveled (2.5 pg study)Table 12: Rearing Frequency (2.5 pg study)Table 13: Expanded mHTT in Cortex in 2.5 pg study (Assay 6: 2B7 / MW1-ST)Table 14: Aggregated mHTT in Cortex in 2.5 pg study (Assay 45: MW8 / 4C9-ST)Table 15: Soluble Expanded mHTT in Striatum 2.5 pg study (Assay 6: 2B7 / MW1-ST)Table 16: Aggregated mHTT in Striatum in 2.5 pg study (Assay 45: MW8 / 4C9-ST)

[0209] Results: As shown in Tables 11 and 12 above, and in Figures 6A and 6B, ASO6 exhibited significant improvements in function in the Total Rearing Frequency and the Total Distance Travelled, and ASO4 exhibited improvement in the Total Rearing Frequency. This improvement in function in the ASO6 treated group correlates well with a reduction of the soluble and aggregated protein in the Cortex (17% and 16% reduction, respectively) and the soluble protein in the striatum of 14% (there was no change in the aggregated protein in the striatum). It is important to note that the ASO6 treated group rearing and total distance traveled is statistically different than the vehicle group, suggesting the ASO6 treatment prevents neurodegeneration that leads to reduced movement behavior. See Figures 7A and 7B. The improvement in function for the ASO4 group also correlates well with the aggregated protein in the Cortex. See Figure 7B.

[0210] Also, as can be seen from both independent chronic dose response studies, the compositions according to embodiments of the present disclosure exhibit significant improvements in functional measures, demonstrating a reversal of phenotype. For example, as shown in Figure 8 (which compares results of both studies), in the higher dose (25 pg) chronic dose response study, the ASO6 treated mice showed a Total Rearing Frequency after treatment of 91 % of WT and no improvement in Total Distance Traveled. And in the lower dose (2.5 pg) chronic dose response study, the ASO6 treated mice showed a Total Rearing Frequency of 97% of WT in addition to a Total Distance Traveled of 81 % of WT.

[0211] Additionally, while both dose response studies indicate significant improvements, the lower dose (2.5 pg) surprisingly and unexpectedly improved the function with respect to both Total Distance Travelled and Total Rearing Frequency in the amount of 25% and 50% relative to the vehicle control group, while the high dose (25 pg) only improved the Total Rearing Frequency relative to the vehicle control group in theamount of 39%. Relative to the WT mice, the Total Rearing Frequency was improved by 97% for the lower dose (2.5 pg) and 91 % for the higher dose (25 pg) - both demonstrating a reversal of the phenotype of the zQ175 mice back to WT as noted above. But the Total Distance Traveled was surprisingly and unexpectedly improved by 81 % relative to the WT group in the lower dose (2.5 pg) study, while no improvement in the Total Distance Travelled relative to WT group (or the vehicle control group) was observed in the high dose (25 pg) study.

[0212] Further, while both studies show statistically significant functional improvements and protein knockdown results, a comparison of the two studies surprisingly and unexpectedly shows that the lower dose (2.5 pg) performed better than the higher dose (25 pg). For example, as seen in a comparison of Figures 5B and 7B, the lower dose (2.5 pg) exhibited greater reduction of the aggregated protein in the cortex in the ASO6 treated group (statistically significant KD of 16%) compared to no statistically significant KD in the cortex in the high dose (25 pg) ASO6 treated group. Additionally, as seen in Figure 5C and 7C, the lower dose (2.5 pg) exhibited greater reduction of the soluble protein in the striatum in the ASO6 treated group (statistically significant KD of 14%) compared to no statistically significant KD in the striatum in the high dose (25 pg) ASO6 treated group. The surprising finding that a dramatically low dose is effective in reducing a target mRNA and protein reduces the risk to patients from general ASO associated toxicity.

[0213] Table 17 below lists the sequences for the siRNAs referenced in the following Examples.

[0214] Table 17 - Sequences for siRNAs 1 -4Example 5. In vitro knockdown of HTT mRNA by siRNAs

[0215] HTT siRNAs were initially evaluated for HTT knockdown efficacy and specificity in vitro, using Iynx1-loop2-derived carrier peptides to mediate delivery into human neuronal cells.

[0216] The key genes involved in Iynx1-loop2-mediated delivery via the nAChRs include the nicotinic receptor alpha? 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.

[0217] 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 of differentiation protocol). Two days later, 30% of the differentiation media was replaced with fresh differentiation media (day 3 of differentiation protocol). siRNAs 1 -6 (SEQ ID NOs:24, 25, 26, 27, 28, 29) were tested with Peptide 1 (SEQ ID NO: 16) or Peptide 4 (SEQ ID NO: 17) as follows.

[0218] 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 pM for Peptide 1 (SEQ ID NO:16):siRNA conjugates and 10 pM-0.77 pM for Peptide 4 (SEQ ID NO:17):siRNA conjugates (10:1 and 13:1 ratio respectively).

[0219] 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 pg 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 NO:24), and Peptide 4 (SEQ ID NO:17):siRNA4 (SEQ ID NO:27); Taqman assay Hs00918128_m1 (HTT2) for Peptide 4 (SEQ ID NO:17):siRNA3 (SEQ ID NO:26); and Taqman assay Hs00240907_m1 (SNCA) for all samples. The qPCR values of the HTT and SNCA genewere 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.

[0220] Resu / ts: Differentiated SHSY5Y cells showed a statistically significant decrease of HTT mRNA expression upon exposure to siRNA3 (SEQ ID NO:26) or siRNA4 (SEQ ID NO:27) 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.

[0221] Table 18 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 9 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:26) was correlated with a 23% (P < .01 ) reduction of HTT mRNA. Exposure to Peptide 1 (SEQ ID NO:16):siRNA4 (SEQ ID NO:27) was correlated with a 16% (P < .05) reduction of HTT mRNA. Exposure to Peptide 4 (SEQ ID NO:17):siRNA3 (SEQ ID NO:26) was correlated with a 37% (P < .05) reduction of HTT mRNA. Exposure to Peptide 4 (SEQ ID NO:17):siRNA4 (SEQ ID NO:27) 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 18In Table 18, “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 NOS:24, 25, 26, 27 and 28).

[0222] Conclusions: Statistically significant and specific knockdown of HTT mRNA by siRNA3 (SEQ ID NO:26) or siRNA4 (SEQ ID NO:27), 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.

[0223] 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).

[0224] 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:26) and siRNA4 (SEQ ID NO:27) when conjugated to Peptide 1 (SEQ ID NO:16).

[0225] The cells showed a decrease in HTT mRNA expression upon exposure to siRNA 3 and siRNA4 (SEQ ID NOS:26 and 27, respectively) when conjugated to Peptide 1 (SEQ ID NO:16).

[0226] Table 19 summarizes the change in measured mRNA levels normalized to SNCA; values in bold highlight statistically significant knockdowns. Figure 10 presents the same data in bar chart form; the error bars represent the standard deviation. As shown in Table 19 and Figure 10, all of the peptide:siRNA conjugates at all ratios exhibited significant knockdown, with the siRNA4 (SEQ ID NO:27) conjugates showing better knockdown than the siRNA3 (SEQ ID NO:26) conjugates. And the siRNA3 (SEQ ID NO:23) conjugates exhibited similar knockdown at the 10:1 ratio across three batches. Also, the siRNA4 (SEQ ID NO:27) 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 the absence of any siRNA or peptide) did not show any statistically significant effect on HTT mRNA levels.Table 19Example 6. In vivo knock-down of mouse HTT mRNA by siRNA with Peptide 4 (SEQ IDNO: 17)

[0227] 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 weresynthesized by Dharmacon (Lafayette, CO). Carrier peptides were synthesized by Lifetein (Hillsborough, NJ).

[0228] siRNA3 (SEQ ID NO:26) or siRNA4 (SEQ ID NO:27) 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.

[0229] 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 5.

[0230] Result: siRNA3 (SEQ ID NO:26) and siRNA4 (SEQ ID NO:27) 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 HTT mRNA levels as normalized against SNCA and relative to the saline control; values in bold highlight statistically significant knockdowns. Figure 11 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:26), 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:27), 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:26 and 27).

[0231] Conclusions: Statistically significant and specific knockdown of HTT mRNA was observed in vivo in wild-type mice treated with siRNA3 (SEQ ID NO:26) or siRNA4 (SEQ ID NO:27), conjugated with Peptide 4 (SEQ ID NO: 17). Based on the data from this study, siRNA3 (SEQ ID NO:26) and siRNA4 (SEQ ID NO:27) were further tested in a humanized HTT mouse model.Example 7. In vivo knock-down of human HTT mRNA in transgenic mice

[0232] 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 6.

[0233] siRNA3 (SEQ ID NO:26) or siRNA4 (SEQ ID NO:27) 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.

[0234] 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 6. cDNA was generated from 1 pg of RNA and qPCR reaction triplicates were run as in Example 6. 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 5.

[0235] Result: siRNA3 (SEQ ID NO:26) and siRNA4 (SEQ ID NO:27) 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.

[0236] Table 20 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 12 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:26), 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:27), and a 49% (P < .01 ), reduction was observed in the ST.Table 20In Table 20, “Peptide 4” corresponds to (SEQ ID NO:17); and “siRNA3” and “siRNA4” respectively correspond to (SEQ ID NOS:26 and 27).

[0237] 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 8: In vivo knock-down of mouse HTT mRNA by siRNA with Peptide 1 (SEQ IDNO: 16) and Peptide 4 (SEQ ID NO: 17)

[0238] Methods: The same methods were used as in the experiment of Example 6, 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:26) was used in all samples.

[0239] 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:26), provided a specific and statistically significant decrease of HTT mRNA expression in the HP and ST of wild-type mice. Table 21 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 13 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:26). 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:26).Table 21

[0240] 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 9. In vivo knock-down of human HTT mRNA in Q175 mice

[0241] 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 6.

[0242] siRNA3 (SEQ ID NO:26) 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 (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.

[0243] 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 6. cDNA was generated from 1 pg of RNA and qPCR reaction triplicates were run as in Example 6. 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).

[0244] Result: siRNA3 (SEQ ID NO:26) 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 showsignificantly 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.

[0245] Tables 22-24 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 knockdowns. Figures 15A, 16A and 17A present the same data in bar chart form; the error bars represent the standard deviation. Tables 25-27 summarize the non-normalized data. Figures 15B, 16B and 17B present the same data in bar chart form; the error bars represent the standard deviation.Table 22 - Knock-down in Striatum (Normalized)Table 23 - Knock-Down in Cortex (Normalized)Table 24 - Knock-down in Hippocampus (Normalized)Table 25 - Knock-Down in Striatum (Non-normalized)Table 26 - Knock-Down in Cortex (Non-normalized)Table 27 - Knock-Down in Hippocampus (Non-normalized)

[0246] 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.

[0247] 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 present disclosure, as well as the appended claims, and equivalents thereof.

[0248] Also, 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 numericalprecision 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 solution with multiple solutes.

[0249] 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.

Claims

WHAT IS CLAIMED IS:

1. A conjugate, comprising: a therapeutic effector agent; and a carrier peptide comprising SEQ ID NO:1, a molar ratio of the therapeutic effector agent to the carrier peptide being 1 : greater than or equal to 10.

2. The conjugate of claim 1 , wherein the therapeutic effector agent comprises an oligomer or oligonucleotide.

3. The conjugate of any of claims 1 and 2, wherein the therapeutic effector agent comprises siRNA or an anti-sense oligonucleotide.

4. The conjugate according to any preceding claim, wherein the carrier peptide comprises SEQ ID NO:4.

5. A conjugate, comprising: a therapeutic effector agent; and a carrier peptide comprising SEQ ID NO:1, a molar ratio of the therapeutic effector agent to the carrier peptide being 1 : lower than or equal to 10.

6. The conjugate of claim 5, wherein the therapeutic effector agent comprises an oligomer or oligonucleotide.

7. The conjugate of any of claims 5 and 6, wherein the therapeutic effector agent comprises siRNA or an anti-sense oligonucleotide.

8. The conjugate according to any of claims 5-7, wherein the carrier peptide comprises SEQ ID NO:4.

9. A pharmaceutical composition for administration to a subject, comprising: the conjugate of any preceding claim; and one or more pharmaceutically acceptable excipients.

10. The pharmaceutical composition of claim 9, wherein a concentration of the therapeutic effector agent is lower than 4,000 pg per kg of body weight of the subject.

11. The pharmaceutical composition of any of claims 9 and 10, wherein a concentration of the therapeutic effector agent is lower than 600 pg per kg of body weight of the subject.

12. The pharmaceutical composition of any of claims 9-11 , wherein a concentration of the therapeutic effector agent is lower than 100 pg per kg of body weight of the subject.

13. The pharmaceutical composition of any of claims 9-12, wherein the carrier peptide of the conjugate comprises SEQ ID NO:4.

14. The pharmaceutical composition of any of claims 9-13, wherein the therapeutic effector agent comprises an oligomer or oligonucleotide.

15. The pharmaceutical composition of and of claims 9-14, wherein the therapeutic effector agent comprises siRNA or an anti-sense oligonucleotide.

16. A pharmaceutical composition for administration to a subject, comprising: a conjugate comprising a therapeutic effector agent conjugated to a carrier peptide comprising SEQ ID NO:1 ; and one or more pharmaceutically acceptable excipients, a concentration of the therapeutic effector agent being lower than 4,000 pg per kg of body weight of the subject.

17. The pharmaceutical composition of claim 16, wherein the concentration of the therapeutic effector agent is lower than 600 pg per kg of body weight of the subject.

18. The pharmaceutical composition of any of claims 16 and 17, wherein the carrier peptide comprises SEQ ID NO:4.

19. A method of administering the conjugate of any of claims 1-8 or the pharmaceutical composition of any of claims 9-18 to a subject, the method comprising: administering an amount of the conjugate or pharmaceutical composition sufficient to deliver an amount of the therapeutic effector agent lower than 4,000 pg per kg of body weight of the subject.

20. The method of claim 19, wherein the amount of the conjugate or pharmaceutical composition is sufficient to deliver an amount of the effector agent lower than 600 pg per kg of body weight of the subject.

21. The method of claim 19 or 20, wherein the amount of the conjugate or pharmaceutical composition is sufficient to deliver an amount of the effector agent lower than 100 pg per kg of body weight of the subject.

22. The method of any of claims 19-21 , wherein the carrier peptide of the conjugate comprises SEQ ID NO:4.

23. The method of any of claims 19-22, wherein the therapeutic effector agent of the conjugate comprises an oligomer or oligonucleotide.

24. The method of any of claims 19-23, wherein the therapeutic effector agent of the conjugate comprises siRNA or an anti-sense oligonucleotide.

25. The method of any of claims 19-24, wherein the subject has been diagnosed with, suffers from, or is at risk of developing a neurodegenerative disorder or disease.

26. The method of any of claims 19-25, where the subject is experiencing or has experienced symptoms of cognitive decline.

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