Apolipoprotein AI nanodiscs for central nervous system delivery

Abstract

This disclosure provides therapeutic nanodiscs comprising a lipid-binding polypeptide, a lipid bilayer, and a therapeutic agent, wherein the therapeutic agent may be used to treat, prevent, or diagnose central nervous system diseases, disorders, injuries, or injuries. The lipid bilayer may be 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), and the therapeutic agent may be a nucleic acid polymer. Furthermore, methods for administering therapeutic nanodiscs to treat, prevent, or diagnose central nervous system diseases, disorders, injuries, or injuries, as well as the use of such therapeutic nanodiscs, are provided.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority rights to U.S. Provisional Patent Application US63 / 472,886 and U.S. Provisional Patent Application US63 / 472,887, both filed on June 14, 2023.

[0002] This disclosure relates to compositions and methods for treating, preventing or diagnosing neurological diseases or disorders. In particular, the compositions include apolipoprotein AI nanodiscs for delivering therapeutic agents to the central nervous system. [Background technology]

[0003] Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder characterized by the onset of motor, cognitive, and psychiatric symptoms. HD is caused by a CAG trinucleotide repeat expansion in the HTT gene, which encodes the Huntington's protein.[1] The resulting mutant Huntington's protein (mHTT) has an elongated polyglutamine region and is associated with the acquisition of toxic function.[2] This leads to progressive degeneration of neurons throughout the brain, particularly cortical and striatal neurons.[3]

[0004] Neuronal loss also occurs, albeit to a lesser degree, in other brain regions, including the hippocampus, substantia nigra, and brainstem nuclei, and correlates with the broad range of symptoms experienced by patients with late-stage HD [4]. Currently approved treatments for Huntington's disease only attempt to alleviate the symptoms of chorea and do not slow or halt its progression.

[0005] One promising therapeutic approach involves reducing mHTT levels in the brain using gene silencing techniques. Previous studies using conditional mHTT knockouts in mice have suggested that downregulating mHTT expression improves pathological and behavioral outcomes of the disease [5]. Further research has demonstrated the therapeutic potential of reducing mHTT using antisense oligonucleotides (ASOs) [6,7] as well as other RNA interference therapies [8-12].

[0006] Due to its high water solubility, low toxicity, long tissue half-life, stability at room temperature, and cellular uptake via endocytosis, ASO is a particularly promising approach to reducing mHTT levels compared to other RNA-silencing therapies [13,14]. However, the therapeutic potential of ASO is limited by its inability to cross the blood-brain barrier (BBB) ​​when administered into the systemic circulation. Therefore, they are administered to the central nervous system (CNS) via direct surgical intervention into the cerebrospinal fluid (CSF) compartment. In mice, intraventricular (ICV) injection of ASO results in broad distribution throughout the CNS and dose-dependent suppression of HTT

[15] .

[0007] However, in non-human primates, intrathecal CSF administration results in limited brain distribution, with higher ASO concentrations in surface areas and lower concentrations in deeper areas such as the basal ganglia [6]. Because HD is a diffusion brain disorder, broad delivery of HTT-reducing agents is necessary to achieve full therapeutic potential for Huntington's depression, with significant delivery to the most affected deep brain regions.

[0008] ASOs have recently been administered via intrathecal (IT) infusion in clinical trials for several CNS disorders, including spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and hemoglobin (HD) [16-20]. The success in terms of therapeutic outcomes for SMA and ALS stems from the fact that these diseases primarily affect the spinal cord, which is particularly well-suited for intrathecal delivery. While ASOs provide a pathway for entry into the CNS, intrathecal delivery is invasive and requires repeated administration. Therefore, this route of administration carries the risk of infection, CSF leakage, and other serious surgical complications in immunocompromised patients

[21] .

[0009] Furthermore, based on previous non-human primate (NHP) studies, it is likely that intrathecal infusion of ASO in patients will result in limited brain distribution, and therefore limited target binding in the brain regions most affected by HD, namely the deeper layers of the cortex and striatum [6]. This medial-lateral concentration gradient is not unique to ASO and has also been observed around the catheter tip after intrathecal infusion of small molecule drugs such as morphine in patients

[22] .

[0010] A Phase III clinical trial in which HD patients received the HTT-targeting ASO tominersen was halted due to worsening patient outcomes and the occurrence of ventricular enlargement in one treatment arm with more frequent dosing

[23] . The increased ventricular volume has been hypothesized to be a result of increased CSF volume, impaired CSF flow and / or neuroinflammation, but a definitive explanation has yet to be determined

[24] . Hydrocephalus has also been demonstrated to occur in rhesus monkeys after IT administration of dextran

[25] and has been reported after intrathecal treatment with the ASO nusinersen in SMA patients

[26] . This raises the question of whether ventricular enlargement may be a consequence of IT delivery, and whether subsequent accumulation of negatively charged fractions into the ventricles may lead to an inflammatory response and impaired CSF flow.

[0011] Intranasal administration routes offer a non-invasive approach for direct delivery of therapeutic macromolecules to the brain that do not cross the blood-brain barrier (BBB). A growing number of substances, including proteins, siRNAs, viral and non-viral vectors, and even stem cells, have been shown to rapidly enter the brain via nasal routes[27-32], some of which have been tested in human clinical trials for CNS disorders[33,34]. Substances administered intranasally acquire uptake into the brain by following olfactory and trigeminal pathways originating from the nasal cavity

[35] . Once in the brain, intranasal biomolecules migrate through the perivascular space (perivascular) and rapidly distribute at the microvessel level, achieving deeper penetration into the brain parenchyma compared to intrathecal delivery

[36] .

[0012] IT infusion of ASO (intrathecal delivery) into cerebrospinal fluid is currently used to deliver HTT-reducing ASO to the brain in HD clinical trials. Recently, IT infusion of the human HTT-targeting ASO, HTT_RX, has been shown to induce a dose-dependent reduction in mHTT concentration in CSF in HD patients, suggesting HTT target binding in the brain

[70] . In non-human primates, ASO delivered by IT infusion is heterogeneously distributed throughout the brain, resulting in higher HTT suppression in surface areas of the cortex and spinal cord and reduced HTT suppression in deeper brain structures such as the basal ganglia [6,7]. In the human brain, this gradient may be exacerbated because the human brain volume is several times larger than that of NHPs

[71] . Furthermore, since ASO degrades over time in vivo, repeated IT infusions are required to maintain the HTT-reducing effect, making this administration route considerably invasive

[14] . However, IT infusion of ASO would also bypass surrounding tissues. The surrounding tissue is also a significant site of pathology in HD

[72] .

[0013] The advantages of intranasal administration are that it bypasses the blood-brain barrier (BBB), directly targets the brain, results in broad brain distribution (particularly in deeper brain structures such as the basal ganglia), and limits peripheral exposure to the therapeutic agent. However, a potential limitation of intranasal administration is that it is an inefficient route of delivery insofar as fractions of the intranasal dose reach the brain

[35] , which can lead to undesirable side effects. Otherwise, it also has advantages in treating surrounding tissues.

[0014] One way to overcome this disadvantage is through the use of a carrier or nanoparticle carrier

[37] which may potentially limit the clearance of macromolecules in the nasal cavity and / or facilitate their ability to penetrate the nasal mucosa and pass through the olfactory epithelium—a rate-limiting step of intranasal administration

[38] . [Overview of the project]

[0015] This application is in part based on the fortunate discovery that apolipoprotein AI nanodiscs (NDs) produced with K133C or E146C ApoA-I lipid-binding polypeptides exhibit significantly improved CNS / brain delivery of ASO compared to wild-type ApoA-I.

[0016] Furthermore, this application demonstrates that NDs formed using intravenously administered apoA-I K133C or E146C show enhanced brain distribution compared to intravenously administered WT apoA-I. It also shows that single intranasal administration of apoA-I K133C ND, which carries the HTT-targeted ASO, results in a significant reduction in mHTT in the cortex and striatum.

[0017] In one embodiment, a therapeutic nanodisc is provided, which comprises (a) a lipid-binding polypeptide, encoded by SEQ ID NO: 3 or 4; (b) a lipid bilayer; and (c) a therapeutic agent.

[0018] According to further embodiments, therapeutic nanodiscs are provided, each comprising (a) a lipid-binding polypeptide, encoded by SEQ ID NO: 3 or 4; (b) a lipid bilayer; and (c) a nucleic acid polymer.

[0019] According to another embodiment, a composition is provided which comprises (a) a lipid-binding polypeptide, encoded by SEQ ID NO: 3 or 4; and (b) a lipid bilayer. The composition may further contain a therapeutic or diagnostic agent.

[0020] According to another embodiment, a method is provided for treating a neurological disorder or disability, the method comprising administering a therapeutic nanodisc described herein to a person in need thereof in such a manner that it contains an effective amount of a therapeutic agent.

[0021] According to another embodiment, a method is provided for treating or diagnosing a neurological disorder or condition, the method comprising administering a composition described herein, comprising an effective amount of a therapeutic agent to a person in need, or a diagnostic agent to a person in need.

[0022] According to another embodiment, the use of the therapeutic nanodiscs described herein for treating neurological disorders or disabilities is provided.

[0023] According to another embodiment, the use of the therapeutic nanodiscs described herein in the manufacture of a pharmaceutical product for treating a neurological disorder or disability is provided.

[0024] According to another embodiment, the use of the compositions described herein for treating neurological disorders or disabilities is provided.

[0025] According to another embodiment, the use of the compositions described herein in the manufacture of a pharmaceutical product for the treatment of a neurological disorder or disability is provided.

[0026] According to another embodiment, a diagnostic method for diagnosing a neurological disorder or disability is provided, the method comprising administering a composition described herein, which contains an effective amount of a diagnostic agent, to a person in need thereof.

[0027] According to another embodiment, a method is provided for administering the composition or therapeutic nanodisc described herein in an effective amount to a person in need.

[0028] The lipid bilayer may be selected from diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside. The lipid bilayer may be dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxy The following may be selected from silates (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (transDOPE), and one or more of their analogues.The lipid bilayer consists of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), cholesterol, and 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine (PEG-C-DMA). The lipid bilayer may be selected from one or more of the following: 1,2-dilinoleyloxy-3-(N,N-dimethyl)aminopropane (DLinDMA), N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadiene-1-yl-1,3-dioxolane-4-ethaneamine (DLin-KC2-DMA), 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadiene-1-yl-10,13-nonadecadiene-1-yl ester (DLin-MC3-DMA), and their analogues. The lipid bilayer may also be a 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer.

[0029] The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 400:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 350:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 300:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 250:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 200:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 190:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 180:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 170:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 160:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 150:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 140:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 130:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 120:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 110:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 105:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 100:1 and 400:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 75:1 and 167:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 61:1 and 124:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 80:1 and 110:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 80:1 and 120:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 70:1 and 120:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 60:1 and 120:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 50:1 and 120:1.The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 40:1 and 120:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be between 100:1 and 105:1. The molar ratio of the lipid bilayer to the lipid-bound polypeptide may be 105:1.

[0030] Therapeutic nanodiscs may have a diameter between approximately 5 nm and 100 nm, as measured by a transmission electron microscope (TEM). Alternatively, therapeutic nanodiscs may have a diameter between approximately 5 nm and 40 nm, as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter between approximately 5 nm and 50 nm, as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter between approximately 5 nm and 60 nm, as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter between approximately 5 nm and 70 nm, as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter between approximately 5 nm and 80 nm, as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter between approximately 5 nm and 90 nm, as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter between approximately 6 nm and 40 nm, as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter of approximately 7 nm to 40 nm as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter of approximately 8 nm to 40 nm as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter of approximately 9 nm to 40 nm as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter of approximately 10 nm to 40 nm as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter of approximately 10 nm to 30 nm as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter of approximately 10 nm to 20 nm as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter of approximately 5 nm to 20 nm as measured by a transmission electron microscope (TEM). Therapeutic nanodiscs may have a diameter of approximately 5 nm to 30 nm as measured by a transmission electron microscope (TEM).

[0031] The therapeutic agent may be an antisense oligonucleotide (ASO), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), aptamer, ribozyme, mRNA (messenger RNA), plasmid, ribonucleoprotein (RNP), or small molecule. The therapeutic agent may be an ASO. The ASO may target mutant Huntington's thyroid hormone (mHTT). The therapeutic agent may be selected from one or more of tominersen (CAS No.: 1709886-74-7), lovanersen, WVE-120102, WVE-003, AMT-130, and Sequence ID No. 5. The therapeutic nanodisc may be for delivering the therapeutic agent to the central nervous system. The therapeutic nanodisc may be for delivering the therapeutic agent systemically. The therapeutic nanodisc may be for delivering the therapeutic agent to the brain. The therapeutic nanodisc may be for delivering the therapeutic agent to the spinal cord. Therapeutic nanodiscs may be used to deliver therapeutic agents to specific or multiple parts of the brain. Therapeutic nanodiscs may be used to deliver therapeutic agents to one or more of the olfactory bulb and prefrontal cortex, striatum, thalamus and hypothalamus, and brainstem and cerebellum.

[0032] Therapeutic nanodiscs may be for the treatment of neurological diseases or disorders. Neurological diseases or disorders may be selected from one or more of the following: Huntington's disease (HD), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM), spinal muscular atrophy (SMA), spinocerebellar degeneration (SCA), spinal muscular atrophy (SMA), Parkinson's disease (PD), Alzheimer's disease (AD), frontotemporal dementia (FTD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), central nucleus myopathy, Alexander disease, cancer, stroke, meningitis, encephalitis, prion diseases, polyneuropathy due to hereditary ATTR amyloidosis (hATTR-PN), and polyneuropathy of hereditary transthyretin-mediated amyloidosis (ATTR). CNS disorders or conditions are selected from one or more of the following: Huntington's disease, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, neuropathy, spinal muscular atrophy, dementia, frontotemporal dementia, cancer, multiple sclerosis, hereditary metabolic disorders, and autoimmune diseases or disorders. Neurological disorders or conditions may also be classified as HD.

[0033] Neurological disorders or conditions may be selected from one or more of the following: Huntington's disease (HD), Duchenne muscular dystrophy (DMD), spinal muscular atrophy (SMA), Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), core muscle disease, Alexander disease, prion disease, polyneuropathy due to hereditary ATTR amyloidosis (hATTR-PN), polyneuropathy due to hereditary transthyretin-mediated amyloidosis (ATTR), and CNS cancers.

[0034] The therapeutic agent may be selected from one or more of the following: tetrabenazine (TBZ), deutetrabenazine (DBZ), olanzapine, risperidone, citalopram, fluoxetine, sertraline, lamotrigine, and carbamazepine. The therapeutic agents include tominersen, WVE-120102 / 120101, WVE-003, (CUG)7, TTX-3360, AMT-130, AAV.shHD2.1, VY-HTT01, branapram, PTC518, TAK-686, ZF-KOX1, pridopidine, laquinimod, fenofibrate, neframapimod, nilotinib, SRX246, varenicline, SAGE-718, PBT2, eteplirsen, SRP-5051, casimersen, renadylsen, golodylsen, viltolarsen, WVE-N531, nusinersen, ION306, ION859, ION464, IONIS-MAPT_Rx_, ATL1102, tofersen, urefnersen, and IONIS The therapeutic agent may be selected from one or more of the following: C9Rx, WVE-004, ION541, DYN101, zirganersen, ION717, aprontersen, inotersen, patisirane, butricirane, giboshiran, lumasirane, inclisiran, and pegaptanib. The therapeutic agent may also be SEQ ID NO: 5.

[0035] Therapeutic nanodiscs may be formulated as an aerosol. Therapeutic nanodiscs may be formulated for intravenous or intranasal administration. Therapeutic nanodiscs may be formulated for intravenous administration. Therapeutic nanodiscs may be formulated for CNS administration. Therapeutic nanodiscs may be formulated for intravenous injection (IV), intranasal inhalation, intraventricular (ICV) injection, lumbar intrathecal (IT) injection, intrathecal (IT) infusion, or cisterna magna injection.

[0036] The compositions described herein may further include therapeutic or diagnostic agents.

[0037] In some embodiments, the lipid may be a neutral lipid, a cationic lipid, or an ionizable lipid. In some embodiments, the neutral lipid may be dimyristoylphosphatidylcholine (DMPC). In some embodiments, the lipid may be deuterated.

[0038] In some embodiments, the nucleic acid delivery particles contain a molar ratio of about 30 moles of lipid component to about 1 mole of ApoA-I component. In some embodiments, the nucleic acid delivery particles contain a molar ratio of about 55 moles of DMPC to about 1 mole of ApoA-I. In some embodiments, the nucleic acid delivery particles contain a molar ratio of about 80 moles of DMPC to about 1 mole of ApoA-I. In some embodiments, the nucleic acid delivery particles contain a molar ratio of about 105 moles of DMPC to about 1 mole of ApoA-I. In some embodiments, the nucleic acid delivery particles contain a molar ratio of about 120 moles of DMPC to about 1 mole of ApoA-I.

[0039] In one embodiment, the nucleic acid polymer may be a DNA oligomer, an RNA oligomer, an antisense DNA oligonucleotide, an antisense RNA oligonucleotide, a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a plasmid, a messenger RNA (mRNA), or an aptamer. In one embodiment, the nucleic acid polymer is an antisense oligonucleotide (ASO). In one embodiment, the nucleic acid delivery particle contains a molar ratio of about 1 mole of ApoA-I to about 5 moles of ASO. In one embodiment, the nucleic acid delivery particle contains a molar ratio of about 1 mole of ApoA-I to about 3 moles of ASO. In one embodiment, the nucleic acid delivery particle contains a molar ratio of about 1 mole of ApoA-I to about 1 mole of ASO.

[0040] In a preferred embodiment, the nucleic acid delivery particles contain a molar ratio of about 1 mole of ApoA-I to about 3 moles of ASO to about 10⁵ moles of DMPC.

[0041] In one embodiment, the nucleic acid polymer component binds to the lipid component and / or the lipid-binding polypeptide component via electrostatic surface interactions. In one embodiment, the nucleic acid polymer component attaches to the lipid component and / or the lipid-binding polypeptide component by conjugation or other means. In one embodiment, the nucleic acid polymer may attach to the lipid component and / or the lipid-binding polypeptide component via a linker.

[0042] In one embodiment, a method is provided for regulating the expression of a target gene, which includes administering nucleic acid delivery particles to silence the expression of the target gene, or, as an alternative, administering nucleic acid delivery particles to induce the expression of the target gene. In one embodiment, a method is provided for regulating the expression of a target gene expressed in cells of the CNS or brain, which includes administering nucleic acid delivery particles to silence the expression of the target gene, or, as an alternative, administering nucleic acid delivery particles to induce the expression of the target gene. In one embodiment, the route of administration of the nucleic acid delivery particles is intravenous, parenteral, or intranasal.

[0043] In one embodiment, a method for treating or preventing a CNS disease in a mammal is provided, which comprises administering an effective amount of nucleic acid delivery particles for the purpose of modulating the expression of a target gene associated with a CNS disease or disorder, wherein delivery of the therapeutic nucleic acid polymer to the brain or CNS is preferred. In a further aspect of the invention, the CNS disease or disorder may include, but is not limited to, Huntington's disease, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, neuropathy, spinal muscular atrophy, dementia, frontotemporal dementia, cancer, multiple sclerosis, hereditary metabolic disorders, autoimmune diseases or disorders.

[0044] In one embodiment, a method is provided for treating or preventing a musculoskeletal or neuromuscular disease or disorder in a mammal, which comprises administering an effective amount of nucleic acid delivery particles, for the purpose of modulating the expression of a target gene associated with a musculoskeletal disease or disorder, wherein delivery of the therapeutic nucleic acid polymer to muscle tissue is preferred. In a further aspect of the invention, the musculoskeletal disease or disorder may include, but is not limited to, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, myotonic muscular dystrophy, limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dryfus muscular dystrophy, distal muscle disorder, spinal muscular atrophy (SMA), muscle atrophy, or myotonia.

[0045] In one embodiment, a method is provided for treating or preventing liver disease or impairment in mammals, which comprises administering an effective amount of nucleic acid delivery particles for the purpose of modulating the expression of a target gene associated with liver disease or impairment, wherein delivery of the therapeutic nucleic acid polymer to the liver is preferred. In a further aspect of the invention, liver disease or impairment may include, but is not limited to, cirrhosis, hepatocellular carcinoma, viral hepatitis, nonviral hepatitis, cholestatic liver disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hepatic fibrosis, and hereditary liver diseases such as hemoglobinosis, Wilson's disease, alpha-1 antitrypsin deficiency, hyperoxaluria, and oxalate deposition.

[0046] In one embodiment, a method is provided for treating or preventing a lung disease or disorder in a mammal, which is intended to modulate the expression of a target gene associated with the lung disease or disorder, comprising administering an effective amount of nucleic acid delivery particles, wherein delivery of the therapeutic nucleic acid polymer to the lungs is preferred. In a further aspect of the invention, the lung disease or disorder may include, but is not limited to, cystic fibrosis, chronic obstructive pulmonary disease, cancer, or pulmonary fibrosis.

[0047] In a further aspect, the pharmaceutical compositions described herein are provided to be used for the treatment, alleviation, or prevention of diseases or disorders of the lungs, muscles, or liver.

[0048] In a further aspect, the pharmaceutical compositions described herein are provided for use in the treatment, alleviation, or prevention of CNS diseases or disorders.

[0049] This summary does not necessarily describe all features of the invention. Other aspects, features, and advantages of the invention will become apparent to those ordinary skill in the art by reviewing the following description of specific embodiments of the invention. [Brief explanation of the drawing]

[0050] [Figure 1]Figure 1 shows that ApoA-I ND successfully forms electrostatic interactions with antisense oligonucleotides. (a) Electrophoretic mobility shift assay (EMSA) demonstrates that when ASO was incubated with different molar amounts of apoA-I ND (stoichiometric ratios from ASO to apoA-I of 1:1 to 3:1) at 37°C for 1 hour, ASO was visible at the same level as apoA-I ND, suggesting a potential electrostatic interaction between ASO and apoA-I. (b) Size exclusion chromatography results show the separation of nucleic acids, proteins, and phospholipids after ApoA-I ND was incubated with ASO at 37°C for 1 hour (molar ratio of 2.5 mol ASO:1 mol apoA-I:105 mol DMPC). ASO eluted in the same fraction as both apoA-I and DMPC. (c) Size exclusion chromatography results performed on each individual component (nucleic acid, protein, and phospholipid) alone (as negative controls). ASO, apoA-I, and DMPC all showed different elution patterns. (d) Oligreen ASO association assay results show that after treatment with the surfactant Triton X-100, the amount of ASO associated with both apoA-I WT and K133C ND was similar to that of untreated ND (two-way ANOVA did not show significance due to ND species, Triton X-100 treatment, or significant interactions). (e) Dynamic light scattering showed a slight shift in the size of NDs prepared using apoA-I WT or apoA-I K133C protein. ApoA-I WT NDs were larger in size compared to NDs formed with apoA-I K133C (n=3; mean diameter ± standard deviation = 10.76 ± 0.05 nm) (mean diameter ± standard deviation = 11.95 ± 0.34 nm). [Figure 2]Figure 2 shows that the lipid-free ApoA-I protein and apoA-I ND successfully reach the brain after intranasal administration. (a) BACHD mice were intranasally administered 150 μg of apoA-I WT (lipid-free), apoA-I K133C (lipid-free), apoA-I WT ND, apoA-I K133C ND, or PBS, and euthanized 2 hours later. Nanodisc-form apoA-I WT resulted in higher whole-brain delivery compared to the lipid-free apoA-I WT protein. Lipid-free K133C achieved considerably higher levels in the brain compared to both apoA-I WT and apoA-I WT ND, while K133C apoA-I ND achieved the highest apoA-I levels in the whole brain. One-way ANOVA: P<0.0001; Tukey's multiple comparison test: *p<0.05, ***p<0.001, ****p<0.0001). (b) Across the entire rostral-caudal brain slab (AD), apoA-I K133C ND yielded significantly higher levels of apoA-I in each region compared to apoA-I WT ND, except in the most caudal brain region D (including the brainstem and cerebellum). Two-way ANOVA shows significance by brain region (P<0.0001), treatment (P<0.0001), and significant interaction between brain region x treatment (P<0.01); Bonferroni's multiple comparison test: **p<0.01, ****p<0.0001. (c) Dorsal external imaging of the brain of BACHD mice 2 hours after intranasal administration of apoA-I K133C ND labeled with PBS or cf750 dye. (d) Ventral external imaging of the brain of BACHD mice 2 hours after intranasal administration of apoA-I K133C ND labeled with PBS or cf750 dye. (e) Representative figure showing the rostral-caudal brain slab used for ELISA (enzyme-linked immunosorbent assay). Section A consists of the olfactory bulb and frontal cortex, B is at the striatal level, C is at the thalamic and hypothalamic level, and D is at the brainstem and cerebellar level. [Figure 3]Figure 3 shows that intranasal administration of ApoA-I ND is unaffected by HD mutations at 2 and 6 months of age. WT and BACHD mice (2(a), 6(b), and 12(c) months of age) were intranasally administered K133C ApoA-I ND and euthanized 2 hours after intranasal administration. (a,b) At both 2 and 6 months of age, there were no significant differences in apoA-I levels across the brain between WT vs BACHD mice. Two-way ANOVA shows significance by brain region (P<0.0001). (c) At 12 months of age, significantly higher levels of apoA-I were measured in the most rostral brain region of A, consisting of the olfactory bulb and prefrontal cortex. Two-way ANOVA shows significance by brain region (P<0.0001); Bonferroni's multiple comparison test: *p<0.05. [Figure 4] Figure 4 shows the cellular localization of apoA-I after intranasal administration of ApoA-I K133C ND. Following intranasal administration of apoA-I K133C ND, apoA-I is deposited in various brain cells throughout the brain. These include NeuN+ neurons (ac), CD31+ capillary endothelial cells (df), Iba1+ microglia (gi), GFAP+ astrocytes (jl), and lectin-labeled choroid plexus cells (mo). Scale bar = 80 μm. [Figure 5]Figure 5 shows that ApoA-I K133C ND CNS rapidly enters the brain after intranasal administration and shows a dose-dependent increase. (ab) BACHD mice were intranasally administered 150 μg of ApoA-I K133C ND and euthanized at 1, 2, 3, 6, and 24 hours post-treatment. (a) The whole-brain mean level of ApoA-I was highest at 2 hours post-intranasal administration. One-way ANOVA: P<0.0001; Tukey's multiple comparison test: 2 hours and 24 hours vs 1 hour, ****p<0.0001. (b) ApoA-I levels were highest in the rostral brain region A at the initial, 1, 2, 3, and 6 hour time points. Across all four rostral-caudal brain regions, ApoA-I levels were consistently and significantly highest at 2 hours post-intranasal treatment. Two-way ANOVA shows significance by brain region (P<0.0001), treatment (P<0.0001), and interaction (P<0.0001); Tukey's multiple comparison test: **p<0.01, ***p<0.001, ****p<0.0001. (cd) BACHD mice were intranasally administered 75, 150, 300, or 500 μg of apoA-I K133C ND and euthanized 2 hours after intranasal administration. (c) The whole-brain mean shows a dose-dependent increase in apoA-I levels, with the highest dose of 500 μg achieving significantly higher levels than the lower doses (75 and 150 μg). One-way ANOVA: p<0.01; Tukey's multiple comparison test, **p<0.01. (d) Dose-dependent increases in ApoA-I levels were also observed along the rostral-caudal axis of the brain. Two-way ANOVA: significance by dose (p<0.01), brain region (p<0.0001), and interaction (p<0.01); Tukey's multiple comparison test, **p<0.01,**p<0.001,****,p<0.0001. [Figure 6]Figure 6 shows that K133C apoA-I ND has minimal distribution to peripheral tissues and is rapidly cleared after intranasal administration. ApoA-I levels were measured in cerebrospinal fluid (a), lung (b), liver (c), and plasma (d) of BACHD mice 1, 2, and 6 hours after intranasal treatment with K133C apoA-I ND. (a) ApoA-I levels in cerebrospinal fluid increased at 1 hour and plateaued between 2 and 6 hours. (b) In the lungs, apoA-I levels were highest in the first 2 hours, and apoA-I was almost completely eliminated from the lungs by 6 hours. (c) In the liver, apoA-I levels remained minimal throughout 6 hours. (d) In plasma, apoA-I levels were low at 1 hour and continued to steadily increase by 6 hours. [Figure 7] Figure 7 shows that intranasal administration of ApoA-I K133C ND results in higher apoA-I levels in the brain compared to intravenous administration of the same dose. (a) BACHD mice received the same dose of 150 μg of apoA K133C-I ND either intranasally or intravenously. Whole-brain ApoA-I levels were significantly higher 2 hours after intranasal administration compared to the intravenous route. Unpaired t-test, *p<0.05. (b) When examining different brain regions along the rostral-caudal axis, intranasal administration resulted in significantly higher levels of apoA-I in the most rostral brain region (A), consisting of the olfactory bulb and prefrontal cortex. Two-way ANOVA shows significance by brain region (P<0.001), treatment (P<0.01), and interaction (P<0.001); Bonferroni's multiple comparison test: ****p<0.0001. [Figure 8]Figure 8 shows that a single intranasal dose of K133C ASO-ND results in a significant reduction in brain mHTT. (a) BACHD mice received PBS or apoA-I K133C ASO-ND and were euthanized 24 hours later. DL-IHC against ASO and NeuN shows that ASO is deposited in NeuN+ cells 24 hours after intranasal administration of ASO-ND. (bd) BACHD mice were intranasally administered PBS, ASO alone, or apoA-I K133C ASO-ND and were euthanized 4 weeks later. (b) The whole-brain mean relative mHTT levels of brain lysates from these mice show a significant reduction in ASO-ND treated mice compared to PBS- and ASO-treated controls. One-way ANOVA shows significance by treatment (P<0.05); Tukey's multiple comparison test, *p<0.05. (c) Representative Western blots showing mHTT and mouse HTT levels in micro-dissected brain regions after intranasal treatment. (d) Examination of relative mHTT levels in micro-dissected brain regions shows significant mHTT reduction in the olfactory bulb, cortex, and striatum of ASO-ND treated mice. Two-way ANOVA shows significance by treatment (P<0.0001); Tukey's multiple comparison test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 9]Figure 9 shows that intranasal administration of two doses of ApoA-I K133C ASO-ND monthly sustains mHTT reduction in deep brain structures. (a) BACHD mice were administered two doses of PBS, ASO, or ASO-ND monthly, and one dose of ASO-ND monthly, and were euthanized 8 weeks after the initial dose. (b) The whole-brain mean shows that both one-dose and two-dose ASO-ND resulted in a significant reduction in mHTT. One-way ANOVA: P<0.0001; Tukey's multiple comparison test: **p<0.01,****p<0.0001 vs PBS-treated controls. (c) Examination of relative mHTT levels in micro-dissected brain regions shows a significant reduction in mHTT in the olfactory bulb, striatum, and brainstem of mice administered two doses of ASO-ND monthly. Two-way ANOVA shows significance with treatment (P<0.0001); Tukey's multiple comparison test: p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 10] Figure 10 shows the in vitro characterization of ApoA-I nanodiscs containing different ratios of lipid and lipid-binding protein components. (A) Native polyacrylamide gel electrophoresis of nanodiscs containing ApoA-I:DMPC in molar ratios ranging from 1:30 to 1:105. DMPC alone and ApoA-I alone are included as control samples. (B) The percentage of ApoA-I incorporated into the nanodiscs when ApoA-I:DMPC is combined in different ratios. [Figure 11] Figure 11 shows the association of antisense oligonucleotides with ApoA-I ND. (A) Results of electrophoretic mobility shift assays of ApoA-I nanodiscs in the presence of model antisense oligonucleotides (ASO alone), ApoA-I nanodiscs alone (ND alone), or specified ratios (ApoA-I:ASO at 1:1, 1:3, and 1:5). (B) Results of size exclusion chromatography showing the relative concentrations of lipids (DMPC), antisense oligonucleotides (ASO), or lipid-binding polypeptides (ApoA-1) in the eluted fractions. [Figure 12]Figure 12 shows that ApoA-I ND, which delivers ASO, enhances HTT suppression in HD patient-derived lymphoblast cells. (A) Western blot analysis of HTT protein expression in HD patient-derived lymphoblast cells exposed to any of the following: media alone, antisense oligonucleotides (ASO) directed to the Huntington gene, DMPC lipid alone, DMPC lipid and ASO, ApoA-I and ASO, or the nanodisc nucleic acid delivery particles of the present invention. (B) Graph showing relative HTT protein levels of wild-type (wtHTT) and mutant HTT (mHTT) proteins following the treatment conditions shown in (A). [Figure 13] Figure 13 shows a model of the ApoA-I "loop belt" conformation in a disc-shaped HDL. [Figure 14] Figure 14 shows the amino acid sequence of ApoA-I, which contains 24 amino acid signal peptides (SEQ ID NO: 1). The signal peptide sequence is shaded at the N-terminus of ApoA-I, and the mutation site described in the present invention is underlined in bold. [Figure 15]Figure 15 shows that ApoA-I mutants can form NDs with different properties. (A) Native PAGE protein gel showing the molecular weight of nanodisc complexes containing either wild-type ApoA-I (ApoA-I WT ND), ApoA-I with a cysteine ​​substitution at amino acid residue 133 (ApoA-I K133C ND), or ApoA-I with a cysteine ​​substitution at amino acid residue 146 (ApoA-I E146C ND). (B) Electron microscope image of nanodisc nucleic acid delivery particles containing either wild-type ApoA-I (ApoA-I WT ND), ApoA-I with a cysteine ​​substitution at amino acid residue 133 (ApoA-I K133C ND), or ApoA-I with a cysteine ​​substitution at amino acid residue 146 (ApoA-I E146C ND). (C) Graph showing the nanometer particle size of nucleic acid delivery particles containing either wild-type ApoA-I (ApoA-I WT ND), ApoA-I with a cysteine ​​substitution at amino acid residue 133 (ApoA-I K133C ND), or ApoA-I with a cysteine ​​substitution at amino acid residue 146 (ApoA-I E146C ND). (D) Dynamic light scattering analysis of nucleic acid delivery particles containing either wild-type ApoA-I (ApoA-I WT ND), ApoA-I with a cysteine ​​substitution at amino acid residue 133 (ApoA-I K133C ND), or ApoA-I with a cysteine ​​substitution at amino acid residue 146 (ApoA-I E146C ND). [Figure 16] Figure 16 shows that ApoA-I in nanodiscs can enter the brain more efficiently than lipid-free ApoA-I. ApoA-I levels as measured by ELISA (enzyme-linked immunosorbent assay) in brain homogenate samples obtained from mice intravenously administered either wild-type ApoA-I or nanodiscs containing wild-type ApoA-I and lipids. [Figure 17]Figure 17 shows that ApoA-I K133C ND enhances CNS entry after systemic administration. ApoA-I levels as measured by ELISA (enzyme-linked immunosorbent assay) in various brain tissues from mice intravenously administered one of the following: a nanodisc containing wild-type Apo-AI and lipids (ApoA-I WT ND), a nanodisc containing ApoA-I with a cysteine ​​substitution at amino acid residue 133 and lipids (ApoA-I K133C ND), or a nanodisc containing ApoA-I with a cysteine ​​substitution at amino acid residue 146 and lipids (ApoA-I E146C ND). [Figure 18] Figure 18 shows the biodistribution of various ND formulations in different regions of the brain (A) and cerebrospinal fluid (CSF) (B) after IV injection. NP F1: Nanodisc containing wild-type ApoA-I; NP F2: Nanodisc containing ApoA-I K133C; NP F3: Nanodisc containing ApoA-I E146C; NP F4: Nanodisc containing both ApoA-I K133C and E146C substitutions; NP F5: Nanodisc containing both ApoA-I R160V (valine substitution) and H162A (alanine substitution) substitutions. [Figure 19] Figure 19 shows (A) external imaging reveals increased CNS entry by ApoA-I K133C ND compared to nanodiscs containing wild-type ApoA-I, and (B) a graphical quantitative representation of imaging results of relative fluorescence in the brain as a percentage of fluorescence observed in the liver. [Figure 20] Figure 20 shows the time-course biological distribution of nanodiscs containing ApoA-I K133C ND or wild-type ApoA-I, indicating rapid entry into the CNS after IV administration. (A) Data showing entry of nanodiscs into the brain. (B) Data showing entry of nanodiscs into the CSF. [Figure 21] Figure 21 shows the dose-dependent increase in ApoA-I K133C ND in the brain (A) and CSF (B) after IV administration. [Figure 22] Figure 22 shows the dose-dependent increase in ApoA-I K133C ND in all brain regions after IV administration. [Figure 23] Figure 23 shows that HD mutation status does not significantly alter CNS entry of ApoA-I K133C ND in wild-type or BACHD animals at various ages (2–12 months old). [Figure 24] Figure 24 shows that ApoA-I WT and ApoA-I K133C ND, which carry ASO, can induce significant mutant HTT suppression in the CNS compared to ASO alone (A: cortex, B: striatum, C: hippocampus, D: cerebellum). [Figure 25] Figure 25 shows that ApoA-I K133C ND increases peripheral exposure after systemic administration. ApoA-I levels as measured by ELISA (enzyme-linked immunosorbent assay) in tissue homogenate samples (A: liver, B: quadriceps femoris, C: kidney) from mice intravenously administered one of the following: a nanodisc containing wild-type Apo-AI and lipids (ApoA-I WT ND), a nanodisc containing K133C-substituted and lipid-containing Apo-AI (ApoA-I K133C ND), or a nanodisc containing E146C-substituted and lipid-containing Apo-AI (ApoA-I E146C). [Figure 26] Figure 26 shows the biodistribution of various ND formulations in peripheral tissues after IV injection (A: quadriceps femoris, B: liver, C: kidney). NP F1: Nanodisc containing wild-type ApoA-I; NP F2: Nanodisc containing ApoA-I K133C; NP F3: Nanodisc containing ApoA-I E146C; NP F4: Nanodisc containing both ApoA-I K133C and E146C substitutions; NP F5: Nanodisc containing both ApoA-I R160V (valine substitution) and H162A (alanine substitution) substitutions. [Figure 27] Figure 27 shows the time course of ApoA-I K133C ND biodistribution (gray squares) or nanodiscs (black circles with wild-type ApoA-I) in liver (A) and quadriceps femoris (B) tissue. [Figure 28] Figure 28 shows the dose-dependent increase in ApoA-I K133C ND in the liver (A) and quadriceps femoris (B) after IV administration. [Figure 29]Figure 29 shows that HD mutation status does not significantly alter the peripheral distribution of ApoA-I K133C ND in the liver (A) or quadriceps femoris (B) in wild-type or BACHD animals at various ages (2–12 months of age). [Figure 30] Figure 30 shows that ApoA-I WT and K133C ND, which carry ASO, can induce significant mutant HTT suppression in the quadriceps femoris (A), liver (B), lungs (C), and heart (D) compared to ASO alone. [Figure 31] Figure 31 shows that ASO modification does not affect its HTT-reducing activity. (A) Lymphoblastic cells from HD patients treated with either PBS, unmodified ASO, ASO modified with a 5'-terminal amine group (aminoASO), or ASO modified with a 5'-terminal amine group and a 4-formylbenzamide moiety (4FB-ASO), and protein HTT levels were quantified by immunoblotting in (A) and (B). [Figure 32] Figure 32 shows that covalent conjugation of ASO to ApoA-I K133C does not affect ND formation. (A) Immunoblot and SDS-PAGE analysis of nanodiscs containing ApoA-I K133C substitution (ApoA-I K133C) or modified ASO (ASO:ApoA-I K133C) conjugated to nanodiscs containing ApoA-I K133C substitution. (B) Native PAGE analysis of increased concentration of modified ASO (ASO:ApoA-I K133C) conjugated to nanodiscs containing ApoA-I K133C substitution. (C) Electron microscopy analysis of modified ASO (ASO:ApoA-I K133C) conjugated to nanodiscs containing ApoA-I K133C substitution. (D) Graph showing the nanometer diameter length of nanodiscs containing ApoA-I K133C substitution (ApoA-I K133C) or modified ASO (ASO:ApoA-I K133C) conjugated to nanodiscs containing ApoA-I K133C substitution. [Figure 33]Figure 33 shows that coupling of ApoA-I K133C ND (ASO: ApoA-I K133C NC ND) with ASO enhances HTT suppression in lymphoblasts derived from HD patients compared to ASO alone. (A) shows immunoblotting analysis, and (B) shows quantitative analysis with illustrations. [Figure 34] Figure 34 shows that ASO:ApoA-I K133C-coupled ND (ASO:ApoA-I K133C NC ND) enhances the reduction of HTT in brain regions (A: cortex, B: striatum, C: hippocampus, D: cerebellum) after intraventricular injection in BACHD mice compared to PBS control or unmodified ASO. [Figure 35] Figure 35 shows that systemic administration of ASO: ApoA-I K133C-coupled ND (ASO: ApoA-I K133C NC ND) results in potent suppression of mHTT in brain regions (A: cortex, B: striatum, C: hippocampus, D: cerebellum) of BACHD mice. [Figure 36] Figure 36 shows that ASO:ApoA-I K133C-coupled ND (ASO:ApoA-I K133C NC ND) enhances the reduction of HTT in peripheral tissues (A: quadriceps femoris, B: liver, C: lung, D: heart) after intracerebroventricular injection in BACHD mice compared to PBS control or unmodified ASO. [Figure 37] Figure 37 shows that systemic administration of ASO: ApoA-I K133C-coupled ND (ASO: ApoA-I K133C NC ND) results in potent suppression of mHTT in peripheral tissues (A: quadriceps femoris, B: liver, C: lung, D: heart) of BACHD mice. Detailed explanation

[0051] The following detailed description will be better understood when read in conjunction with the attached figures and tables. For illustrative purposes, the figures and tables illustrate embodiments of the present invention. However, the present invention is not limited to the exact arrangements, examples, and fixtures shown.

[0052] In this invention, we consider the use of apolipoprotein AI nanodiscs as transport vehicles for ASO for intranasal administration to the brain. Nanodiscs are biocompatible disc-shaped complexes composed of phospholipids and scaffold proteins such as apolipoprotein AI (apoA-I). These nanodiscs spontaneously form in the presence of apoA-I and phospholipids, and the phospholipids are assembled into a bilayer surrounded by two apoA-I molecules

[39] . The apoA-I scaffold stabilizes the disc and shields the hydrophobic lipid acyl chains of the phospholipid bilayer from the hydrophilic environment

[40] . One of the main advantages of these apoA-I nanodiscs is their similarity to nascent high-density lipoprotein (HDL) and thus their lack of immunogenicity due to their ability to evade recognition by macrophages

[41] .

[0053] ApoA-I nanodiscs (NDs) have been used as in vivo drug delivery vehicles for hydrophobic drugs including amphotericin B, total trans retinoic acid, and curcumin [42-44]. ApoA-I NDs have also been shown to bind to siRNA and promote gene silencing in cultured cells

[45] . Most importantly, recombinant human apoA-I has been demonstrated to enter the brain after intravenous administration in mice by crossing the blood-CSF barrier in a dose- and time-dependent manner

[46] . Here we present the results of testing two ND formulations using two forms of the apoA-I protein, the wild-type protein and the K133C mutant. ApoA-I K133C was chosen because the insertion of a cysteine ​​residue was found to create a disulfide bond between two antiparallel apoA-I molecules, tightly tethering apoA-I around the ND, potentially providing greater cell penetration and distribution

[47] .

[0054] ApoA-I ND has several properties that make it an ideal drug delivery vehicle: 1) it is similar to nascent HDL particles and therefore biocompatible; 2) it is spontaneously formed in the presence of ApoA-I and lipids and is easy to generate; and 3) under the correct stoichiometry of ApoA-I:lipids, it forms monodisperse particles with a diameter of <50 nm, limiting clearance detection by the immune and lymphoid systems

[94] .

[0055] The present invention provides lipid NDs for non-invasive targeted delivery to the CNS of nucleic acid cargoes for the treatment of HD and other CNS-related diseases or disorders. The lipid NDs of the present invention comprise (a) lipids, (b) lipid-binding polypeptides, and (c) nucleic acid polymers.

[0056] Lipid nanodiscs As used herein, therapeutic nanodiscs (NDs) refer to lipid-binding polypeptides, lipid bilayers, and therapeutic agents, the lipid-binding polypeptides being encoded by SEQ ID NOs: 3 or 4. NDs are disc-shaped particles composed of a phospholipid bilayer surrounded by recombinant ApoA-I, which acts as a membrane scaffold protein, organizing lipids and shielding hydrophobic lipid acyl chains from the hydrophilic environment [84,85]. In particular, NDs can self-assemble into particles of controlled size depending on the lipid composition and the ApoA-I:lipid molar ratio. Structurally, NDs are similar to nascent HDL particles before esterified cholesterol loading and before transfer to spherical particles

[86] . NDs have been widely used to study the structure and function of integral membrane proteins [87-90] and have also been used as delivery platforms for bioactive compounds such as total trans retinoic acid and amphotericin B [42,44,91]. The lipid NDs described herein consist of lipid-binding polypeptides and lipid bilayers and include any therapeutic or diagnostic agents. The therapeutic agent may optionally be selected from antisense oligonucleotides (ASOs), short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), aptamers, ribozymes, mRNAs (messenger RNAs), plasmids, ribonucleoproteins (RNPs); or small molecules. The therapeutic agent may also be a nucleic acid polymer. The nucleic acid may be an antisense oligonucleotide (ASO), a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), an aptamer, a ribozyme, an mRNA (messenger RNA), or a plasmid. The nucleic acid may be an antisense oligonucleotide (ASO), a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), an aptamer, a ribozyme, an mRNA (messenger RNA), a plasmid, or may form part of a ribonucleoprotein (RNP).

[0057] The lipid bilayer consists of dimyristoyl phosphatidylcholine (DMPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- The following may be selected: ruboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (transDOPE), and their analogues.

[0058] Alternatively, the lipid bilayer can consist of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), cholesterol, and 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristilocypropylamine (PEG-C The following may be selected from -DMA), 1,2-dilinoleyloxy-3-(N,N-dimethyl)aminopropane (DLinDMA), N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadiene-1-yl-1,3-dioxolane-4-ethanolamine (DLin-KC2-DMA), 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadiene-1-yl-10,13-nonadecadiene-1-yl ester (DLin-MC3-DMA), and their analogues.

[0059] In certain embodiments, lipid ND includes neutral lipids. The term “neutral lipid” refers to any of several lipid species that exist in uncharged or neutral amphoteric forms at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. Exemplary lipids include, for example, dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cycline This includes hydroxyhexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (transDOPE). In one embodiment, the neutral lipid is dimyristoylphosphatidylcholine (DMPC).

[0060] In certain embodiments, lipid ND includes apolipoproteins as lipid-binding polypeptide components. Apolipoproteins are proteins that bind to lipids (lipid-soluble substances such as fats and cholesterol) to form lipoproteins. They transport lipids through the lymphatic and circulatory systems. Apolipoprotein A1 (ApoA-I) is encoded by the APOA1 gene and plays a role in lipid metabolism. Apolipoprotein AI is the major protein component of high-density lipoprotein (HDL) particles in plasma. HDL is involved in the retrograde transport of cholesterol from peripheral tissues to the liver (RCT), where it is excreted or recycled

[73] . ApoA-I is one of the most abundant apolipoproteins in the CSF [74-76] and brain [77,78]. However, APOA-I mRNA (messenger RNA) levels are very low in the brain throughout human development and adulthood

[79] . This suggests that the source of ApoA-I present in the CNS is primarily derived from plasma produced by the liver and intestines

[80] . There is also evidence that brain microvascular endothelial cells may also produce and secrete ApoA-I

[81] . In one embodiment, ApoA-I is modified to promote disulfide bridge formation between ApoA-I molecules of lipid ND. The modification may include the introduction of at least one cysteine ​​residue. The ApoA-I modification may include the introduction of one or more cysteine ​​residues at amino acid positions 133(K) or 146(E). The lipid-binding polypeptide may be encoded by SEQ ID NO: 3 or 4, or by a sequence having 90% sequence identity to either SEQ ID NO: 3 or 4. Preferably, the lipid-binding polypeptide is encoded by SEQ ID NO: 3 or 4. More preferably, the lipid-binding polypeptide is encoded by SEQ ID NO: 3. Furthermore, the lipid-binding polypeptide used herein may include both modified and unmodified amino acids. The modification may be beneficial in one or more of the following ways: loading of the therapeutic agent (including nucleic acid polymers) or diagnostic agent; delivery of the therapeutic agent or diagnostic agent; targeting of the therapeutic agent or diagnostic agent; bioavailability of the therapeutic agent or diagnostic agent; half-life of the therapeutic agent or diagnostic agent; or minimization of undesirable effects of the therapeutic agent or diagnostic agent.

[0061] nucleic acid polymers The lipid nanoparticles of the present invention are useful for targeted CNS delivery of nucleic acid polymers such as ASOs. As used herein, the term “nucleic acid polymer” is intended to include any oligonucleotide or polynucleotide. Fragments containing up to 50 nucleotides are generally called oligonucleotides, and longer fragments are called polynucleotides. In certain embodiments, the oligonucleotides of the present invention are 8 to 50 nucleotides long. In certain embodiments, the oligonucleotides of the present invention are 8 to 150 nucleotides long. In certain embodiments, the oligonucleotides of the present invention are 8 to 500 nucleotides long. Oligonucleotides are classified as deoxyribooligonucleotides or ribooligonucleotides. Deoxyribooligonucleotides are composed of a 5-carbon sugar called deoxyribose, which is covalently bonded to phosphate at the 5' and 3' carbons of this sugar to form alternating unbranched polymers. Ribooligonucleotides are composed of similar repeating structures where the 5-carbon sugar is ribose. The nucleic acids present in the lipid nanoparticles of the present invention include any known form of nucleic acid. The nucleic acids used herein may be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. In one embodiment, the polynucleic acid is an antisense oligonucleotide.

[0062] While various embodiments of the present invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in the art. Such modifications involve the substitution of known equivalents for any aspect of the invention and achieve substantially the same results in substantially the same manner. The following examples are provided for illustrative purposes only and are not intended to limit the claimed invention.

[0063] This disclosure provides compositions for treating, preventing, or diagnosing central nervous system conditions, diseases, disorders, injuries, or damages using therapeutic nanodiscs, wherein the therapeutic nanodiscs comprise a lipid-binding polypeptide, a lipid bilayer, and a therapeutic agent. Alternatively, the therapeutic nanodiscs comprise a lipid-binding polypeptide, a lipid bilayer, and a nucleic acid polymer.

[0064] A non-limiting list of nucleic acid polymers that may be used for neurological disorders and disorders, as well as other disorders, and for their treatment is provided in Table 1 below.

[0065] Table 1: Nucleic acid polymers used in the treatment of central nervous system diseases or disorders. JPEG2026521709000002.jpg188159

[0066] As used herein, therapeutic agents are intended to include antisense oligonucleotides (ASOs), short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), aptamers, ribozymes, mRNAs (messenger RNAs), plasmids, or small molecules.

[0067] As used herein, nucleic acid polymers are intended to include antisense oligonucleotides (ASOs), short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), aptamers, ribozymes, mRNAs (messenger RNAs), or plasmids. The nucleic acid polymer may be selected from one or more of the nucleic acid polymers listed in Table 1.

[0068] As used herein, diagnostic agents are intended to include substances used to examine the body in order to detect impairment of the normal functioning of the body. Diagnostic agents described herein may also include radiopharmaceuticals and contrast agents for use in imaging technologies (including X-ray, magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), computed tomography (CT) (including volumetric quantification CT (vQCT), high-resolution CT (hrCT), and micro-CT (μCT)), positron emission tomography (PET), single-photon emission computed tomography (SPECT), thermography, electrostimulation imaging (ESI), digital mammography, tactile imaging, magnetic source imaging (MSI), medical optical imaging, ultrasound and electrical impedance tomography (EIT), diagnostic ultrasound, PET / CT hybrid, three-dimensional ultrasound computed tomography (3D USCT), and simultaneous PET / MRI).

[0069] Table 2: Unofficial Sequence List JPEG2026521709000003.jpg185159

[0070] (Signal sequence) +=LNA modification and *=phosphorothioate modification -C represents positions 133 and 146 in sequence 3 and 4, respectively.

[0071] As used herein, the terms “messenger RNA” or “mRNA(messenger RNA)” refer to polynucleotides that encode and express secreted proteins.

[0072] The nucleic acid polymers used herein include both modified and unmodified ones. In one embodiment, the nucleic acid polymer comprises one or more coding and uncoding regions. The nucleic acid polymer may be purified from natural resources, produced using recombinant expression systems, optionally purified, or chemically synthesized. Modification of nucleic acid polymers can improve immunogenicity, stability, and translation efficiency and fidelity of the nucleic acid polymer, and thus improve desired activity.

[0073] The nucleic acid polymer may contain nucleotide analogs. The nucleic acid polymer may contain modified internucleoside bonds. The modified internucleoside bonds may be peptide-nucleic acid bonds, morpholino bonds, N3' to P5' phosphoramide bonds, methylphosphonate bonds, or phosphorothioate bonds. The nucleic acid polymer may have one or more modified sugar moieties. The modified sugar moieties may be 2'-O-alkyloligoribonucleotides. The nucleic acid polymer may be a gapmer. The nucleic acid polymer may have a 2'MOE gapmer modification. The nucleic acid polymer may have a modified nucleic acid base. The modified nucleic acid base may be 5-methylpyrimidine or 5-propynylpyrimidine. One or more nucleotide analogs may contain loc nucleic acid (LNA). The LNA unit includes beta-D-oxy-LNA monomer. Nucleic acid analogs include Loc nucleic acid (LNA), MOE, 2'-O-(2-methoxyethyl), BNA, 2',4'-crosslinked nucleic acid, N-Me-aminooxyBNA, 2'-N-(methyl)-4'-C-aminooxymethylene 2',4'-crosslinked nucleic acid, N-Me-aminooxy 2',4'-crosslinked nucleic acid, 2'-N-(methyl)-4'-C-aminooxymethylene 2',4'-crosslinked nucleic acid, and 2',4'-BNA. NC [NMe], 2'-O,4'-C-(N-methyl)aminomethylene 2',4'-crosslinked nucleic acid, 2'-O,4'-C-aminomethylene 2',4'-crosslinked nucleic acid (2',4'-BNA), and 2',4'-BNA NC One or more of these may be selected. As used herein, the non-nucleotide analog nucleic acid may be a standard nucleic acid, but the nucleic acid polymer may still have modified or unmodified bonds.

[0074] The nucleic acid polymer may further contain modified internucleoside bonds. The modified internucleoside bonds may be peptide-nucleic acid bonds, morpholino bonds, N3' to P5' phosphoramide bonds, methylphosphonate bonds, or phosphorothioate bonds. The nucleic acid polymer may further contain modified sugar moieties. The modified sugar moieties may be 2'-O-alkyl oligoribonucleotides. The nucleic acid polymer may further have 2'MOE gapmer modifications. The ASO may further have 2'OMe gapmer modifications. The nucleic acid polymer may further contain modified nucleic acid bases. The modified nucleic acid bases may be 5-methylpyrimidine or 5-propynylpyrimidine.

[0075] In embodiments in which nucleic acid polymers are chemically synthesized, the nucleic acid polymer may include nucleoside analogs such as analogs having chemically modified bases or chemically modified sugars, and / or backbone modifications. In some embodiments, the nucleic acid polymer may be a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine), a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fu Luorauridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phospholamidate bonds), or containing them.

[0076] The mRNA (messenger RNA) of this disclosure may be synthesized according to any of the various known methods. For example, the mRNA (messenger RNA) in a particular embodiment may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template comprising a promoter, a pool of ribonucleotide triphosphates, a buffer system which may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNAse inhibitor. While mRNA (messenger RNA) from an in vitro transcription reaction may be desirable in a particular embodiment, other sources of mRNA (messenger RNA), such as mRNA (messenger RNA) produced from bacteria, fungi, plants, and / or animals, should also be considered.

[0077] In some embodiments, the nucleic acid polymer includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements (e.g., iron-responsive elements) that affect the stability or translation of the nucleic acid polymer. In some embodiments, the nucleic acid polymer may be about 15 to 500 nucleotides or longer.

[0078] Route of administration In some embodiments, the therapeutic nanodiscs or compositions described herein may be part of a pharmaceutical composition. The pharmaceutical composition may provide prophylactic, mitigating, or therapeutic benefits. The pharmaceutical composition may be administered in any appropriate dose. The type of administration used to introduce the therapeutic or diagnostic agent may include any route for delivering the therapeutic nanodiscs or composition to a central nervous system (CNS) site or systemically. Alternatively, delivery may be to the extracellular space including the interstitium and / or cerebrospinal fluid. The site may include the brain and / or spinal cord. Methods of administration include, but are not limited to, intravenous injection (IV), intranasal inhalation, intraventricular (ICV) injection, lumbar intrathecal (IT) injection, intrathecal (IT) infusion, cisterna magna injection, and the use of a catheter (e.g., an intrathecal catheter) for introducing the therapeutic nanodiscs or composition into the brain or spinal cord. The therapeutic nanodiscs or compositions described herein may be administered to a subject. As used herein, “subject” may include humans, non-human primates, rats, mice, cattle, horses, pigs, sheep, goats, dogs, cats, and the like. The subject is suspected of having, or at risk of having, a central nervous system disorder, injury, or damage, and is known to a person with ordinary skill in the art. Some examples are listed in Table 1.

[0079] Methods and materials ApoA-I expression and purification The ApoA-I wt, K133C, and E146C plasmids were a gift from Michael Oda and optimized for bacterial expression. Plasmid expression and purification were performed as described by Ryan et al.

[48] and Oda et al.

[49] . Briefly, BL21(DE3)pLysS cells transformed with pNFXex-apoA-I WT and K133C plasmids (Martin et al.

[39] ) were expressed in NCZYM containing 50 μg / ml ampicillin. TM Media (Sigma) TM In the catalog (#N3643), the cells were cultured at 37°C. Expression was observed when the culture reached 0.6 OD600 of isopropyl-thiogalactoside (IPTG).TM This was induced by adding B-PER (Thermo Fisher) to a final concentration of 0.5 mM. After 3 hours of incubation, the bacteria were centrifuged at 10,000 g for 15 minutes. TM Lysozyme (final concentration 1 mg / ml) and DNase I (1 μl per 10 ml) were added to (#78248) and dissolved. The mixture was then sonicated on ice and centrifuged at 20,000 g for 30 minutes at 4°C. The supernatant fraction was mixed with an equal volume of 2x column loading buffer (40 mM NaH2PO4, 1 M NaCl, 6 M guanidine HCl, pH 7.4) and frozen at -20°C for 1-3 days before purification.

[0080] For purification, the lysate was thawed, centrifuged at 5,000 g for 10 minutes at 4°C, and added to a pre-prepared Ni-NTA agarose column through a 0.22 μm filter. The flow-through was passed through the column two additional times. The column was washed with 25 mL of 1x column loading buffer, and then washed with wash buffer (20 mM NaH2PO4, 0.5 M NaCl, 20 mM imidazole, 0.05% Tween-20, pH 7.4) until A280 reached 0. Finally, the column was eluted in 1 mL fractions with 25 mL of elution buffer (20 mM NaH2PO4, 0.5 M NaCl and 0.5 M imidazole, pH 7.4). Protein was detected by A280 (imidazole corrected), and the sample was dialyzed at 4°C with PBS containing 1 mM benzamidine and 1 mM EDTA. Alternatively, the protein-containing fraction can be pooled, dialyzed with PBS at 4°C for 24 hours, and filtered through a 0.2 μm syringe filter (Pall TM It was filter-sterilized using catalog #4612 and stored at -80°C until use.

[0081] Nanodisc preparation For nanodisk preparation, apoA-I proteins (i.e., wt;K133C; and E146C) were slowly thawed on ice to reduce precipitation, then filtered and sterilized using a 0.2 μm filter, and used for DC protein absorption assay (Bio-Rad).TM , the concentrations were measured using catalog #5000113 and 5000114). For in vitro applications, apoA-I was diluted to the desired concentration with Dulbecco's phosphate-buffered saline (PBS; Sigma-Aldrich TM , catalog #D8537). For in vivo applications, the apoA-I protein was concentrated using a centrifugal filter unit with a 10 kDa nominal molecular weight cut-off (MWCO, Millipore TM , catalog #MRCPRT010), filter sterilized using a 0.2 μm syringe filter, and adjusted to the desired concentration with PBS. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) suspended in chloroform was brought to room temperature before use at 25 mg / mL (Avanti Polar Lipids TM , catalog #850345C or NC0845353), transferred to a v-bottom glass vial, dried under an argon stream, and then placed at 37 °C for 2 minutes to equilibrate the temperature in a biosafety cabinet. The dried lipid was then resuspended in the apoA-I solution at a molar ratio of 105:1, gently co-crushed for 1 minute, and then incubated at 22 °C for 16 hours to spontaneously form into nanodiscs (NDs).

[0082] For ASO-ND preparation, the nanodiscs were first prepared as described above. The next day, the ASO was slowly thawed on ice and the desired amount was mixed with the nanodiscs. The ASO-nanodisc mixture was incubated at 37 °C for 1 hour and gently mixed before use.

[0083] Antisense oligonucleotide (ASO) targeting human HTT In this study, as previously described, we used an ASO gapmer targeting intron 22 of the human HTT transcript with a fully modified phosphorothioate backbone and lock nucleic acid (LNA) wing modification (+T*+A*+A*+T*+A*C*G*T*A*A*G*T*G*T*+A*+C*+A*+C*+A*+A;+=LNA modification and *=phosphorothioate modification - MW=6416 g / mol)[22, 50, 98]. The same HTT ASO was synthesized with a reactive amine group modification attached to the 5' terminal nucleotide base having a 6-carbon linker to enable conjugation to apoA-I K133C (amino-HTT ASO, MW=6596 g / mol). The ASO was commercially synthesized (Qiagen) TM ), HPLC-purified, Dulbecco's phosphate-buffered saline (PBS; Sigma-Aldrich TM It was resuspended in (catalog #D8537). Sterile PBS was used to resuspend the ASO and then incubated at 37°C for 2 hours. The ASO concentration was calculated using the following formula: concentration (M) = OD260 x dilution factor / ε260, where ε260 = extinction coefficient of the oligo at 260 nm. For ASO treatment, the ASO was diluted to the concentration indicated in sterile PBS or prepared on apoA-I nanodiscs and administered as follows.

[0084] Conjugation of HTT ASO to apoA-I K133C For the modification of ApoA-I K133C, aliquot-resolved proteins were thawed on ice, quantified by DC assay, and then diluted to 5 μg / μL in 1× modification buffer (10× modification buffer: 1.0 M Na3PO4, 1.5 M NaCl, pH 8.0). Zeba Spin with 7 kDa MWCO TM Desalting column (Thermo Scientific) TMUsing catalog #89892, the buffer for apoA-I K133C was completely replaced with I× modified buffer. After the buffer exchange, the apoA-I K133C protein concentration was requantified by DC assay, and this value was used to modify the protein with succinimidyl-6-hydrazinonicotinamide (S-HyNic) at a molar substitution ratio (MSR) of 3, according to the manufacturer's protocol. TM Vector Laboratories TM The amount of fresh S-HyNic (catalog #S-1002-010) was calculated for each reaction. TM The calculated volume of anhydrous N,N-dimethylformamide (DMF) from Sigma-Aldrich TM The protein was resuspended in catalog #227056 (100 ml), added to the desalted protein, vortexed at room temperature for 3 hours, and incubated. After incubation, the HyNic-modified apoA-I K133C protein was desalted in 1 × conjugated buffer (10 × conjugated buffer: 1.0 M Na3PO4, 1.5 M NaCl, pH 6.0) using a Zeba Spin desalting column. The modified protein was then frozen and stored at -80°C until further use.

[0085] For the modification of amino-HTT ASO, the molecule was resuspended in nuclease-free dH2O, and then diluted to 0.5 OD / μL by adding 10× modification buffer to a final concentration of 1×. (Succinimidyl-4-formylbenzamide (s-4FB. Vector Laboratories)) TM (Catalog #S-1004-105) was resuspended fresh in anhydrous DMF after each use, and the modification of amino-HTT ASO was carried out at room temperature for 3 hours according to the manufacturer's protocol. After incubation, 4FB-modified HTT ASO (4FB-HTT ASO) was diluted 10-fold with 1× conjugated buffer and Amicon Ultra with 3 kDa MWCO was used. TM 0.5 mL Centrifugal Separation Filter Unit Column (Amicon) TMThe 4FB-HTT ASO was loaded in 500 μL aliquot units (catalog #UFC500324) and concentrated at 14,000 × g for 20-30 minutes at 4°C. After concentration, the 4FB-HTT ASO was washed three times with 1 × conjugated buffer, and after each wash, it was centrifuged at 14,000 × g for 20-30 minutes at 4°C, and stored at -80°C until use.

[0086] For the conjugation of HyNic-modified apoA-I K133C and 4FB-HTT ASO, the reagents were thawed on ice, briefly centrifuged to remove precipitates, and then subjected to DC assay and Nanodrop assays, respectively. TM Quantitative analysis was performed. MSRs were measured for both modified apoA-I K133C and HTT ASO according to the manufacturer's protocol. In glass vials, the calculated volumes of HyNic-apoA-I K133C and P4FB-HTT ASO were measured using 1×TurboLink. TM Catalyst buffer (10 × TurboLink TM Combine with catalyst buffer (100 mM aniline, 100 mM Na3PO4, 150 mM NaCl, pH 6.0), incubate at room temperature for 2 hours, then desalinate using a Zeba Spin column. TM Desalting was performed in PBS using [method]. The MSR of HTT ASO + apoA-I K133C conjugated was quantified according to the manufacturer's protocol. Conjugation was confirmed by immunoblotting and electrophoretic mobility shift assay (EMSA) as described below. ND was generated with HTT ASO + apoA-I K133C conjugated and DMPC in a molar ratio of 1:105, based on the apoA-I concentration as described above.

[0087] Electrophoretic mobility shift assay (EMSA) ApoA-I ND is prepared as described above using 37.37 nmol (25.33 μg) DMPC and 355.87 pmol (10 μg) apoA-I, and then incubated at 37°C for 1 hour with 355.36 pmol (2.28 μg), 1.01 nmol (6.8 μg), or 1.78 nmol (11.4 μg) ASO (MW = 6417.12 g / mol), which corresponds to a stoichiometric range of 1:1 to 1:5 apoA-I:ASO. The ND formulation is run on a 2% agarose gel at 100V for approximately 45 minutes, and the gel is cooled with OliGreen TM (Invitrogen TM ASO was visually identified by staining with #O7582. For further verification of HTT ASO + apoA-I K133C conjugation, 5 μg (0.78 nmol) of HTT ASO and the molar equivalent of HTT ASO + apoA-I K133C conjugation were used on a 2% agarose gel (SyberSafe TM The gels were separated at 130V for 30 minutes using a GelDoc system (BioRad). TM Imaging was performed using ). For comparison of apoA-I ND migration in plasma, samples from animals injected with PBS or either apoA-I WT ND or apoA-I K133C ND were prepared in 50 mM Tris pH 8, 150 mM NaCl, and 1% IGEPAL. TM and protease inhibitors (Roche TM The sample was diluted 1:25 with a buffer containing (catalog #11836145001). The sample was then 2× Native Tris-Glycine Sample Buffer (Invitrogen). TM Diluted with LC2673, and run repeatedly on 4-20% Tris-glycine gel (Invitrogen, XP04200BOX), and Tris-glycine native run buffer (Invitrogen TM It was run for 2 hours at 130V using an LC2672. NativeMark TM Protein Standard (Invitrogen) TMLC0725 was used as the size standard. The gel was then cut in half and all proteins and apoA-I were visualized. One half was used for Coomassie R-250. TM (Bio-Rad TM The samples were stained according to the manufacturer's protocol (catalog #161-0400). The other half was NuPAGEd to 0.2 μm nitrocellulose. TM Transport buffer (Invitrogen TM The gels were transported at 18V for 2 hours using catalog #NP00061 and probed for apoA-I as described above. Both gels and blots were obtained using the LI-COR Odyssey imaging system. TM Imaging was performed using [the specified method].

[0088] Size exclusion chromatography assay Sephadex TM G-50 Medium Beads (Pharmacia TM The 17-0043-01) was rehydrated according to the manufacturer's instructions, loaded onto a 15 ml column, and washed three times with PBS. The ASO ND formulation was prepared using 37.37 nmol (25.33 μg) DMPC, 355.87 pmol (10 μg) apoA-I, and 1.01 nmol (6.8 μg) ASO in a total volume of 200 μL, slowly added to the top of the bead bed, allowed to settle for 2 minutes, and then 3 mL of PBS was added. For the measurement of DNA, protein, and lipid concentrations, approximately 60–65 50 μL fractions were collected in each fraction in a 96-well plate. DNA concentration was measured using NanoDrop. TM Protein concentration is measured using a spectrophotometer (ssDNA), and lipid concentration is measured using a DC protein absorption assay (Sigma). TM Measured using catalog #MAK122.

[0089] Oligreen ASO Association Assay ApoA-I ND was prepared as described above, incubated with ASO (37°C for 1 hour), and then dialyzed to remove free, unbound ASO. The dialyzed preparation was then treated with Triton X-100. TM (Final concentration 1%) was used for treatment and incubated at 37°C for 15 minutes. Oligreen TM The working solution is prepared with 1xTE buffer (pH 7.5) and Oligreen. TM Prepared by diluting the reagents and kept in the dark until use. Triton X-100 TM The treated ND was then diluted 1:20 with 1xTE buffer. 100 μl of each sample and standard was pipetteed into a 96-well plate, and 100 μl of Oligreen was added. TM The working solution was added to each well and incubated in the dark at room temperature for 3 minutes. Absorbance was measured at 520 nm.

[0090] Optical density (OD) measurement DMPC monoformulation was prepared using 25.3 μg (37.4 nmol) DMPC resuspended in 10 μL PBS and incubated at 22°C for 16 hours. ApoA-I ND was prepared using 25.3 μg (37.4 nmol) DMPC and 10 μg (356 pmol) apoA-I in 10 μL total volume PBS as described above. OD measurement was performed using NanoDrop. TM 2000 Spectrophotometer (Thermo Fisher Scientific TM ) was executed at 3x speed at 590nm.

[0091] Dynamic Light Scattering Analysis (DLS) ApoA-I ND was prepared as described above using 25.3 μg (37.4 nmol) DMPC and 10 μg (356 pmol) apoA-I in a total volume of 100 μL PBS. The sample was diluted 5-fold with PBS immediately before loading into the cuvette, and DLS sizing was performed using a Malvern Zetasizer. TM (Malvern Panalytical TMThe procedure was performed according to the manufacturer's instructions using [a specific tool / method]. The mean particle size and polydispersity index (PDI) are shown from five readings from each sample or from the first peak.

[0092] Native polyacrylamide gel electrophoresis (PAGE) The nanodiscs were prepared as described above and mixed with 15% glycerol at the desired concentration before loading, and Novex Tris-Glycine 4-20% WedgeWell TM The gel was loaded. The gel was run at 120V at room temperature (RT) for 120 minutes in 1X Tris-glycine native run buffer. The gel was removed and stored in the dark in a Syber Safe container. TM Incubated for 60 minutes (1xTBE at 1:4000), then rinsed 3 times with 1xTBE, Bio-Rad TM The gel was scanned using an XR+ imager. The gel was then rinsed three times with 1x PBS, stained at room temperature for 30 minutes with Coomassie Blue (0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid), rinsed three times with 1x TBE, and then subjected to Odyssey infrared imaging. TM System (LI-COR Biosciences) TM The gel was scanned using ). Alternatively, the ND formulation was prepared using the molar ratio of DMPC and apoA-I calculated from the published molecular weights (MW; DMPC = 677.93 g / mol, apoA-I = 28,100 g / mol) as described above. For DMPC:apoA-I stoichiometric studies, the molar ratio of DMPC used in each formulation was calculated based on 10 μg (356 pmol) apoA-I. For a comparison of the electrophoretic mobility of apoA-I WT and K133C ND, 25.3 μg (37.4 nmol) DMPC and 10 μg (356 pmol) apoA-I (corresponding to a 105:1 DMPC:apoA-I molar ratio) were used in a total volume of 10 μL. Samples were prepared using 2 × Native Tris-Glycine Sample Buffer (Invitrogen). TM Diluted with LC2673, 4-20% Tris-glycine gel (InvitrogenTM Loaded into XP04200BOX, Tris-glycine native execution buffer (Invitrogen TM It was run for 2 hours at 130V with LC2672. High MW ladder (GE TM GE17-0445-01) was loaded into the same gel and used as a size standard. The gel was then loaded with Coomassee R-250 (Bio-Rad) according to the manufacturer's protocol. TM Stained with (catalog #161-0400), LI-COR Odyssey imaging system TM (LI-COR Biosciences TM The images were taken using ).

[0093] Transmission electron microscope (TEM) Negative-Steining TEM of the apoA-I ND formulation was performed as previously described

[95] . Briefly, apoA-I ND was prepared using 38 μg (56 nmol) DMPC and 15 μg (534 pmol) apoA-I in 200 μL total volume PBS as described above. 10 μL of each formulation was added to a carbon-coated grid, and after 20 seconds, excess liquid was wiped off. A small droplet of 2% potassium phosphotungstate (pH 6.5) was added to the grid and incubated for 10 seconds. Excess dye was then removed, and the grid was allowed to air dry. Samples were taken using a Hitachi H7600. TM TEM (Hitachi TM Imaging was performed at 80kV using 500,000-600,000× direct magnification, acquired with an XR51 camera with exposure=800msec, gain=1, and bin=1. The diameter of the apoA-I ND was measured in Fiji TM (ImageJ TM The analysis is performed using ) and the size in nm is presented.

[0094] Brain microvascular endothelial cell (BMEC) differentiation GM03621 fibroblast cells were obtained from the Nigomus Human Gene and Cell Repository at the Coriel Institute for Medical Research. The fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) using the episomal non-integrated reprogramming protocol described in

[96] with OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA. pCXLE-hOCT3 / 4-shp53(Addgene TM Plasmid #27077), pCXLE-hSK (Addgene TM Plasmid #27078), and pCXLE-hUL (Addgene TM Plasmid #27080) was a gift from Shinya Yamanaka

[96] . Pluripotency markers were validated using qPCR and immunofluorescence, and a normal karyotype was confirmed. BMECs were differentiated from GM03621 iPSCs as described in

[97] , with the following modifications: GM03621 iPSCs to Matrigel TM (Corning TM , 6-well plates coated with catalog #354277) daily with mTESR1 (StemCell TM (Catalog #85850) Cells were grown with a media change until they reached 50% confluence. Cells were then subjected to 20% knockout serum replacement. TM (Gibco TM (Catalog #10828010), 1% MEM Non-essential amino acids (Gibco) TM (Catalog #11140050), 1% GlutaMax TM (Gibco TM (Catalog #35050061), 1% Pen / Strep (Gibco) TM The cells were switched to DMEM / F12 media containing β-mercaptoethanol (catalog #15140122) for 5 days with daily media changes. On day 6 of differentiation, the cells were given 1% platelet-poor serum (Sigma-Aldrich TM , catalog #P2918) and human basic FGF (Prospec TM, complete endothelial serum-free medium (EM. Gibco, catalog #CYT-218) TM , catalog #11111044), was switched over 2 days with daily medium changes.

[0095] Immunofluorescence (IF) BMECs were seeded in complete EM at a density of 25,000 cells / cm TM on coverslips coated with 400 μg / mL collagen IV (Sigma-Aldrich, catalog #C5533) and 100 μg / mL fibronectin (Sigma-Aldrich TM , catalog #F4759), and the medium was changed daily until confluence was reached. The medium was then removed, and the cells were washed twice with PBS and then fixed with 4% paraformaldehyde in PBS for 12 minutes at room temperature. The cells were washed twice with PBS and then permeabilized with 0.2% Triton X-100 in PBS for 15 minutes at room temperature. BMECs were then washed twice with PBS and blocked with blocking buffer containing 5% FBS (Gibco 2 in PBS for 2 hours with rabbit anti-PECAM-1 (1:50. Developmental Studies Hybridoma Bank TM , catalog # P2B1) and mouse anti-claudin-5 (1:500. Invitrogen TM , catalog #35-2500) or rabbit anti-occludin (1:250. Invitrogen TM , catalog #71-1500) and mouse anti-GLUT-1 (1:1000. Abcam TM , catalog #ab40084) in antibody dilution buffer containing 1% FBS + 0.1% Triton X-100 in PBS at room temperature for 2 hours. The cells were washed three times with blocking buffer, and the primary antibody was goat anti-rabbit Alexa Fluor 488 TM (1:500. Invitrogen TM (1:500. Invitrogen TM, Catalog #A32731) and Goat anti-Mouse Alexa Fluor 594 TM (1:500. Invitrogen TM , Catalog #A-11005) secondary antibody combination was used for detection at room temperature for 1 hour in antibody dilution buffer. Coverslips were then washed three times with blocking buffer and mounted on slides with ProLong Gold Antifade with DAPI TM (Thermo Fisher Scientific TM , Catalog #P36931).

[0096] Trans-epithelial cell permeability experiment BMECs were plated at a minimum density of 25,000 cells / cm in complete EM in 1.12 cm Transwell permeable inserts (Corning 2 TM TM , Catalog #3401) coated with 400 μg / mL collagen IV and 100 μg / mL fibronectin. Media was changed daily until confluence was reached. Prior to each trans-epithelial cell permeability experiment, trans-epithelial electrical resistance (TEER) was measured in triplicate for each Transwell using an epithelial volt / Ω meter (World Precision Instruments 2 TM TM , Catalog #EVOM2). ApoA-I ND formulations composed of 38 μg (56 nmol) DMPC and 15 μg (534 pmol) apoA-I in 100 μL PBS were generated as described above. Equivalent amounts (534 pmol) of lipid-free apoA-I WT and apoA-I K133C were prepared in 100 μL PBS. Complete EM media from the insert (apical chamber) and basolateral chamber were replaced with Hank’s Balanced Salt Solution containing 10 mM HEPES and 1% FBS TM (Gibco TM ​​​​The transport buffer was replaced with the one specified in catalog #14025092. At time = 0 min, 100 μL of lipid-free apoA-I or apoA-I ND was added to the insert, and 200 μL aliquots were taken at 15-minute intervals, replaced with fresh transport buffer to maintain the quantified volume of the basal outer lumen. ApoA-I concentrations were measured using human apoA-I ELISA (enzyme-linked immunosorbent assay) as described below.

[0097] Animal treatment The study was conducted in BACHD mice

[51] in strict accordance with a protocol approved by the Animal Care Board of the University of British Columbia (A16-0130, A20-0107).

[0098] Nearly equal numbers of male and female mice were used in all experiments. The animals were kept in a clean barrier facility under a 12-hour light-to-12-hour dark cycle and given ad-lib access to food and water. For intranasal procedures, mice were anesthetized with ketamine and xylazine (100 / 10 mg / kg, ip), then placed in a supine position with their noses erect and their heads flat on the surface. A 10 μl Hamilton syringe with a blunt 30-gauge needle was administered every 2 minutes, alternating sides, until a total volume (60-90 μl) was reached. TM The treatment was administered in 2.5 μl increments through the nostril using a syringe.

[0099] The mice remained in a supine position for 30 minutes after intranasal administration.

[0100] For intravenous procedures, the animals received the same dose / volume via tail vein injection.

[0101] Animal collection and tissue processing Animals assigned to biochemical analysis were subjected to 2,2,2-tribromoethanol (Avertin TM Sigma-Aldrich TMTerminal anesthesia was performed using (catalog #T48402). For selected experiments, terminal CSF was collected from the cisterna magna, and whole blood was then collected via cardiac puncture for plasma separation as previously described

[98] . Alternatively, CSF was collected as previously described [22, 50]. Briefly, in a 50 μL Hamilton with a 12°C bevel. TM The syringe is inserted into the large chamber using an appropriate stereotactic device, Micro4 TM UltraMicroPump with controller TM This was used to extract CSF. For apoA-I ELISA (enzyme-linked immunosorbent assay) studies only, animals were perfused with PBS for 4 minutes prior to collection to flush out any loosely associated apoA-I. Brains were then harvested whole or microdissected into individual brain regions, and peripheral tissues were also collected. Samples were rapidly frozen in liquid N2 and stored at -80°C until use.

[0102] Animals assigned to external imaging, histology, and immunohistochemistry (IHC) were terminally anesthetized with 2,2,2-tribromoethanol, transcardiac perfused with ice-cold PBS for 4 minutes, followed by transcardiac perfused with 4% paraformaldehyde (PFA) in PBS for 4 minutes. The brain was removed and post-fixed in 4% PFA in PBS at 4°C for 24 hours, then transferred to PBS for external imaging, 70% ethanol for histology, or 30% sucrose containing 0.01% NaN3 until equilibrated for IHC. Longitudinal plasma collection was performed using EDTA-coated microbed tubes (Sarstedt). TM The procedure was performed on mice by bleeding from the saphenous vein using catalog #16.444.100) and kept on ice. The blood was then centrifuged at 4000×g for 10 minutes at 4°C to separate the plasma, which was transferred to a clean microcentrifuge tube and rapidly frozen in liquid N2. Plasma from animals that received apoA-I ND was collected longitudinally from the saphenous vein as described above. The samples were diluted 2-fold in PBS and tested on the Mouse Cytokine 44-Plex Discovery Assay. TM(Eve Technologies Corporation TM The samples were sent for analysis using [method name]. Sample values ​​that fell below the detection limit were specified as 0 pg / mL. LIF levels fell below the detection limit for all samples and were not reported. Statistical outliers identified using the ROUT method were excluded from the analysis with a cutoff Q value set to 1%. Plasma apoA-I concentrations were measured by ELISA (enzyme-linked immunosorbent assay) as described above, and plasma PK characteristics were measured using PhoenixWinNonlin [method name]. TM Software (Certara TM Modeled using ).

[0103] IV tail vein injection The animals are weighed, and then a mouse tail illuminator (Bioseb) is used. TM The tail was restrained with a catalog #MTI. The tail was heated on an illuminator for 2-3 minutes to highlight the tail vein, sterilized with 70% ethanol, and a 31 gauge needle (BD). TM Insulin syringes (catalog # 320440) were used to deliver the formulation via tail vein as a bolus injection in volumes up to 10 mL / kg. For biodistribution and tolerability studies, dose calculations for apoA-I ND were performed based on mg apoA-I / kg animal body weight and delivered in doses ranging from 5 to 60 mg / kg (178 to 2136 nmol / kg). For mHTT target binding experiments, dose calculations for the molar equivalent of HTT ASO as HTT ASO ND were performed based on HTT ASO or mg ASO / kg animal body weight and delivered in doses ranging from 1.67 to 10 mg / kg (260 to 1559 nmol / kg).

[0104] ICV injection Animals received a unilateral ICV bolus injection as previously described [71,72]. For mHTT target binding studies, 5 μg (0.78 nmol) or 15 μg (2.34 nmol) of HTT ASO or its molar equivalent was delivered by ICV injection as HTT ASO ND.

[0105] tissue preparation For immunohistochemistry, animals were anesthetized with 2,2,2-tribromoethanol (Avertin™) and disposed of by transcardiac perfusion with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. The brain was removed and fixed in 4% PFA for 24 hours, then placed in 30% sucrose for 2–4 days. The brain was then cryosectioned at 25 μm, and the sections were stored at 4°C in PBS containing 0.01% NaN3. For biochemical analysis, mice were anesthetized with 2,2,2-tribromoethanol (Avertin) and CSF was collected. Blood was then collected via cardiac puncture for plasma separation. The brain was removed and placed on ice for approximately 1 minute to increase tissue stiffness. A mouse brain matrix was used to divide the brain into left and right hemispheres. The right hemisphere was then micro-dissected into different regions (e.g., striatum, cortex, hippocampus, cerebellum), and the left hemisphere was dissected into four rostral-caudal slabs using the brain matrix (Figure 2e). All brain samples were then frozen in liquid N2 and stored at -80°C until use. For ELISA (enzyme-linked immunosorbent assay) and immunoblotting, samples were homogenized in SDP lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1% IGEPAL, 40 mM β-glycerophosphate, and 10 mM NaF) using protease inhibitors (1X cOmplete protease inhibitor cocktail, 1 mM phenylmethanesulfonyl fluoride in isopropanol, 1 mM sodium bisulfite, and 5 μM Z-VAD-FMK) and 0.1% sodium dodecyl sulfate (SDS) as described above

[52] .

[0106] HD lymphoblast culture and treatment GM04724 Lymphoblastic Cells at Coriel Medical Institute TM The cells were obtained from the Nigomus Human Gene Cell Repository. Lymphoblasts were collected using RPMI-1640 media (Gibco). TMThe cells were propagated supplemented with 10% FBS (catalog #11875093). For all treatment experiments, GM04724 lymphoblasts were co-pulverized into single-cell suspensions, counted using a hemocytometer, and seeded with 5 × 10⁵ cells in 1 mL RPMI-1640 per well of a 12-well plate. For activity studies, HTT ASO, amino-HTT ASO, 4FB-HTT ASO, and HTT ASO ND were diluted to 3 μM in pre-warmed RPMI-1640 media. The treatment was then added to wells containing lymphoblasts at a final concentration of 1 μM, the cells were co-pulverized into single-cell suspensions, and incubated for 120 hours before collection.

[0107] histology For tolerability assessment, fixed tissues were rinsed three times with PBS and then placed overnight in 70% ethanol. The tissues were subsequently processed, embedded in paraffin, sectioned (5 μm), and stained with hematoxylin and eosin (H&E) (BC Children's Hospital Histology Core Laboratory). H&E slides were prepared using an Olympus BX61. TM Microscope (Olympus Scientific Files) TM The tiles were scanned using ( ) and a 10x air objective lens was used.

[0108] Immunohistochemistry (IHC) For the evaluation of cellular tropism, brains equilibrated with 30% sucrose were frozen on dry ice, and Tissue-TEK OCT implanted compound (Sakura TM Mounted in catalog #4583, Cryostat (Leica TM The striatum was cut into 25 μm coronal sections with free suspension in PBS + 0.01% NaN3 via CM3050S. A series of sections spanned the striatum and separated at 200 μm intervals were then treated with rabbit anti-apoA-I antibody (1:1000, Abcam). TM (Catalog #ab52945) and mouse anti-NeuN (1:1000. Sigma-Aldrich) TM(Catalog #MAB377) or Tomato Lectin DyLight 649 TM (1:1000. Vector Laboratories) TM Co-staining was performed with either of the following (catalog #DL-1178-1). The primary antibody was goat anti-rabbit AlexaFluor-488. TM (1:500, Invitrogen TM (Catalog #A-11008) and goat anti-mouse AlexaFluor-568 TM (1:500, Invitrogen TM (Catalog #A-11004) was detected with a secondary antibody. The section then contained ProLong Gold Antifade with DAPI. TM It was mounted using

[0109] ApoA-I enzyme-linked immunosorbent assay (ELISA) Human apoA-I quantification is performed using ELISA (Enzyme-linked immunosorbent assay) Pro: Human apoA-I Kit (Mabtech) TMThe assay was performed using (3710-1HP-10) according to the manufacturer's instructions. The final volume of 100 μl / well was maintained throughout the assay. Pre-coated plates were washed five times with wash buffer before use and between all steps. All subsequent steps were performed at room temperature with continuous stirring. Samples were diluted with Apo ELISA (enzyme-linked immunosorbent assay) buffer, and samples, standards, and background controls were added to the wells. The plates were then incubated for 2 hours. Next, detection antibodies were added to each well and incubated for 1 hour. Streptavidin-HRP was then added to each well, and the plates were incubated for 1 hour. TMB substrates were added to the wells and incubated for 10–15 minutes until full color development occurred, protected from direct sunlight. The reaction was stopped by adding stop solution to each well. Absorbance was measured at 450 nm, and apoA-I concentrations were normalized to the total protein mg in the sample. The whole-brain mean was calculated by averaging normalized apoA-I levels across all four rostral-caudal brain slabs shown in Figure 2e. Alternatively, apoA-I concentrations in culture media, tissue lysates, and biofluids were measured using a human apoA-I ELISA (enzyme-linked immunosorbent assay) kit (Mabtech). TM Quantification was performed according to the manufacturer's protocol using catalog #3710-1HP-2. Absorbance measurements were taken using a POLARstar Omega TM Plate reader (BMG LabTech) TM) were acquired at 450 nm and 600 nm using ). Dilutions were tested for each sample type to ensure that measurements fell within the linear range of the assay. Standard curves generated using purified human recombinant apoA-I standards (provided in the ELISA (enzyme-linked immunosorbent assay) kit) at concentrations ranging from 0.6 to 40 ng / mL were included in all experimental plates to calculate absolute apoA-I concentrations (not shown). Comparisons of detection using equivalent concentrations of purified recombinant apoA-I WT and apoA-I K133C were performed before running experimental samples. Detection of both apoA-I proteins was nearly equivalent, with absorbance values ​​falling within the linear range of the assay (not shown). Tissues and biofluids from PBS-treated mice were included in all experimental plates, and the average absorbance values ​​of identical tissues / biofluids were removed from the experimental sample values ​​(unless otherwise shown) to provide apoA-I concentrations against a background. The ApoA-I concentration is presented for culture medium (ng / mL), tissue (apoA-I ng / lysate mg), or biofluid (apoA-I ng / biofluid μL).

[0110] Fluorescent dye labeling of ApoA-I For external imaging research, the near-infrared CF750 fluorescent dye (Sigma-Aldrich TM Labeling of apoA-I WT and apoA-I K133C proteins (catalog #SCJ4600059) was carried out according to the manufacturer's protocol under the following conditions. Briefly, apoA-I WT or apoA-I K133C protein was diluted to 5 mg / mL (356 nmol / mL) in PBS, and 0.66 mL (3.3 mg) of each protein was adjusted to pH 8.3 with a final concentration of 0.1 M NaHCO3 for labeling. For each apoA-I protein, 1 μmol CF750 succinimimidyl ester was dissolved in 100 μL dH2O at a concentration of 10 mM, and the dye was added dropwise to the apoA-I solution. The labeling reaction was incubated at room temperature for 1 hour under quantitative stirring. Excess dye was then removed using a centrifugation filter unit (Amicon) with a 10 kDa MWCO. TMRemoved using catalog #UFC5010.

[0111] External imaging Two-month-old BACHD mice were assigned for external imaging and received 250 μg of CF750-labeled apoA-I ND intravenously. The animals were perfused two hours post-injection as described above. The entire or sectioned brain, fixed and along the frontal axis, was scanned using the AMI HTX Imaging System. TM (Spectral Instruments Imaging TM Images were obtained using a 745 / 790nm excitation / emission filter, power=10%, exposure=2s, and bin=1. Average fluorescence intensity values ​​(photons / s / cm²) were obtained. 2 / sr) is Aura Imaging Software (Version 2.0.1, Spectral Instruments Imaging TM The individual regions of interest in each brain were determined by plotting them using ) and intensity values ​​from PBS-injected brains were removed to provide a fluorescent signal on top of the background. Fire lookup tables (LUTs) were created in ImageJ TM It was applied using [a specific method], and the fluorescence intensity was highlighted.

[0112] Immunoblotting Regarding the verification of HTT ASO + apoA-I K133C conjugate, using a 4×NuPAGE LDS Sample Buffer. TM (Invitrogen TM (Catalog # NP0007) and 50 mM dithiothreitol (DTT) were added to 10 μg (356 pmol) of the molar equivalents of apoA-I K133C and ASO+apoA-I K133C conjugate, and the samples were denatured at 70°C for 10 minutes. The samples were then processed using NuPAGE. TM MOPS SDS execution buffer (Invitrogen TM (Catalog #NP0001), NuPAGE TM 12%, bis-Tris gel (Invitrogen TMThe process was repeated (catalog #NP0341) and run at 175V for 45-50 minutes. The gel was then cut in half for imaging of both ASO and apoA-I. One half was used for Quant-iT OliGreen. TM ssDNA reagent (1:10,000. Invitrogen) TM The samples were incubated at room temperature for 30 minutes (catalog #O7582) and imaged using GelDoc. The other half was imaged using NuPAGE. TM Transport buffer (Invitrogen TM The membrane was transferred to 0.2 μm nitrocellulose (catalog #NP00061) at 18 V for 2 hours. The membrane was then transferred to 0.05% Tween-20 in PBS containing 5% skim milk at room temperature. TM (PBST TM Blocked in a stirrer for 30 minutes, washed three times with PBST, and rabbit anti-apoA-I antibody (1:1000, Abcam) TM The sample (catalog #ab52945) was incubated overnight in a locker at 4°C with 5% BSA in PBST containing 0.01% NaN3. After incubation, the membrane was washed three times with PBST and treated with goat anti-rabbit IgG(H+L) Alexa Fluor 680. TM (1:5000. Invitrogen) TM The membrane (catalog #A-21076) was incubated in 5% skim milk / PBST at room temperature for 1 hour. Finally, the membrane was washed four times with PBST and then infused into the LI-COR Odyssey Imaging System. TM Imaging was performed using

[52] . Quantitative immunoblotting was performed as described above to measure HTT levels in cells and tissue lysates.

[52]

[0113] Double-label immunohistochemistry Free-floating coronal sections (25 μm thick) of mouse brains were blocked for 1 hour in a solution containing 10% goat normal serum (NGS), 10% donkey normal serum (ND), or both, in PBS. The sections were then blocked with ApoA-1 (rabbit anti-human apoA-I; Abcam) in a solution containing 5% NDS, 5% NGS, or both, in PBS. TMab52945;1:250) and GFAP (rat anti-GFAP, Invitrogen TM 13-0300;1:1000), NeuN (mouse anti-NeuN;Millipore TM MAB377;1:1000), Ionized calcium-binding adapter molecule 1 (Iba1; goat anti-Iba1, Abcam TM AB107159;1:1000), Tomato lectin-conjugated Dylight 649 TM (Vector Laboratories TM , #DL-1178-1;1:1000), or rabbit anti-ASO antibody (Ionis Therapeutics) TM The samples were incubated overnight at 4°C with primary antibodies (1:2000) from both sources. The sections were then incubated for 1 hour with the corresponding secondary antibody diluted 1:500 in PBS. The secondary antibody used was goat anti-rabbit Alexa Fluor 488 for apoA-I detection. TM (Invitrogen TM (A11008) or Donkey Anti-Rabbit Alexa Fluor 488 TM GFAP detection for goat anti-rat Alexa Fluor 568 TM (Invitrogen TM (A11077), NeuN Anti-Goat Mouse Alexa Fluor 633 for Detection TM (Invitrogen TM (A-21052), and Iba1 detection for donkey anti-goat Alexa Fluor 594 TM (Invitrogen TM It was A11058).

[0114] The section is mounted on a slide and is Fluoromount-G (Southern Biotech TM Coverslip was applied using ).

[0115] Huntington low-bis-Western blot Full-length HTT alleles were degraded using allele isolation immunoblotting as described above

[46] . Briefly, 50 μg of total protein was loaded onto a 10% bis:acrylamide (1:200 bis:acrylamide) gel. The protein was transferred to a 0.45 μm nitrocellulose membrane and bled with mouse 1CU-4C8 anti-HTT antibody (Fisher Scientific). TM , #MAB2166) and rabbit anti-calnexin antibody (Sigma TM Incubated with IR dye 800CW goat anti-mouse (Rockland). TM , #610-131-007) and AlexaFluor TM 680 Goat-Rabbit (Molecular Probes) TM Secondary antibody (#A2107) was used. Odyssey infrared imaging. TM The system (LI-COR) is used for blot scanning, and Image Studio Lite TM v5.2 was used to perform concentration measurements to quantify band intensity. The IOD values ​​of the HTT band were normalized to the IOD of the loaded control calnexin, and then normalized to the same allele for PBS-treated mice on the same membrane.

[0116] Microscopic examination Labeled sections were imaged using a Leica SP8 STED confocal microscope with a 40x objective lens. The image format was 2048 × 2048 pixels at a scan speed of 600 Hz. All images for each set of markers were acquired with the same laser and gain settings to allow for comparison. Z-stacks (257 nm) were imaged using Hygens Professional Deconvolution. TM Software was used to deconvolute the images. HD BMEC and brain sections for tropical region studies were taken with a Leica TCS SP5 with 40x and 63x oil immersion objective lenses. TM Laser scanning confocal inverted microscope (Leica Microsystems) TM), and Leica Application Suite Advanced Fluorescence TM The images were imaged using software.

[0117] Experimental design and statistical analysis All statistical analysis is done in GraphPad Prism TM (Version 8 or 9 GraphPad) TM The analysis was performed using the following methods. Group comparisons of normally distributed data with similar variances were analyzed using two-tail Student's t-tests (two groups) or ANOVA (three or more groups). Post-hoc analyses of ANOVA were performed using Tukey or Sidak tests for multiple comparison corrections. Linear regression was performed to assess dose-response. Bar graphs were presented as scatter plots to show the complexity and variability of the data. Group data from the mean of multiple animals were presented as mean ± standard error of the mean. Experiments involving animals included approximately equal numbers of males and females. The number of animals used in each experiment (biological replicates; N) is reported in the figure legend. Alpha values ​​<0.05 were considered significant for all analyses. The p-values ​​for each statistical test are presented in the text and the corresponding figure legend, where *=p<0.05, **=p<0.01, ***=p<0.001, and ****=p<0.0001.

[0118] Examples Example 1: ApoA-I ND forms an electrostatic interaction with antisense oligonucleotides.

[0119] First, we sought to determine whether apoA-I nanodiscs have the ability to bind to antisense oligonucleotides (ASOs) designed to promote the degradation of human HTT transcripts

[50] . The secondary structure of apoA-I contains an amino-terminal globule region spanning residues 1–43 and 10 amphiphilic α-helices encompassing residues 44–243, with helices 5, 6, and 7 containing polar faces and positively charged residues [53–55]. We hypothesized that the positively charged faces within the secondary structure of apoA-I could form electrostatic interactions with negatively charged ASOs. To test this, we performed electrophoretic mobility shift assays (EMSA) incubated with quantified amounts of ASO in molars of apoA-I ND (stoichiometric ratio 1:1–1:3 from apoA-I to ASO) for 1 hour at 37°C. ASO was visualized at the level of apoA-I ND, suggesting a potential electrostatic interaction between ASO and apoA-I (Figure 1a).

[0120] The interaction between apoA-I ND and ASO was further investigated using size exclusion chromatography (SEC). ApoA-I ND was incubated with increasing molar amounts of ASO at 37°C for 1 hour, and the formulation was applied to the column. At a molar ratio of 2 mol of ASO:1 mol of apoA-I:10⁵ mol of DMPC, ASO consistently eluted in the same fraction as both apoA-I and DMPC, suggesting an interaction between ASO and ND (Figure 1b). As a negative control, each individual component was run through the column alone, and fractions were collected for analysis. It was observed that ASO, apoA-I, and DMPC eluted in different patterns (Figure 1c).

[0121] OliGreen is used to quantify oligonucleotides and single-stranded DNA in solution in order to quantify the proportion of ASO incorporated into apoA-I ND. TM The assay using fluorescent dyes was optimized. Both ApoA-I WT and K133C ND were incubated with ASO (37°C for 1 hour) and then dialyzed to remove free, unbound ASO. The dialyzed formulations were then treated with the surfactant Triton X-100.TM Processed by, OilChange TM The amount of oligonucleotides associated with apoA-I ND was quantified after incubation. If the oligonucleotides are located / associated along the inside of apoA-I ND, OliGreen TM The signal is from Triton X-100. TM We hypothesized that the Triton X-100 would be superior to apoA-I ND, which is not treated with Triton X-100, because it would release ASO from inside the disc due to the destruction of ND by the surfactant after processing. The results were as follows: TM In the absence of [specific factor], approximately 83–84% of ASOs were associated with both apoA-I WT and K133C ND. In particular, Triton X-100 processing did not dramatically alter the proportion of ASOs associated with either form of apoA-I ND (Figure 1d). Therefore, these data suggest that ASOs can be associated along the apoA-I externality of apoA-I ND.

[0122] Using dynamic light scattering (DLS; Figure 1e), we found that apoA-I WT NDs were larger in size (mean diameter ± standard deviation = 11.95 ± 0.34 nm) and had a higher polydispersity index (PDI = 0.086) compared to NDs formed with apoA-I K133C (n=3; mean diameter ± standard deviation = 10.76 ± 0.05 nm, PDI = 0.067). Both ApoA-I WT and K133C NDs are well within the ideal size range for intranasal administration to the brain and subsequent perivascular distribution.

[0123] To determine if there was a difference between the association of K133C ND with apoA-I WT and ASO, native PAGE was performed, with the gel stained with SYBR safe to visualize ASO, followed by Coomassie staining for total protein to visualize ND (not shown). The signals from both stains showed similar patterns at the ND level, confirming the association of ASO with ND. Signal intensity was quantified and found to be similar for both apoA-I WT ND and apoA-I K133C ND (not shown).

[0124] Example 2: ApoA-I K133C ND shows enhanced CNS penetration after intranasal administration.

[0125] The initial in vivo goal was to determine whether NDs formed from apoA-I WT or apoA-I K133C could enter the CNS after intranasal administration. BACHD mice were treated with 150 μg doses of lipid-free apoA-I WT, lipid-free apoA-I K133C, apoA-I WT ND, or apoA-I K133C ND (in 30 μl PBS) via intranasal administration. Brain apoA-I levels were quantified 2 hours after intranasal administration using a human apoA-I-specific ELISA (enzyme-linked immunosorbent assay) as previously described

[46] . ApoA-I levels were higher in the brain after intranasal administration of apoA-I WT ND compared to lipid-free apoA-I WT, suggesting that ND formulations may improve CNS entry. Whole-brain apoA-I levels were higher after intranasal administration of lipid-free apoA-I K133C compared to lipid-free apoA-I WT (Figure 2a). One-way ANOVA p<0.001; Tukey's multiple comparisons (****p<0.0001), and for apoA-I K133C ND compared to apoA-I WT ND (****p<0.0001).

[0126] To evaluate the CNS distribution of mutant apoA-I K133C after intranasal administration, mice were euthanized 2 hours after intranasal administration, and their brains were microdissected into four rostral-caudal brain slabs (AD; Figure 2e). ApoA-I K133C ND achieved significantly higher CNS penetration across the three most rostral brain regions AC (Figure 2b). Two-way ANOVA shows significance by treatment (p<0.0001), brain region (p<0.0001), and interaction (p<0.0001); Tukey's multiple comparison test for K133C vs K133C ND - A:***p<0.001, B:****p<0.0001, C:**p<0.01, D:ns; WT ND vs K133C ND - A:****p<0.0001, B:****p<0.0001, C:*p<0.05).

[0127] Furthermore, by performing external brain imaging 2 hours after intranasal administration, the total brain distribution of apoA-I-1 labeled with cf750 dye was evaluated after intranasal administration of apoA-I K133C ND. When examining the dorsal brain (Figure 2c), high concentrations were achieved throughout the brain, particularly in the olfactory bulb, cortex, and cerebellum. On the ventral side (Figure 2d), high concentrations were also observed in the olfactory bulb, the central layer of the cortex, around the circle of arteries, and in the hypothalamus, pons, and medulla oblongata.

[0128] Example 3: The HD mutation does not significantly alter the CNS entry and distribution of ApoA-I K133C ND.

[0129] The next objective was to determine whether the HD mutation in BACHD mice affects the amount of apoA-I ND delivered to the brain after intranasal administration, given that blood-brain barrier integrity has been shown to be affected in both HD mice [56,57] and patients

[58] . Separate groups of BACHD mice and their wild-type siblings aged 2, 6, and 12 months received the same dose of apoA-I K133C ND via intranasal administration (150 μg of apoA-I in 30 μl of PBS). The animals were euthanized 2 hours after intranasal administration, and the brains were dissected into four rostral-caudal slabs AD as shown in Figure 2e. In both the 2-month and 6-month groups, there was no significant difference in apoA-I delivery efficiency across all four brain regions (Figures 3a and 3b; two-way ANOVA showed significance by brain region (p<0.0001) but not by genotype, p=0.4482 and p=0.8395, respectively, or by interaction, p=0.7308 and p=0.9755, respectively). However, in the 12-month group, region A (composed of the olfactory bulb and prefrontal cortex) had significantly higher apoA-I levels in BACHD mice compared to their wild-type siblings. There were no significant differences between the two groups across the other three brain regions. (Figure 3c, two-way ANOVA shows significance by brain region (p<0.0001), but not by genotype (p=0.3135) or interaction (p=0.0522); Bonferroni's multiple comparison test, *p<0.05 for region A). These results provide important evidence that apoA-I delivery to the brain after intranasal administration of apoA-I K133C ND is not a result of anatomical changes due to HD mutations (at 2 and 6 months). For the remainder of the study, 2-month-old BACHD mice were continued to be used to rule out potential variability with aged mice.

[0130] Example 4: After intranasal administration of ApoA-I K133C ND, ApoA-I localizes to multiple cell types throughout the brain.

[0131] To determine the cellular distribution of ND, ApoA-I K133C ND was administered intranasally to 2-month-old BACHD mice, and the animals were euthanized 2 hours later. Immunohistochemical staining of human apoA-I and different cellular markers was performed in sagittal sections to determine the cell types to which apoA-I was localized at this time point. Extensive co-localization of apoA-I was observed throughout the brain with both CD31+ capillary endothelial cells and NeuN+ neurons. Less frequently, apoA-I also co-localized with GFAP+ astrocytes and Iba1+ microglia (Figure 4). These localizations were not specific to any particular brain region, but rather occurred across the entire sagittal axis of the section. Furthermore, apoA-I co-localized extensively with lectin-stained choroid plexus cells.

[0132] Example 5: ApoA-I K133C NDCNS rapidly enters the brain and CSF after intranasal administration.

[0133] To determine the optimal dose parameters for apoA-I K133C ND, we conducted a study evaluating the time course of apoA-I distribution in the brain and CSF after intranasal administration. ApoA-I K133C ND was administered intranasally to five groups of mice, and harvested at 1, 2, 3, 6, and 24 hours post-intranasal administration (Figures 5a and 5b). The highest apoA-I levels were found across all four brain regions AD at 2 hours post-intranasal administration. At the 1-hour time point, levels were highest in region A (composed of the olfactory bulb and prefrontal cortex), but decreased over time. Simultaneously, levels were lowest in region D (composed of the cerebellum and brainstem) at the 1-hour time point, but continued to rise over time (Figure 5b; two-way ANOVA shows significance by time point (p<0.0001), brain region (p<0.0001), and significant interaction (p<0.0001); Tukey's multiple comparison tests**p<0.01,***p<0.001,...p<0.0001). Furthermore, apoA-I levels were measurable across all four brain regions (over the background) 24 hours after intranasal administration. The whole-brain mean showed the same trend, with apoA-I levels peaking at 2 hours after intranasal administration and decreasing to approximately 17% of the peak level at 24 hours (Figure 5a; one-way ANOVA shows significance by time point (p<0.0001); Tukey's multiple comparison tests****p<0.0001 compared to 1 hour).

[0134] Example 6: ApoA-I K133C ND shows a dose-dependent increase in brain activity after intranasal administration.

[0135] Next, we sought to determine the relationship between the dose of intranasal-administered apoA-I K133C ND and the apoA-I levels detected in the brain. To answer this question, groups of mice received ascending doses of apoA-I K133C ND (75, 150, 300, or 500 μg apoA-I). Since apoA-I levels were found to peak in the brain 2 hours after intranasal administration, animals for this study were harvested 2 hours after intranasal administration of apoA-I K133C ND, and tissues were analyzed by ELISA (enzyme-linked immunosorbent assay) for human apoA-I (Figures 5c and 5d). When comparing the whole-brain mean apoA-I levels in each treatment group, there was a linear trend between doses of 150–500 μg apoA-I K133C ND, and the whole-brain mean apoA-I level doubled with dose doubling (r 2 (=0.9843). The 500 μg dose was significantly higher than both the 75 μg and 150 μg doses (Figure 5c; one-way ANOVA shows significance by dose (p<0.01); Tukey's multiple comparison test, 500 μg vs 75 and 150 μg**p<0.01).

[0136] Following intranasal administration of ND, a dose-dependent increase in apoA-I levels was observed along the rostral-caudal axis of the brain (Figure 5d; two-way ANOVA shows significance by dose (p<0.01), brain region (p<0.0001), and interaction (p<0.01)). Two hours after intranasal administration, this dose-dependent increase was evident in the most rostral brain region A, consisting of the olfactory bulb and prefrontal cortex, where apoA-I levels were highest, significantly highest after a 500 μg dose, followed by a 300 μg dose (Figure 5d; Tukey's multiple comparison test; **p<0.01, **p<0.001, ****p<0.0001). A similar trend was observed in medial brain region B spanning the striatum, with higher apoA-I levels measured after a 500 μg dose of ND. Simple linear regression confirmed that apoA-I levels significantly increased with increasing dose in regions A and B (non-zero slope, p<0.05). No significant trend was observed in the more caudal brain regions C and D at this time point.

[0137] Example 7: ApoA-I K133C ND has minimal distribution to peripheral tissues and is rapidly cleared after intranasal administration.

[0138] Since intranasal administration delivers macromolecules directly to the brain with limited peripheral exposure, we sought to determine the time course of apoA-I distribution in peripheral tissues after intranasal administration of a 150 μg dose of apoA-I K133C ND. ApoA-I levels were measured in CSF, lung, liver, and plasma at 1, 2, and 6 hours post-intranasal administration. At 1 hour post-intranasal administration, apoA-I had already accessed the CSF compartment and maintained a steady state between 2 and 6 hours post-treatment (Figure 6a). In the lung, apoA-I levels increased at 1 and 2 hours post-intranasal administration but were completely eliminated from the lung at 6 hours (Figure 6b). This suggests that some of the intranasally administered dose is inhaled into the lungs by ND and eliminated over the course of 6 hours. One hour after intranasal administration, apoA-I had not yet reached the liver, but levels increased to 10 ng apoA-I / mg protein in the next time period and showed a tendency toward elimination at the 6-hour time point (Figure 6c). These results indicate that intranasal administration primarily bypasses the first-pass effect, resulting in the minimum amount of apoA-I distributed to the liver during the 6-hour time period.

[0139] In plasma, apoA-I levels were extremely low one hour after intranasal administration and continued to rise over the next five hours (Figure 6d). This suggests a slow exchange of apoA-I redistributed from the lungs and CSF into the plasma over a six-hour period.

[0140] Example 8: Intranasal administration of ApoA-I K133C ND results in increased CNS levels in the brain compared to equivalent intravenous administration.

[0141] Since we have previously demonstrated that apoA-I K133C ND can be accessed in the brain after intravenous administration, we sought to compare the efficiency of brain delivery after both intranasal and intravenous administration. Animals for this study received the same dose (300 μg) of apoA-I K133C ND either intravenously or intranasally and were harvested 2 hours post-administration. ELISA (enzyme-linked immunosorbent assay) results showed that mice that received ND intranasally had significantly higher whole-brain mean apoA-I levels compared to those that received ND intravenously. In fact, whole-brain mean apoA-I was almost four times higher in the intranasal group compared to the intravenous group (Figure 7a, unpaired t-test, *p<0.05). In the most rostral brain region examined, consisting of the olfactory bulb and frontal cortex, ApoA-I levels were significantly higher in mice that received apoA-I K133C ND intranasally compared to those that received intravenous administration. There were no significant differences between the treatment groups in the caudal brain region (BD) examined (Figure 7b; two-way ANOVA shows significance by administration route (P<0.01), brain region (P<0.001), and interaction (P<0.001); region A: Bonferroni multiple comparison test ***p<0.0001).

[0142] Example 9: A single intranasal dose of ASO K133C ND resulted in potent suppression of cortical and striatal mHTT in BACHD mice.

[0143] To visually confirm whether apoA-I K133C ND can effectively deliver ASO to the brain, PBS or ASO-loaded apoA-I K133C ND (or ASO-ND) was administered intranasally to 2-month-old BACHD mice, and the animals were perfused 2 hours later. Double-labeled IHC of ASO and NeuN showed that ASO was deposited in neurons throughout the brain 24 hours after intranasal administration (Figure 8a).

[0144] To determine whether apoA-I K133C ND can effectively deliver ASO to the brain at concentrations sufficient to achieve target binding, single doses of either PBS, ASO alone (300 μg ASO), or ASO-loaded apoA-I K133C ND (or ASO-ND; 300 μg ASO:660 μg apoA-I) were administered intranasally to 2-month-old BACHD mice, and the animals were harvested 4 weeks later. HTT Western blot results showed a significant reduction of approximately 30% in relative mHTT throughout the whole brain of ASO-ND treated mice compared to PBS-treated controls (Figure 8b; one-way ANOVA shows significance by treatment (P<0.05); Tukey's multiple comparison test, *p<0.05). Relative mHTT levels were significantly lower in the olfactory bulb, cortex, and striatum of ASO-ND treated mice compared to PBS-treated controls. In all other brain regions examined, a relative decrease of approximately 20–25% in mHTT levels was observed (Figure 8d; two-way ANOVA shows significance with treatment (P<0.0001); Tukey's multiple comparison test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0145] Next, we wanted to determine whether this mHTT reduction continued over the 8-week period and whether a second dose after 4 weeks helped maintain the mHTT suppression. To evaluate these questions, BACHD mice received PBS, two doses of ASO (300 μg ASO given at 4-week intervals), one dose of ASO-ND (300 μg ASO:660 μg apoA-I), or two doses of ASO-ND (300 μg ASO:660 μg apoA-I given at 4-week intervals) and were euthanized 8 weeks after the initial treatment (Figure 9a). The mean relative mHTT levels in the whole brain of mice treated intranasally with two doses of ASO-ND were significantly lower by approximately 10% compared to mice treated with one dose of ASO-ND (Figure 9b; one-way ANOVA shows significance by brain region (P<0.0001); Tukey's multiple comparison test, **p<0.01, ****p<0.0001).

[0146] In microanatomically dissected brain regions, the second ASO dose after the first four weeks did not achieve a significant reduction in mHTT in any brain region. However, two ASO-ND doses achieved a significant reduction in mHTT in the olfactory bulb, striatum, and brainstem compared to the PBS-treated control. The reduction in mHTT was similar in all brain regions after intranasal administration of one or two doses of ASO-ND, except for the brainstem, where two ASO-ND doses resulted in a significant reduction in mHTT compared to a single ASO-ND dose. (Figure 9c; two-way ANOVA shows significance by brain region (P<0.05) and treatment (P<0.0001); Tukey's multiple comparison test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0147] Example 10: In vitro characterization of ApoA-I nanodiscs

[0148] Recently, it has been demonstrated that ApoA-I can efficiently enter the CNS after intravenous administration in mice via the blood-cerebrospinal fluid barrier (BCSFB)

[46] and the blood-brain barrier (BBB)

[82] . ApoA-I has been shown to transepithelial cell permeability throughout brain microvascular endothelial cells of the BBB via receptor-mediated endocytosis through scavenger receptor B1 (SR-B1)

[83] . Multiple studies have demonstrated that the apolipoprotein AI can interact with charged and / or neutral lipid bilayer vesicles to form disc-shaped complexes known as nanodiscs (ND)[84,85]. Optimization of ApoA-I wild-type (WT) concentration and neutral lipids, 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), was performed to identify formulations in which the majority of apoA-I is incorporated into un. NDs were generated with a quantified mole of apoA-I and an increasing mole of DMPC, and these formulations were subjected to native polyacrylamide electrophoresis (PAGE) (Figure 10A). A ratio of 10⁵ moles of DMPC to 1 mole of ApoA-I resulted in over 99% of free ApoA-I being incorporated into the nanodiscs (NDs) (Figure 10B). This DMPC lipid to ApoA-I ratio was used for ND formation in all subsequent studies.

[0149] Example 11: Antisense oligonucleotides associate with ApoA-I nanodiscs

[0150] Recently, ApoA-I ND has been demonstrated to be usable to deliver chemotherapeutic agents to glioblastoma to induce tumor regression

[92] . Furthermore, ApoA-I ND can bind to siRNA to promote gene silencing in cultured cells

[93] . To determine whether ApoA-I ND has the ability to bind to an antisense oligonucleotide (ASO) designed to target HTT mRNA (messenger RNA) for RNase H1-mediated degradation, an ASO that had previously been shown to potently suppress HTT in a mouse model of HD was used

[50] . This synthetic ASO contains a phosphorothioate-modified backbone, with a lock nucleic acid (LNA)-modified nucleotide wing flanking an unmodified gap (gapmer), giving the molecule a purely negative charge. The secondary structure of ApoA-I contains an amino-terminal globule region spanning residues 1–43 and 10 amphipathic α-helices containing residues 44–243, with helices 5, 6 and 7 containing polar faces and positively charged residues [53–55]. To test whether the positively charged planes within the secondary structure of apoA-I can form electrostatic interactions with negatively charged ASO, an electrophoretic mobility shift assay (EMSA) was performed.

[0151] ApoA-I ND was produced (as described above) and incubated at 37°C for 1 hour with increasing molar amounts of ASO (apoA-I:ASO molar ratio of 1:1 to 1:5). A size shift was observed in the ASO-incubated ApoA-I ND formulation, with the strength and size of the formulation increasing with increasing levels of ASO, suggesting an interaction between ASO and apoA-I ND (Figure 11A).

[0152] Because EMSA requires formulations exposed to an electric field, the integrity of the electrostatic interaction between negatively charged ASO and positively charged apoA-I residues on ND may be altered. To investigate the interaction between ApoA-I ND and ASO, a complementary method, size exclusion chromatography (SEC), was used.

[0153] ApoA-I ND was generated and incubated with increasing molars of ASO at 37°C for 1 hour, and the formulation was applied to a column containing G-50 Sephadex beads (data not shown). Multiple fractions were collected and quantified for nucleic acid, protein, and lipid levels. ASO consistently eluted in the same fractions as apoA-I and DMPC, suggesting an interaction between ASO and ND. As a negative control, each component was mixed immediately before the SEC assay, and each component eluted in a different fraction. Notably, the molar ratio of 3 mol ASO:1 mol ApoA-I:105 mol DMPC was the optimal ratio for this complex under these conditions (Figure 11B).

[0154] Example 12: ApoA-I nanodiscs carrying ASO enhance HTT suppression in HD patient-derived cells.

[0155] To evaluate whether ApoA-I ND can improve the delivery of ASO to cells, different ND formulations were compared to ASO alone in HD patient lymphoblasts using HTT levels as a readout for delivery efficiency. Previous studies have shown that ASO can enter lymphoblasts in the absence of carriers after bus application. ASO apoA-I WT ND (53%) significantly increased HTT suppression compared to ASO alone (25%) after 96 hours of treatment (Figure 12A and 12B. Two-way ANOVA treatment p<0.0001, HTT p=0.4413, interaction p=0.9973. Bonferroni post-test: *p<0.05, **p<0.01, ***p<0.001 compared to NT, #p<0.05 compared to ASO alone). The ASO used in this experiment induced non-selective suppression of both wild-type and mutant Huntington's Streptococcus huntington No significant difference was observed in the magnitude of mHTT suppression compared to post-treatment wild-type HTT (wtHTT), which was consistent with non-selective HTT reduction (Figure 12B).

[0156] Example 13: ApoA-I variant forms nanodiscs

[0157] Based on amino acid scanning and electron paramagnetic resonance, it has been previously proposed that ApoA-I adapts to a “loop-belt” conformation on reconstituted disc-shaped HDL particles, with the loop spanning amino acids 133–146

[39] . This loop structure partially overlaps with the lecithin-cholesterol-acyl-transferase (LCAT) activation site, spanning amino acids 143–165 of ApoA-I

[95] . LCAT is required for the esterification of free cholesterol. Upon esterification, cholesterol migrates from the surface to the hydrophobic core of the nascent disc-shaped HDL, resulting in the formation of spherical HDL. Within each disc-shaped HDL particle, two apoA-I molecules are oriented head-to-tail (antiparallel) and the apoA-I helix lies perpendicular to the phospholipid acyl chain [96,97]. Cysteine ​​substitution mutations of amino acids along the loop region (133-146), and adjacent amino acids including the LCAT site (123-165), can promote disulfide bridge formation between two apoA-I molecules, crossing the loop, fixing them in place, and preventing conversion from discoid to spherical HDL (Figure 13, modified with permission from

[39] ). apoA-I with cysteine ​​mutations at selected sites may limit cholesterol loading to ND, thus reducing involvement in the RCT pathway and promoting increased biodistribution to the CNS. To test this, a series of apoA-I mutants with amino acid substitutions at specific sites in the protein sequence were generated, including apoA-I mutants with cysteine ​​substitutions at key positions in the loop, K133C and / or E146C (Figure 14). Additional apoA-I mutants were produced with substitutions at other amino acid residues R160V (valine substitution) and H162A (alanine substitution) (Figure 14).

[0158] It has been previously shown that cysteine ​​substitution at selected residues of ApoA-I does not impair its ability to form nanodiscs

[39] . Using an optimized molar ratio of 10⁵ moles of DMPC: 1 mole of apoA-I, we found that both ApoA-I K133C and ApoA-I E146C nanodiscs form higher-order complexes on native PAGE (Figure 15A). To further characterize the morphology and size of mutant NDs, electron microscopy (EM) was performed on ApoA-I WT, ApoA-I K133C, and ApoA-I E146C NDs. Each of the ApoA-I WT, ApoA-I K133C mutant, and ApoA-I E146C mutant formed particles of different disk shapes (Figure 15B). Quantification of these particles revealed that ApoA-I WT NDs had a significantly smaller diameter than NDs formed with ApoA-I K133C (Figure 15C, one-way ANOVA p<0.0001; mean diameter: 15.67 nm vs 18.32 nm; Tukey's multiple comparison test p<0.0001) and ApoA-I E146C (Figure 15C, mean diameter: 15.67 nm vs 19.10 nm; Tukey's multiple comparison test p<0.0001). Furthermore, dynamic light scattering was performed to further evaluate the particle size and dispersibility of ApoA-I WT, K133C, and E146C NDs. Consistent with native PAGE and EM data, ApoA-I WT NDs were found to be smaller in size than NDs formed with apoA-I variants, K133C, and E146C (Figure 15D).

[0159] Example 14: ApoA-I K133C ND enhances CNS entry after systemic administration.

[0160] Lipid-free ApoA-I has been shown to be able to enter the CNS after intravenous administration [46,82]. To evaluate whether ND formed from ApoA-I WT can enter the CNS, brain ApoA-I levels were compared 2 hours after intravenous administration of 30 mg / kg of lipid-free ApoA-I WT or ApoA-I WT ND. ApoA-I WT ND levels were significantly higher in the brain compared to lipid-free ApoA-I, suggesting that ND improves CNS entry (Figure 16, unpaired t-test, p=0.0282). ApoA-I levels were quantified using human ApoA-I-specific ELISA (enzyme-linked immunosorbent assay) as previously described

[46] .

[0161] To determine whether NDs formed with ApoA-I mutants could improve CNS entry compared to ApoA-I WT NDs, BACHD mice received a 20 mg / kg bolus intravenous injection of ApoA-I WT NDs, ApoA-I K133C NDs, and ApoA-I E146C NDs (Figure 17). The animals were euthanized 2 hours after injection, and their brains were microdissected into the cortex, striatum, hippocampus, cerebellum, and brainstem. Significant effects of the ApoA-I variant and brain regions were observed in K133C, showing a significant increase in all brain regions compared to WT (striatum: *p=0.03, hippocampus: ****p<0.0001, cortex: *p=0.0361, cerebellum: **p=0.0001, and brainstem: **p=0.0057), and in all brain regions except the striatum, compared to E146C (hippocampus: ####p<0.0001, cortex: ##p=0.0064, cerebellum: ####p<0.0001, and brainstem: ###p=0.0001) (Figure 17, bidimensional ANOVA, brain region p=0.0271, ApoA-I p<0.0001, and interaction p=0.1221). To determine whether K133C and E146C NDs improve CNS entry compared to other apoA-I mutant NDs, the amounts of various apoA-I ND formulations in the brain and CSF were determined (Figure 18A and B). Based on these findings, ApoA-I K133C was identified as a lead mutant for subsequent studies.

[0162] External imaging was performed to verify the finding that ApoA-I K133C ND can enter the brain more efficiently than ApoA-I WT ND. ApoA-I WT and ApoA-I K133C proteins were labeled with the cf750 near-infrared dye, and it was confirmed that this coupling did not alter ApoA-I's ability to form un (data not shown). The cf750 succinimimidyl ester labels primary amines found at the amino terminus or basic residues of proteins. Because the K133C mutant has one less lysine residue than ApoA-I WT, the degree of cf750 labeling differed for each protein, making direct comparison difficult. Absolute fluorescence was found to be higher in cf750-labeled ApoA-I K133C ND compared to ApoA-I WT ND (Figure 19A), but relative fluorescence in the brain was quantified as a percentage of fluorescence in the liver. We measured significantly higher levels of ApoA-I K133C ND fluorescence in the brain compared to ApoA-I WT ND, consistent with ELISA (enzyme-linked immunosorbent assay) data (Figure 19B, 10% vs 5% of liver fluorescence, unpaired t-test p=0.0051).

[0163] Example 15: The time course of biological distribution of ApoA-I K133C nanodiscs shows rapid entry into the CNS after IV administration.

[0164] To evaluate the biodistribution profile of ND over time, BACHD mice received a bolus intravenous injection of either ApoA-I WT ND or ApoA-I K133C ND at 20 mg / kg. Brain and CSF were harvested at different time intervals after injection. In the brain, significantly higher levels of ApoA-I K133C ND were measured at 2 and 4 hours post-injection compared to WT ND (Figure 20A; two-way ANOVA; ApoA-I p<0.0001, time point p=0.1523, and interaction p=0.2058; Sidak multiple comparisons; 2 hours:***p=0.0004, 4 hours:*p=0.0151). No significant effect of the time interval after injection on apoA-I levels in the brain was found. ApoA-I K133C ND levels peaked at 2 hours post-injection, while ApoA-I WT ND levels peaked at 1 hour post-injection. In CSF, significantly higher levels of ApoA-I K133C ND were detected at 1 and 2 hours post-injection compared to ApoA-I WT ND (Figure 20B, two-way ANOVA: ApoA-I p<0.0001, time point p<0.0001, and interaction p<0.0001; Sidak multiple comparisons: 1 hour: ***p=0.0004, 2 hours: ****p<0.0001). There was also a significant effect of the time interval after injection on ApoA-I levels in CSF, with both ApoA-I WT and ApoA-I K133C ND levels peaking at 2 hours post-injection.

[0165] Example 16: Dose-dependent increase in ApoA-I K133C nanodiscs in the brain after IV administration

[0166] To evaluate whether increasing the dose of injected ApoA-I K133C ND leads to increased CNS entry, BACHD mice were subjected to dose-response experiments receiving 10–60 mg / kg of ApoA-I K133C ND in a single IV injection. ApoA-I K133C ND levels were found to peak in the brain 2 hours post-injection, and tissue was harvested at this time point for quantification using human ApoA-I ELISA (enzyme-linked immunosorbent assay). A significant dose-dependent increase in ApoA-I K133C in the brain was observed (Figure 21A, one-way ANOVA p<0.0001; Tukey's multiple comparison test: ***p<0.05 compared to 10 mg / kg, ##p<0.05 compared to 20 mg / kg; linear regression R²=0.7594, slope non-zero p<0.0001). No dose-dependent increase in ApoA-I in CSF 2 hours post-injection was observed (Figure 21B, one-way ANOVA p=0.1082; linear regression R²=0.2172, slope non-zero p=0.1745).

[0167] To obtain resolution for the regional distribution of ND within the dose range in the brain, BACHD mice received 5–30 mg / kg of ApoA-I K133C ND via a single IV injection (Figure 22). The animals were euthanized 2 hours after injection, and the brains were micro-dissected into brain regions (striatum, cortex, hippocampus, cerebellum, and brainstem) for quantification of ApoA-I by ELISA (enzyme-linked immunosorbent assay). Significant dose-response responses were observed in all brain regions (linear regression striatum R2=0.6402, non-zero slope p<0.0001; cortex R2=0.4971, non-zero slope p=0.0011; hippocampus R2=0.5155, non-zero slope p=0.0005; cerebellum R2=0.5773, non-zero slope p=0.0002; brainstem R2=0.5492, non-zero slope p=0.0003). There were no significant differences in ApoA-I levels between brain regions (two-way ANOVA brain region 0.5176, dose p<0.0001, interaction 0.4233).

[0168] Example 17: HD mutation does not significantly alter CNS entry of ApoA-I K133C nanodiscs.

[0169] The blood-brain barrier has been shown to be impaired in HD mice and patients [57,58]. To investigate whether the HD mutation may affect the concentration of ApoA-I ND entering the brain after IV injection, we performed an experiment in which BACHD and wild-type sibling controls received a single IV injection of 10 mg / kg of ApoA-I K133C ND at 2, 6, and 12 months of age. Although no BBB damage has been reported in the striatum of BACHD mice up to 12 months of age

[98] , this experiment served as an important control to rule out the possibility that the HD mutation was responsible for the brain exposure levels observed with ApoA-I K133C ND. Relative ApoA-I levels are presented rather than absolute values ​​because different cohorts were collected and analyzed at different times. No significant effect of genotype on brain ApoA-I levels was observed at any given age (Figure 23, two-way ANOVA: genotype 0.8201, age p=0.3699, interaction p=0.3699).

[0170] Example 18: ASO-carrying apoA-I WT and K133C nanodiscs can induce modest mutant HTT suppression in the CNS.

[0171] To evaluate the potential of ApoA-I ND as an ASO carrier in vivo, 2-month-old BACHD mice received a single IV injection of either PBS or ASO, ASO-ApoA-I WT ND, or ASO-ApoA-I K133C ND (300 μg), and HTT target binding in the CNS was measured after 4 weeks. Significant reductions in mHTT levels were observed in the cortex (Figure 24A, one-way ANOVA, p=0.0195), striatum (Figure 24B, one-way ANOVA, p=0.0309; Tukey's multiple comparisons* p<0.05 compared to PBS), and hippocampus (Figure 24C, one-way ANOVA, p=0.0166; Tukey's multiple comparisons** p<0.01 compared to PBS), but not in the cerebellum (Figure 24D, one-way ANOVA, p=0.8781).

[0172] Example 19: ApoA-I K133C ND increases peripheral exposure after systemic administration.

[0173] Lipid-free ApoA-I has been shown to be able to enter the CNS after intravenous administration [46,82]. To evaluate whether NDs formed with ApoA-I mutants alter the biodistribution in peripheral tissues compared to ApoA-I WT NDs, BACHD mice received a 20 mg / kg bolus intravenous injection of ApoA-I WT, ApoA-I K133C ND, and ApoA-I E146C ND. The animals were euthanized 2 hours after injection, and the liver, quadriceps femoris, kidney, and brain were collected for analysis. In the liver, significantly higher levels of ApoA-I K133C were measured compared to ApoA-I WT and ApoA-I E146C (Figure 25A, one-way ANOVA p<0.0001; Tukey's multiple comparison**p=0.0022 K133C vs WT, ###p<0.0001 K133C vs E146C). Similarly, in the quadriceps femoris muscle, significantly higher levels of ApoA-I K133C were measured compared to ApoA-I WT and ApoA-I E146C (Figure 25B, one-way ANOVA p=0.0002; Tukey's multiple comparison**p=0.0025 K133C vs WT, ###p=0.0002 K133C vs E146C). In the kidney, no significant difference was measured for ApoA-I, but there was a strong trend towards increase for both ApoA-I K133C and ApoA-I E146C compared to the WT (Figure 25C, one-way ANOVA p=0.0533; Tukey's multiple comparison p=0.0523 K133C vs WT, p=0.0827 E146C vs WT). To determine whether ApoA-I K133C ND(NP F2) and ApoA-I E146C(NP F3)ND could improve distribution to peripheral tissues compared to other ApoA-I mutant NDs, the amounts of various ApoA-I ND formulations were determined in the quadriceps femoris, liver, and kidney (Figure 26A-C). Based on these findings, ApoA-I K133C(NP F2) was identified as a lead mutant for subsequent studies.

[0174] Example 20: Time course of biological distribution of ApoA-I K133C nanodiscs after IV administration

[0175] To evaluate the biodistribution profile of ND over time, BACHD mice received a bolus intravenous injection of either ApoA-I WT ND or ApoA-I K133C ND at 20 mg / kg. Liver and quadriceps femoris were harvested at different time intervals after injection. In the liver, significantly higher levels of ApoA-I K133C ND were detected compared to WT ND at 0.5, 1, 2, and 4 hours post-injection (Figure 27A. Two-way ANOVA: apoA-I p<0.0001, time point p=0.0010, and interaction p=0.0103. Sidak's multiple comparisons: 0.5 hour: **** p<0.0001, 1 hour: ** p=0.0020, 2 hour: *** p=0.0001, 4 hour: *** p=0.0005). There was also a significant effect of the time interval after injection on hepatic ApoA-I levels; ApoA-I K133C ND levels peaked 30 minutes after injection, while ApoA-I WT ND levels peaked 2 hours after injection.

[0176] Furthermore, there was a significant increase in ApoA-I K133C ND in the quadriceps femoris muscle at 1, 2, and 4 hours post-injection compared to WT ND (Figure 27B. Two-way ANOVA: apoA-I p<0.0001, time point p=0.6290, and interaction p=0.6958. Sidak's multiple comparisons: 1 hour: *p=0.0125, 2 hours: ***p=0.0001, 4 hours: ***p=0.0002). There was no significant effect of the time interval after injection on ApoA-I levels in the quadriceps femoris muscle. ApoA-I K133C ND levels peaked at 4 hours post-injection, while ApoA-I WT ND levels peaked at 2 hours post-injection. ApoA-I K133C levels remained high in the quadriceps femoris muscle (>6000 ng / mg of ApoA-I) up to 24 hours post-injection.

[0177] Example 21: Dose-dependent increase in ApoA-I K133C nanodiscs in the quadriceps femoris muscle after IV administration.

[0178] To evaluate whether increasing doses of injected ApoA-I K133C ND lead to increased levels in peripheral tissues, BACHD mice underwent dose-response experiments receiving single IV injections of ApoA-I K133C ND ranging from 10 to 60 mg / kg. Tissues were harvested 2 hours post-injection and quantified using human ApoA-I ELISA (enzyme-linked immunosorbent assay). A significant dose-dependent increase in apoA-I K133C was observed in the quadriceps femoris muscle (Figure 28B, one-way ANOVA p<0.0001; Tukey's multiple comparison test: *** p<0.05 compared to 10 mg / kg, ### p<0.05 compared to 20 mg / kg, & p<0.05 compared to 40 mg / kg; linear regression R²=0.9308, slope non-zero p<0.0001). In the liver, a strong trend toward dose-dependent increase was observed, but this did not reach statistical significance (Figure 28A, one-way ANOVA p=0.0531; linear regression R2=0.4335, slope non-zero p<0.0041).

[0179] Example 22: HD mutation does not significantly alter the peripheral distribution of ApoA-I K133C nanodiscs.

[0180] To determine whether the HD mutation may affect the distribution of ApoA-I ND in peripheral tissues, relative ApoA-I levels in the liver and quadriceps femoris were determined (Figure 29A and B). BACHD and wild-type sibling controls received a single IV injection of 10 mg / kg of ApoA-I K133C ND at ages 2, 6, and 12 months. Relative ApoA-I levels are presented as absolute values ​​rather than absolute values ​​because different cohorts were collected and analyzed at different times.

[0181] Example 23: ApoA-I WT and K133C nanodiscs carrying ASO can induce modest mutant HTT suppression in peripheral tissues.

[0182] To evaluate the potential of ApoA-I ND as an ASO carrier in vivo, 2-month-old BACHD mice received a single IV injection of either PBS or 300 μg of either ASO, ASO-ApoA-I WT ND, or ASO-ApoA-I K133C ND, and HTT target binding was measured after 4 weeks. Significant reductions in quadriceps mHTT were observed in peripheral mice treated with ASO, ASO-ApoA-I WT ND, and ASO-ApoA-I K133C ND compared to PBS (Figure 30A, p<0.0001; Tukey's multiple comparisons****p<0.0001 is compared to PBS). Significant reductions in mHTT in the liver were also observed with ASO-ApoA-I WT ND and ASO-ApoA-I K133C ND compared to PBS and ASO treatment (Figure 30B, p<0.0001; Tukey's multiple comparisons: p<0.0001 compared to PBS, p<0.01 compared to ASO). In the lungs, there was a significant reduction in mHTT in all treatment conditions compared to PBS (Figure 30C, p<0.0001; Tukey's multiple comparisons: p<0.0001 compared to PBS). Significant reductions in mHTT were observed in the heart with ASO-ApoA-I WT ND treatment compared to PBS and ASO monotherapy (Figure 30D; p<0.0001; Tukey's multiple comparisons: p<0.0001 compared to PBS, p<0.05 compared to ASO).

[0183] Example 24: Covalent conjugation of ASO to ApoA-I K133C does not affect nanodisk formation.

[0184] Optimizing the conditions for covalent coupling of ApoA-I K133C to ASO designed to target human huntington (HTT) increases the likelihood of ASO remaining attached to ND in vivo and potentially leverages the enhanced brain entry observed in ApoA-I K133C mutants. To couple ASO to ApoA-I, a 6-carbon linker with an amine group was initially added to the 5' end of ASO. For ASO and ApoA-I coupling, the amine group of ASO was modified with a 4-formylbenzamide (4FB) moiety, and the primary amine of the ApoA-I K133C protein was modified with a 6-hydrazinonicotinamide (HyNic) moiety. The resulting coupling with a covalent linker was stable over a range of pHs and temperatures. Prior to optimizing the coupling conditions, the addition of an amino group to ASO and the modification of ASO with the 4FB moiety were tested to determine whether the overall activity of ASO changed compared to unmodified ASO alone. Lymphoblasts derived from HD patients (Coriell NIGMS GM04724) were treated with PBS, unmodified ASO, aminoASO, or 4FB-ASO at 1 μM in culture medium for 120 hours, and HTT target binding was measured by quantitative immunoblotting (Figure 31A). Previous studies have shown that bath application of ASO in culture medium (in the absence of transfection reagents) results in HTT suppression in lymphoblasts. In this example, all treatment conditions resulted in a significant decrease in HTT (Figure 31B, one-way ANOVA p<0.0001; Tukey's multiple comparison test p<0.0001), while there was no significant difference in the magnitude of HTT reduction among the formulations, suggesting that ASO modification did not affect its activity.

[0185] To verify the conjugation of ASO to ApoA-I K133C, ApoA-I K133C and its conjugate were run on SDS-PAGE, stained with Coomassie to detect the protein, and Sybr Safe to detect the nucleic acid. TM Stained with (Figure 32A). Figure 32A shows that ASO was coupled to the ApoA-I K133C protein. HyNic per ApoA-I K133C molecule TMDue to the possible range of modifications, multiple conjugate bands corresponding to different molar ratios of ASO and ApoA-I K133C were observed. The formation of ASO:ApoA-I K133C conjugate NDs was verified using native PAGE (Figure 32B) and EM (Figure 32C). In particular, ASO:ApoA-I K133C non-cleavable conjugated (NC) NDs formed NDs with a similar morphology to APoA-I K133C NDs, but were significantly larger in diameter (Figure 32D).

[0186] Example 25: ASO:ApoA-I K133C conjugate nanodiscs enhance HTT suppression in HD patient-derived lymphocytes, and suppress HTT after ICV injection in BACHD mice.

[0187] We evaluated ASO:ApoA-I K133C NC ND to determine whether it could enhance HTT suppression in HD patient-derived lymphocytes compared to ASO alone. Cells were treated with PBS, ASO alone, or 1 μM ASO:ApoA-I K133C NC ND in culture medium for 120 hours, and HTT suppression was measured quantitatively by immunoblotting (Figure 33A). Both ASO alone and ASO:ApoA-I K133C NC ND resulted in a significant reduction in HTT levels (Figure 33B, ASO = 60% reduction, ASO:ApoA-I K133C ND = 72% reduction; one-way ANOVA, p<0.0001). Treatment with ASO:ApoA-I K133C NC ND significantly increased the degree of HTT suppression compared to ASO alone (t-test, p=0.0277). These data suggest that ASO:K133C ND may enhance intracellular entry compared to ASO alone.

[0188] We compared the suppression of hypertension (HTT) in the brain after ICV injection of PBS, ASO, or ASO:ApoA-I K133C NC ND. A single ICV injection administered either 15 μg of ASO or a molar equivalent of ASO:ApoA-I K133C NC ND, and animals were collected 4 weeks later for quantification of HTT target binding. Significant reductions were observed in all brain regions with both ASO alone and ASO:ApoA-I K133C NC ND (Figure 37A-D; one-way ANOVA p<0.0001 in all brain regions; Tukey multiple comparisons ****p<0.0001 compared to PBS). In particular, ASO:ApoA-I K133C NC ND significantly increased the degree of HTT suppression compared to ASO alone in both the cortex (Figure 34A, Tukey multiple comparison test #p=0.0136) and the striatum (Figure 34B, Tukey multiple comparison test #p=0.0313).

[0189] Peripheral HTT suppression was evaluated after ICV administration of either PBS, ASO, or ASO:ApoA-I K133C NC ND. A significant reduction in HTT in the quadriceps was observed with ASO:ApoA-I K133C NC ND treatment, but not with ASO alone (Figure 35A, one-way ANOVA p=0.0101; Tukey multiple comparison test: *p=0.0141 compared to PBS, #p=0.0426 compared to ASO). Furthermore, in the liver (Figure 35B, one-way ANOVA p=0.0233; Tukey multiple comparison: p=0.1575 compared to PBS, p=0.0190 compared to ASO) and lungs (Figure 35C, one-way ANOVA p=0.0043; Tukey multiple comparison test: p=0.2018 compared to PBS, p=0.0030 compared to ASO), HTT reduction by ASO:ApoA-I K133C NC ND treatment was significant compared to ASO alone, but not significant compared to PBS. In the heart (Figure 35D, one-way ANOVA p=0.1500), no significant HTT reduction was observed with either ASO alone or ASO:apoA-I K133C NC ND.

[0190] Example 26: Achieving Potent mHTT Suppression in the Striatum and Peripheral Tissues of BACHD Mice by Systemic Administration of ASO:ApoA-I K133C Conjugated Nanodisks

[0191] To evaluate the potential of ASO:ApoA-I K133C NC ND to suppress HTT in the CNS, 2-month-old BACHD mice were intravenously injected once with either PBS or 300 μg of ASO:ApoA-I K133C NC ND, and HTT target binding in the CNS was evaluated 4 weeks after injection. In the brain, significant mHTT reduction was measured in the cortex (Figure 36A, unpaired t-test *p = 0.0234), striatum (Figure 36B, unpaired t-test **p = 0.0064), and hippocampus (Figure 36C, unpaired t-test *p = 0.0386), but not in the cerebellum (Figure 36D, unpaired t-test p = 0.0818).

[0192] To evaluate the potential of ASO:ApoA-I K133C NC ND to suppress HTT peripherally, 2-month-old BACHD mice were intravenously injected once with either PBS or 300 μg of ASO:ApoA-I K133C NC ND, and target binding was evaluated 4 weeks after injection. Peripherally, ASO:ApoA-I K133C NC ND treatment resulted in significant mHTT reduction in the quadriceps (Figure 37A, unpaired t-test ***p = 0.0001), liver (Figure 37B, unpaired t-test ****p < 0.0001), lung (Figure 37C, unpaired t-test ***p = 0.0006), and heart (Figure 37D, unpaired t-test ***p = 0.0001).

[0193] Discussion These studies have demonstrated that apoA-I nanodiscs, or therapeutic nanodiscs, can enhance intranasal or intravenous administration of therapeutic agents to the CNS. In particular, they enable the delivery of mHTT-reducing antisense oligonucleotides (ASOs) to the brain, as well as their improved distribution and target binding. Intranasal administration overcomes some of the major limitations associated with ASO delivery, including bypassing the blood-brain barrier and liver, avoiding the invasiveness of intrathecal delivery, limiting systemic exposure, while ensuring target binding in the deep brain structures most susceptible to Huntington's disease. Similarly, systemic administration of ASOs shows potent suppression of mHTT in the striatum and peripheral tissue organs.

[0194] apoA-I NDs assembled with mutants K133C and E146C were able to penetrate the brain more effectively than both apoA-I WT NDs and lipid-free apoA-I protein, demonstrating that this mutation in the apoA-I protein improves delivery to the CNS. Since both apoA-I WT and K133C proteins form NDs of similar size

[59] , it is unlikely that the improved brain penetration is attributable to the size of the NDs. The enhanced delivery of the K133C mutant may be due to the innate helical registry of apoA-1, which tightly opposes cysteine ​​residues to one another, enabling the formation of disulfide bonds that tightly anchor the apoA-I molecule around the nanodisk

[47] . This may result in the K133C type of apoA-I showing a significant improvement in recognition by the scavenger receptor SR-B1, for which apoA-I is a known ligand. SR-B1 is expressed on the surface of several cell types that make up the neurovascular unit, including neurons, astrocytes, and capillary endothelial cells, and promotes the uptake of HDL into cells

[60] .

[0195] Furthermore, it has been reported that SR-B1 is expressed in the nasal mucosa of mice

[61] . Tsuzuki et al. confirmed the mRNA (messenger RNA) and protein expression of the SR-B1 receptor in nasal mucosa tissue and demonstrated the immunohistochemical expression of the SR-B1 receptor in the olfactory epithelium

[61] . It has been demonstrated that the difference in the tertiary structure of apoA-I significantly affects the recognition by this receptor

[62] . Further characterization of the tertiary structure and conformation of apoA-I K133C ND would be useful in clarifying the properties that enhance delivery to the brain, especially after intranasal administration.

[0196] Ex vivo imaging confirmed a widespread distribution of ApoA-I K133C ND throughout the brain after intranasal administration. This was characterized by a high level of dependence on olfactory and trigeminal terminals in the perivascular region of the circle of Willis for entry into the brain and perivascular distribution via the intranasal route

[63] .

[0197] Since the mutant HTT transgene is known to cause anatomical changes in the brains of multiple transgenic mouse models of Huntington's disease, including blood-brain barrier damage, in both R6 / 2 mice and YAC128 mice

[56] , it was sought to determine whether the enhanced entry of apoA-I K133C ND into the brain was a result of the damaged blood-brain barrier in BACHD mice. The results of this study showed that there was no significant difference in the amount of apoA-I K133C measured in the brains of BACHD mice after intranasal administration of ND compared to WT mice at both 2 and 6 months of age. Therefore, apoA-I K133C ND can penetrate into the brain after intranasal administration, regardless of the anatomical changes of the animals as a result of their phenotypic outcomes.

[0198] However, in 12-month-old BACHD mice, higher levels of apoA-I were observed in the olfactory bulb and A region of the prefrontal cortex compared to WT mice. This increase may be due to changes in the olfactory mucosa in the nasal cavity of BACHD mice (improved entry into the brain along the trajectory of olfactory nerve terminals), or even anatomical changes in the olfactory bulb itself (the latter being observed in Huntington's disease patients [64, 65]). Whatever the cause, improved delivery to the CNS may prove beneficial in older patients in the later stages of the disease.

[0199] Multiple studies on therapeutic drug delivery via intranasal administration have demonstrated the preferential perivascular deposition of substances in the brain, as this route results in the distribution of substances in the perivascular space throughout the brain [36, 37, 66]. Therefore, the physicochemical properties of therapeutic drugs and / or their delivery vehicles are important factors in ensuring delivery to intended cellular targets. In this study, we initially hypothesized that apoA-I ND would preferentially deposit in capillary endothelial cells, neurons, and astrocytes, due to the expression of the SR-B1 receptor on the surface of these cells, as apoA-I is a known ligand for this receptor

[60] . Indeed, immunohistochemical analysis demonstrated that after intranasal administration of apoA-I ND, apoA-I was extensively deposited in both capillary endothelial cells and neurons throughout the brain. Co-localization of apoA-I with astrocytes was also observed, but significantly lower than in the aforementioned cells, regardless of SR-B1 receptor expression.

[0200] Similarly, apoA-I deposition in microglia was observed to a lower degree. Two hours after intranasal administration, there was significant co-localization of apoA-I with leptin-stained choroid plexus cells. This is consistent with the findings of Stukas et al., who demonstrated that intravenously administered apoA-I enters the CNS via the choroid plexus

[46] . This cell distribution pattern was similar in both WT and K133C apoA-I ND, but more apoA-I-positive cells were observed throughout the brain after intranasal treatment of apoA-I K133C ND.

[0201] Because IHC staining was performed only 2 hours after intranasal administration, the question remains whether some apoA-I from pulmonary plasma exchange still accessed the brain via the choroid plexus, or whether apoA-I from intranasal administration into the perivascular space entered the brain parenchyma via the choroid plexus. Since apoA-I levels in cerebrospinal fluid were higher than those in plasma 1 and 2 hours after intranasal administration, and since exchange from cerebrospinal fluid and lungs to plasma is the more likely direction, this hypothesis suggests that the latter case is the actual reality.

[0202] Consistent with most intranasal administration studies, apoA-I was already delivered to the brain within one hour of intranasal administration. Since brain distribution is a dynamic process after intranasal administration, a time-series study was conducted to determine when apoA-I levels were highest in the brain and to assess the appearance of changes in regional distribution at different time points after treatment. Results from this study showed that delivery was highest at two hours post-intranasal administration across all four anterior-posterior axial brain regions. Furthermore, levels remained highest in the most rostral brain region A (composed of the olfactory bulb and prefrontal cortex), but began to shift to the most caudal brain regions over the course of 24 hours post-treatment. These results present two important findings. The first is that if this rapid entry into the CNS is translated to human administration, patients do not need to be immobile or anesthetized (as occurs in mice, with their limited nasal volume and forced nasal breathing), and nasal spray or nasal drops would suffice for humans. A second important finding is that apoA-I remains detectable in the brain even after 24 hours, and therefore rapid clearance is not a limiting factor for the delivery of ASOs by this mode.

[0203] The observed dose-dependent increase in apoA-I levels is also promising for conversion to Huntington's disease patients, potentially leading to patient-directed dosing by increasing the frequency of administration for specific patients based on disease progression and severity. The plateau reached at the highest dose measured in this study is most likely due to limitations in nasal cavity size and volume in rodents, which are significantly larger in humans and associated with longer residence times

[67] .

[0204] One of the primary advantages of intranasal administration is its ability to deliver therapeutic agents to the brain while bypassing the first-stage effects. Indeed, the results of this study confirmed this to be true for apoA-I ND, with minimal liver exposure detected over a 6-hour post-treatment course and showing a trend toward complete clearance. In the lungs, higher levels of apoA-I were detected 1 hour after intranasal administration but were completely cleared by 6 hours. This is most likely because the mice inhaled part of the dose due to the limited volume of their nasal cavities. The rapid clearance of apoA-I from the lungs and liver over a course of several hours, as well as the fact that empty WT apoA-I ND did not produce cytotoxic effects in vitro

[42] , and that neither apoA-I WT nor K133C ND has been shown to produce an immune response in vivo (data not shown), suggests that this mode of delivery would not result in long-term negative effects. From this perspective, it would be important to examine the immunogenicity and / or off-target effects of apoA-I ND after repeated, long-term intranasal treatment in vivo.

[0205] Generally, in gene therapy targeting the CNS, minimal peripheral exposure results in fewer side effects. The results of this study effectively demonstrated improved delivery of apoA-I ND to the CNS via intranasal administration compared to intravenous administration at the same dose. At 2 hours, the difference in whole-brain apoA-I levels between intranasally administered animals and intravenously administered mice was primarily due to increases in the olfactory bulb and prefrontal cortex. However, since brain distribution after intranasal administration is a dynamic process (as demonstrated in Figure 5b), it is natural that a large amount of apoA-I measured in the most rostral brain region (A) would be distributed along the anterior-posterior brain axis over the following several hours. In Huntington's disease, where mHTT is expressed peripherally, including throughout the body, it may be possible to individually adjust different doses to the brain via intranasal administration and to the periphery via intravenous administration, as needed, enabling more comprehensive target binding throughout the body.

[0206] This study successfully demonstrated that a single intranasal dose of apoA-I K133C ASO-ND significantly reduced mHTT levels in the striatum, cortex, and olfactory bulb (this reduction was maintained over an 8-week period). A second dose, administered 4 weeks after the initial intranasal dose, achieved a reduction of over 25% in mHTT in all brain regions except the cerebellum. In contrast, the intrathecal route, the current preferred method of ASO delivery to the CNS, produces a concentration gradient with high levels of ASO delivery to the outer layers of the cortex, resulting in minimal distribution to deeper brain regions such as the striatum [6]. The results of this study demonstrate the therapeutic potential of intranasal administration of apoA-I ND as a mode of ASO delivery to the brain. The only other study demonstrating successful ASO delivery after intranasal administration in mice required a daily dose of 100 μg of ASO (another type of synuclein-reduced ASO coupled to indatraline to target mononuclear amine neurons) over a 28-day course to achieve significant target binding

[68] , and this was not sustained with shorter treatment paradigms

[69] . The fact that the apoA-I delivery mode in this study is not only completely non-invasive but also capable of resulting in sustained and significant target binding in deeper brain structures is a major advantage and improvement over current ASO delivery methods.

[0207] In conclusion, these studies successfully demonstrated the therapeutic potential of intranasal administration of apoA-I K133C ND for the delivery of antisense oligonucleotides to the brain. This study showed that this approach successfully delivers ASO to the brain and can significantly reduce mHTT expression in the cortex and striatum, the areas most affected by Huntington's disease. This delivery mode will overcome obstacles that have limited the clinical efficacy of mHTT-reducing ASOs to date and will allow for easier and more accessible dose setting for patients. This mode is not only completely non-invasive but also, due to its ability to bypass the blood-brain barrier and deliver and distribute ASO to deeper brain tissue, is an ideal approach for the treatment of Huntington's disease and other neurological disorders and disorders. Most importantly, these studies demonstrate both the utility and potential of apoA-I nanodiscs for delivering macromolecules (i.e., antisense oligonucleotides (ASOs), short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), aptamers, ribozymes, mRNAs (messenger RNAs), plasmids, or small molecules) to the brain via intranasal routes or systemic injection. Combining non-invasive intranasal routes with the non-immunogenic delivery vehicle of apoA-I NDs enables broad applications for the delivery of ASOs and other macromolecules for the treatment of various CNS diseases and disorders.

[0208] Regardless of the various embodiments of the invention disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of persons ordinary skill in the art. Such modifications include the substitution of known equivalents for any aspect of the invention and achieving the same results in substantially the same manner. Numerical ranges include the numerical values ​​that define the range. The term “including” is used herein as substantially equivalent to the phrase “including, but not limited to,” indicating an open termination condition, and the term “including” has the corresponding meaning. As used herein, the singular “a,” “an,” and “the” include plural referents unless the context explicitly indicates otherwise. Thus, for example, a reference to “thing” includes multiple such things. Citations of references herein do not constitute an acknowledgment that such references are prior art to embodiments of the invention. The invention includes all embodiments and variations, substantially as described above, and in relation to the examples and drawings.

[0209] References [1] ME MacDonald, et al., A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell. 72 (1993) 971-983. https: / / doi.org / 10.1016 / 0092-8674(93)90585-E. [2] CA Ross, SJ Tabrizi, Huntington's disease: from molecular pathogenesis to clinical treatment. Lancet Neurol. 10 (2011) 83-98. https: / / doi.org / 10.1016 / S1474-4422(10)70245-3. [3] H.J. Waldvogel, et al., The Neuropathology of Huntington’s Disease. in: H.H.P. Nguyen, M.A. Cenci (Eds.). Behav. Neurobiol. Huntingt. Dis. Park. Dis., Springer, Berlin, Heidelberg, 2015: pp. 33-80. https: / / doi.org / 10.1007 / 7854_2014_354. [4] H.L. Paulson, R.L. Albin, Huntington’s Disease: Clinical Features and Routes to Therapy. in: D.C. Lo, R.E. Hughes (Eds.). Neurobiol. Huntingt. Dis. Appl. Drug Discov., CRC Press / Taylor & Francis, Boca Raton (FL), 2011. http: / / www.ncbi.nlm.nih.gov / books / NBK56000 / (Accessed on November 30, 2022). [5] A. Yamamoto, J.J. Lucas, R. Hen, Reversal of Neuropathology and Motor Dysfunction in a Conditional Model of Huntington’s Disease. Cell. 101 (2000) 57-66. https: / / doi.org / 10.1016 / S0092-8674(00)80623-6. [6] H.B. Kordasiewicz, et al., Sustained therapeutic reversal of Huntington’s disease by transient repression of huntingtin synthesis. Neuron. 74(6) (2012) 1031-1044. https: / / doi.org / 10.1016 / j.neuron.2012.05.009. [7] A.L. Southwell, et al., Huntingtin suppression restores cognitive function in a mouse model of Huntington’s disease. Sci. Transl. Med. 10 (2018) eaar3959. https: / / doi.org / 10.1126 / scitranslmed.aar3959. [8] M. DiFiglia, et al., Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits. Proc. Natl. Acad. Sci. 104 (2007) 17204-17209. https: / / doi.org / 10.1073 / pnas.0708285104. [9] S.Q. Harper, et al., RNA interference improves motor and neuropathologicalabnormalities in a Huntington’s disease mouse model. Proc. Natl. Acad. Sci. 102 (2005) 5820-5825. https: / / doi.org / 10.1073 / pnas.0501507102.

[10] S.B. Thomson, et al., AAV5-miHTT-mediated huntingtin lowering improves brain health in a Huntington’s disease mouse model. Brain. (2022) awac458. https: / / doi.org / 10.1093 / brain / awac458.

[11] E.A. Spronck, et al., AAV5-miHTT Gene Therapy Demonstrates Sustained Huntingtin Lowering and Functional Improvement in Huntington Disease Mouse Models. Mol. Ther. Methods Clin. Dev. 13 (2019) 334-343. https: / / doi.org / 10.1016 / j.omtm.2019.03.002.

[12] N.S. Caron, et al., Potent and sustained huntingtin lowering via AAV5 encoding miRNA preserves striatal volume and cognitive function in a humanized mouse model of Huntington disease. Nucleic Acids Res. 48 (2020) 36-54. https: / / doi.org / 10.1093 / nar / gkz976.

[13] J.K. Watts, D.R. Corey, Silencing disease genes in the laboratory and the clinic, J. Pathol. 226 (2012) 365-379. https: / / doi.org / 10.1002 / path.2993.

[14] R.S. Geary, et al., Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv. Drug Deliv. Rev. 87 (2015) 46-51. https: / / doi.org / 10.1016 / j.addr.2015.01.008.

[15] A.L. Southwell, et al., In Vivo Evaluation of Candidate Allele-specific Mutant Huntingtin Gene Silencing Antisense Oligonucleotides. Mol. Ther. 22 (2014) 2093-2106. https: / / doi.org / 10.1038 / mt.2014.153.

[16] Ionis Pharmaceuticals, Inc., A Phase 1, Double-Blind, Placebo-Controlled, Dose- Escalation Study of the Safety, Tolerability, and Pharmacokinetics of ISIS 333611 Administered Intrathecally to Patients With Familial Amyotrophic Lateral Sclerosis Due to Superoxide Dismutase 1 Gene Mutations. clinicaltrials.gov, 2012. https: / / clinicaltrials.gov / ct2 / show / NCT01041222 (Accessed on October 25, 2022).

[17] Ionis Pharmaceuticals, Inc., A Randomized, Double-blind, Placebo-controlled Study to Evaluate the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of Multiple Ascending Doses of Intrathecally Administered ISIS 443139 in Patients With Early Manifest Huntington’s Disease. clinicaltrials.gov, 2019. https: / / clinicaltrials.gov / ct2 / show / NCT02519036 (Accessed on October 25, 2022).

[18] Biogen, An Open-Label Study to Assess the Efficacy, Safety, Tolerability, and Pharmacokinetics of Multiple Doses of ISIS 396443 Delivered Intrathecally to Subjects With Genetically Diagnosed and Presymptomatic Spinal Muscular Atrophy. clinicaltrials.gov, 2021. https: / / clinicaltrials.gov / ct2 / show / NCT02386553 (Accessed on October 25, 2022).

[19] Hoffmann-La Roche, An Open-Label Adaptive Multiple-Dose Study to Investigate the Pharmacokinetics and Pharmacodynamics of RO7234292 in CSF and Plasma, and Safety and Tolerability Following Intrathecal Administration in Patients With Huntington’s Disease. clinicaltrials.gov, 2022. https: / / clinicaltrials.gov / ct2 / show / NCT04000594 (Accessed on October 25, 2022).

[20] Hoffmann-La Roche, A Randomized, Multicenter, Double-Blind, Placebo-Controlled, Phase III Clinical Study to Evaluate the Efficacy and Safety of Intrathecally Administered RO7234292 (RG6042) in Patients With Manifest Huntington’s Disease. clinicaltrials.gov, 2022. https: / / clinicaltrials.gov / ct2 / show / NCT03761849 (Accessed on October 25, 2022).

[21] E.M. Delhaas, F.J.P.M. Huygen, Complications associated with intrathecal drug delivery systems. BJA Educ. 20 (2020) 51-57. https: / / doi.org / 10.1016 / j.bjae.2019.11.002.

[22] N.S. Caron, et al., Cerebrospinal fluid mutant huntingtin is a biomarker for huntingtin lowering in the striatum of Huntington disease mice. Neurobiol. Dis. 166 (2022) 105652. https: / / doi.org / 10.1016 / j.nbd.2022.105652.

[23] K. Kingwell, Double setback for ASO trials in Huntington disease. Nat. Rev. Drug Discov. 20 (2021) 412-413. https: / / doi.org / 10.1038 / d41573-021-00088-6.

[24] M.E. Rook, A.L. Southwell, Antisense Oligonucleotide Therapy: From Design to the Huntington Disease Clinic. BioDrugs. 36 (2022) 105-119. https: / / doi.org / 10.1007 / s40259-022-00519-9.

[25] J.P. Dufour, et al., Hydrocephalus after Intrathecal Administration of Dextran to Rhesus Macaques (Macaca mulatta). Comp. Med. 68 (2018) 227-232. https: / / doi.org / 10.30802 / AALAS-CM-17-000096.

[26] Biogen, SPINRAZA (nusinersen) [Prescribing Information] injection, for intrathecal use. (2018).www.spinraza.com / content / dam / commercial / specialty / spinraza / caregiver / en_us / pdf / spin raza-prescribing-information.pdf.

[27] R.G. Thorne, et al., Delivery of insulin-like growth factor-I to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration. Neuroscience. 127 (2004) 481-496. https: / / doi.org / 10.1016 / j.neuroscience.2004.05.029.

[28] I.-D. Kim, et al., Intranasal Delivery of HMGB1 siRNA Confers Target Gene Knockdown and Robust Neuroprotection in the Postischemic Brain. Mol. Ther. 20 (2012) 829-839. https: / / doi.org / 10.1038 / mt.2011.291.

[29] R. Draghia, et al., Gene delivery into the central nervous system by nasal instillation in rats. Gene Ther. 2 (1995) 418-423.

[30] L. Danielyan, et al., Therapeutic Efficacy of Intranasally Delivered Mesenchymal Stem Cells in a Rat Model of Parkinson Disease. Rejuvenation Res. 14 (2011) 3-16. https: / / doi.org / 10.1089 / rej.2010.1130.

[31] A.E.-E. Aly, et al., Intranasal Delivery of pGDNF DNA Nanoparticles Provides Neuroprotection in the Rat 6-Hydroxydopamine Model of Parkinson’s Disease. Mol. Neurobiol. 56 (2019) 688-701. https: / / doi.org / 10.1007 / s12035-018-1109-6.

[32] V. Sava, et al., Enriched chitosan nanoparticles loaded with siRNA are effective in lowering Huntington’s disease gene expression following intranasal administration. Nanomedicine Nanotechnol. Biol. Med. 24 (2020) 102119. https: / / doi.org / 10.1016 / j.nano.2019.102119.

[33] S. Craft, et al., Intranasal Insulin Therapy for Alzheimer Disease and Amnestic Mild Cognitive Impairment: A Pilot Clinical Trial. Arch. Neurol. 69 (2012) 29-38. https: / / doi.org / 10.1001 / archneurol.2011.233.

[34] A.J. Guastella, et al., Intranasal Oxytocin Improves Emotion Recognition for Youth with Autism Spectrum Disorders. Biol. Psychiatry. 67 (2010) 692-694. https: / / doi.org / 10.1016 / j.biopsych.2009.09.020.

[35] S.V. Dhuria, et al., Intranasal delivery to the central nervous system: Mechanisms and experimental considerations. J. Pharm. Sci. 99 (2010) 1654-1673. https: / / doi.org / 10.1002 / jps.21924.

[36] J.J. Lochhead, et al., Rapid Transport within Cerebral Perivascular Spaces Underlies Widespread Tracer Distribution in the Brain after Intranasal Administration. J. Cereb. Blood Flow Metab. 35 (2015) 371-381. https: / / doi.org / 10.1038 / jcbfm.2014.215.

[37] A.E.-E. Aly, et al., Intranasal delivery of hGDNF plasmid DNA nanoparticles results in long-term and widespread transfection of perivascular cells in rat brain. Nanomedicine Nanotechnol. Biol. Med. 16 (2019) 20-33. https: / / doi.org / 10.1016 / j.nano.2018.11.006.

[38] M.M. Migliore, et al., Brain delivery of proteins by the intranasal route of administration: A comparison of cationic liposomes versus aqueous solution formulations. J. Pharm. Sci. 99 (2010) 1745-1761. https: / / doi.org / 10.1002 / jps.21939.

[39] D.D.O. Martin, et al., Apolipoprotein A-I Assumes a “Looped Belt” Conformation on Reconstituted High Density Lipoprotein*. J. Biol. Chem. 281 (2006) 20418-20426. https: / / doi.org / 10.1074 / jbc.M602077200.

[40] R.A.G.D. Silva, et al., A Mass Spectrometric Determination of the Conformation of Dimeric Apolipoprotein A-I in Discoidal High Density Lipoproteins. Biochemistry. 44 (2005) 8600-8607. https: / / doi.org / 10.1021 / bi050421z.

[41] D.A. Bricarello, et al., Reconstituted Lipoprotein: A Versatile Class of Biologically-Inspired Nanostructures. ACS Nano. 5 (2011) 42-57. https: / / doi.org / 10.1021 / nn103098m.

[42] B.L. Burgess, et al., A phospholipid-apolipoprotein A-I nanoparticle containing amphotericin B as a drug delivery platform with cell membrane protective properties. Int. J. Pharm. 399 (2010) 148-155. https: / / doi.org / 10.1016 / j.ijpharm.2010.07.057.

[43] M. Ghosh, et al., Curcumin nanodisks: formulation and characterization. Nanomedicine Nanotechnol. Biol. Med. 7 (2011) 162-167. https: / / doi.org / 10.1016 / j.nano.2010.08.002.

[44] A.T.K. Singh, et al., All trans retinoic acid nanodisks enhance retinoic acid receptor mediated apoptosis and cell cycle arrest in mantle cell lymphoma. Br. J. Haematol. 150 (2010) 158-169. https: / / doi.org / 10.1111 / j.1365- 2141.2010.08209.x.

[45] M. Rui, et al., Recombinant High Density Lipoprotein Nanoparticles for Target-Specific Delivery of siRNA. Pharm. Res. 30 (2013) 1203-1214. https: / / doi.org / 10.1007 / s11095-012-0957-4.

[46] S. Stukas, et al., Intravenously injected human apolipoprotein A-I rapidly enters the central nervous system via the choroid plexus. J. Am. Heart Assoc. 3 (2014) e001156. https: / / doi.org / 10.1161 / JAHA.114.001156.

[47] A.L. Cooke, et al., A thumbwheel mechanism for APOA1 activation of LCAT activity in HDL. J. Lipid Res. 59 (2018) 1244-1255. https: / / doi.org / 10.1194 / jlr.M085332.

[48] R.O. Ryan, T.M. Forte, M.N. Oda, Optimized bacterial expression of human apolipoprotein A-I. Protein Expr. Purif. 27 (2003) 98-103. https: / / doi.org / 10.1016 / S1046-5928(02)00568-5.

[49] M.N. Oda, et al., Cysteine Substitutions in Apolipoprotein A-I Primary Structure Modulate Paraoxonase Activity. Biochemistry. 40 (2001) 1710-1718. https: / / doi.org / 10.1021 / bi001922h.

[50] A.L. Southwell, et al., Ultrasensitive measurement of huntingtin protein in cerebrospinal fluid demonstrates increase with Huntington disease stage and decrease following brain huntingtin suppression. Sci. Rep. 5 (2015) 12166. https: / / doi.org / 10.1038 / srep12166.

[51] M. Gray, et al., Full-Length Human Mutant Huntingtin with a Stable Polyglutamine Repeat Can Elicit Progressive and Selective Neuropathogenesis in BACHD Mice. J. Neurosci. 28 (2008) 6182-6195. https: / / doi.org / 10.1523 / JNEUROSCI.0857-08.2008.

[52] N.S. Caron, et al., Reliable Resolution of Full-Length Huntingtin Alleles by Quantitative Immunoblotting. J. Huntingt. Dis. 10 (2021) 355-365. https: / / doi.org / 10.3233 / JHD-200463.

[53] D.W. Borhani, et al., Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation. Proc. Natl. Acad. Sci. U. S.A. 94 (1997) 12291-12296. https: / / doi.org / 10.1073 / pnas.94.23.12291.

[54] J.P. Segrest, et al., The amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function. J. Lipid Res. 33 (1992) 141-166.

[55] R.T. Nolte, D. Atkinson, Conformational analysis of apolipoprotein A-I and E-3 based on primary sequence and circular dichroism. Biophys. J. 63 (1992) 1221-1239.

[56] S. Franciosi, et al., Age-dependent neurovascular abnormalities and altered microglial morphology in the YAC128 mouse model of Huntington disease. Neurobiol. Dis. 45 (2012) 438-449. https: / / doi.org / 10.1016 / j.nbd.2011.09.003.

[57] A. Di Pardo, et al., Impairment of blood- brain barrier is an early event in R6 / 2 mouse model of Huntington Disease. Sci. Rep. 7 (2017) 41316. https: / / doi.org / 10.1038 / srep41316.

[58] J. Drouin-Ouellet, et al., Cerebrovascular and blood-brain barrier impairments in Huntington’s disease: Potential implications for its pathophysiology. Ann. Neurol. 78 (2015) 160-177. https: / / doi.org / 10.1002 / ana.24406.

[59] A.L. Cooke, The Molecular Interaction of Apolipoprotein A-I and Lecithin: Cholesterol Acyl Transferase. University of Cincinnati, 2018. https: / / etd.ohiolink.edu / apexprod / rws_olink / r / 1501 / 10?clear=10&p10_accession_num=uc in1543580636448357 (Accessed on December 1, 2022).

[60] C. Borras, et al. HDL- like-Mediated Cell Cholesterol Trafficking in the Central Nervous System and Alzheimer’s Disease Pathogenesis. Int. J. Mol. Sci. 23 (2022) 9356. https: / / doi.org / 10.3390 / ijms23169356.

[61] S. Tsuzuki, et al. A novel role for scavenger receptor B1 as a contributor to the capture of specific volatile odorants in the nasal cavity. Biomed. Res. Tokyo Jpn. 39 (2018) 117-129. https: / / doi.org / 10.2220 / biomedres.39.117.

[62] M.C. de Beer, et al. Apolipoprotein A-I conformation markedly influences HDL interaction with scavenger receptor BI. J. Lipid Res. 42 (2001) 309-313. https: / / doi.org / 10.1016 / S0022-2275(20)31693- X.

[63] J.J. Lochhead, T.P. Davis, Perivascular and Perineural Pathways Involved in Brain Delivery and Distribution of Drugs after Intranasal Administration. Pharmaceutics. 11 (2019) 598. https: / / doi.org / 10.3390 / pharmaceutics11110598.

[64] B. Highet, et al. Huntingtin Aggregates in the Olfactory Bulb in Huntington’s Disease. Front. Aging Neurosci. 12 (2020). https: / / www.frontiersin.org / articles / 10.3389 / fnagi.2020.00261 (Accessed on September 9, 2022).

[65] J. Patino, et al. Olfactory Dysfunction in Huntington’s Disease. J. Huntingt. Dis. 10 (n.d.) 413-422. https: / / doi.org / 10.3233 / JHD- 210497.

[66] N.N. Kumar, et al. Passive immunotherapies for central nervous system disorders - current delivery challenges and new approaches., Bioconjug. Chem. 29 (2018) 3937-3966. https: / / doi.org / 10.1021 / acs.bioconjchem.8b00548.

[67] W. Hofmann, B. Asgharian, The effect of lung structure on mucociliary clearance and particle retention in human and rat lungs, Toxicol. Sci. Off. J. Soc. Toxicol. 73 (2003) 448-456. https: / / doi.org / 10.1093 / toxsci / kfg075.

[68] D. Alarcon-Aris, et al. Anti-α-synuclein ASO delivered to monoamine neurons prevents α-synuclein accumulation in a Parkinson’s disease-like mouse model and in monkeys, EBioMedicine. 59 (2020) 102944. https: / / doi.org / 10.1016 / j.ebiom.2020.102944.

[69] D. Alarcon-Aris, et al. Selective α-Synuclein Knockdown in Monoamine Neurons by Intranasal Oligonucleotide Delivery: Potential Therapy for Parkinson’s Disease, Mol. Ther. 26 (2018) 550-567. https: / / doi.org / 10.1016 / j.ymthe.2017.11.015.

[70] Tabrizi, S.J., et al., Targeting Huntingtin Expression in Patients with Huntington’s Disease. N Engl J Med, 2019.

[71] Donahue, C.J., et al., Quantitative assessment of prefrontal cortex in humans relative to nonhuman primates. Proc Natl Acad Sci U S A, 2018. 115(22): p. E5183-E5192.

[72] van der Burg, J.M., M. Bjorkqvist, and P. Brundin, Beyond the brain: widespread pathology in Huntington’s disease. Lancet Neurol, 2009. 8(8): p. 765-74.

[73] Duong, P.T., et al., Characterization of nascent HDL particles and microparticles formed by ABCA1-mediated efflux of cellular lipids to apoA-I. J Lipid Res, 2006. 47(4): p. 832- 43.

[74] Roheim, P.S., et al., Apolipoproteins in human cerebrospinal fluid. Proc Natl Acad Sci U S A, 1979. 76(9): p. 4646-9.

[75] Koch, M., et al., Apolipoproteins and their subspecies in human cerebrospinal fluid and plasma. Alzheimers Dement (Amst), 2017. 6: p. 182-187.

[76] Koch, S., et al., Characterization of four lipoprotein classes in human cerebrospinal fluid. J Lipid Res, 2001. 42(7): p. 1143-51.

[77] Harr, S.D., et al., Brain expression of apolipoproteins E, J, and A-I in Alzheimer’s disease. J Neurochem, 1996. 66(6): p. 2429-35.

[78] Huang, J.T., et al., Independent protein-profiling studies show a decrease in apolipoprotein A1 levels in schizophrenia CSF, brain and peripheral tissues. Mol Psychiatry, 2008. 13(12): p. 1118-28.

[79] Elliott, D.A., C.S. Weickert, and B. Garner, Apolipoproteins in the brain: implications for neurological and psychiatric disorders. Clin Lipidol, 2010. 51(4): p. 555-573.

[80] Glickman, R.M. and P.H. Green, The intestine as a source of apolipoprotein A1. Proc Natl Acad Sci U S A, 1977. 74(6): p. 2569-73.

[81] Weiler-Guttler, H., et al., Synthesis of apolipoprotein A-1 in pig brain microvascular endothelial cells. J Neurochem, 1990. 54(2): p. 444-50.

[82] Zhou, A.L., et al., Apolipoprotein A-I crosses the blood-brain barrier through clathrin- independent and cholesterol-mediated endocytosis. J Pharmacol Exp Ther, 2019.

[83] Fung, K.Y., et al., SR-BI Mediated Transcytosis of HDL in Brain Microvascular Endothelial Cells Is Independent of Caveolin, Clathrin, and PDZK1. Front Physiol, 2017. 8: p. 841.

[84] Bayburt, T.H., Y.V. Grinkova, and S.G. Sligar, Self-Assembly of Discoidal Phospholipid Bilayer Nanoparticles with Membrane Scaffold Proteins. Nano Letters, 2002. 2(8): p. 853-856.

[85] Denisov, I.G., et al., Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size. J Am Chem Soc, 2004. 126(11): p. 3477-87.

[86] Zannis, V.I., A. Chroni, and M. Krieger, Role of apoA-I, ABCA1, LCAT, and SR-BI in the biogenesis of HDL. J Mol Med (Berl), 2006. 84(4): p. 276-94.

[87] Bayburt, T.H. and S.G. Sligar, Self-assembly of single integral membrane proteins into soluble nanoscale phospholipid bilayers. Protein Sci, 2003. 12(11): p. 2476-81.

[88] Bayburt, T.H. and S.G. Sligar, Single-molecule height measurements on microsomal cytochrome P450 in nanometer-scale phospholipid bilayer disks. Proc Natl Acad Sci U S A, 2002. 99(10): p. 6725-30.

[89] Banerjee, S., T. Huber, and T.P. Sakmar, Rapid incorporation of functional rhodopsin into nanoscale apolipoprotein bound bilayer (NABB) particles. J Mol Biol, 2008. 377(4): p. 1067-81.

[90] Denisov, I.G. and S.G. Sligar, Nanodiscs for structural and functional studies of membrane proteins. Nat Struct Mol Biol, 2016. 23(6): p. 481-6.

[91] Nelson, K.G., et al., Nanodisk-associated amphotericin B clears Leishmania major cutaneous infection in susceptible BALB / c mice. Antimicrob Agents Chemother, 2006. 50(4): p. 1238-44.

[92] Kadiyala, P., et al., High-Density Lipoprotein-Mimicking Nanodiscs for Chemo- immunotherapy against Glioblastoma Multiforme. ACS Nano, 2019. 13(2): p. 1365-1384.

[93] Ghosh, M., et al., Cationic lipid nanodisks as an siRNA delivery vehicle. Biochem Cell Biol, 2014. 92(3): p. 200-5.

[94] Prokop, A. and J.M. Davidson, Nanovehicular intracellular delivery systems. J Pharm Sci, 2008. 97(9): p. 3518-90.

[95] Sorci-Thomas, M.G., et al., The hydrophobic face orientation of apolipoprotein A-I amphipathic helix domain 143-164 regulates lecithin:cholesterol acyltransferase activation. J Biol Chem, 1998. 273(19): p. 11776-82.

[96] Segrest, J.P., et al., A detailed molecular belt model for apolipoprotein A-I in discoidal high density lipoprotein. J Biol Chem, 1999. 274(45): p. 31755-8.

[97] Koppaka, V., et al., The structure of human lipoprotein A-I. Evidence for the “belt” model. J Biol Chem, 1999. 274(21): p. 14541-4.

[98] Mantovani, S., et al., Motor deficits associated with Huntington’s disease occur in the absence of striatal degeneration in BACHD transgenic mice. Hum Mol Genet, 2016. 25(9): p. 1780-91.

[99] L. Zhang, H. Tong, M. Garewal, G. Ren, Optimized negative-staining electron microscopy for lipoprotein studies, Biochim. Biophys. Acta 2013 (1830) 2150-2159.

[0100] K. Okita, Y. Matsumura, Y. Sato, A. Okada, A. Morizane, S. Okamoto, H. Hong, M. Nakagawa, K. Tanabe, K. Tezuka, T. Shibata, T. Kunisada, M. Takahashi, J. Takahashi, H. Saji, S. Yamanaka, A more efficient method to generate integration-free human iPS cells, Nat. Methods 8 (2011) 409-412.

[0101] E.S. Lippmann, S.M. Azarin, J.E. Kay, R.A. Nessler, H.K. Wilson, A. Al-Ahmad, S. P. Palecek, E.V. Shusta, Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells, Nat. Biotechnol. 30 (2012) 783-791.

[0102] N.S. Caron, R. Banos, C. Yanick, A.E. Aly, L.M. Byrne, E.D. Smith, Y. Xie, S.E. P. Smith, N. Potluri, H. Findlay Black, L. Casal, S. Ko, D. Cheung, H. Kim, I. S. Seong, E.J. Wild, J.J. Song, M.R. Hayden, A.L. Southwell, Mutant huntingtin is leared from the brain via active mechanisms in Huntington disease, J. Neurosci. 41 (2021) 780-796.

Claims

1. A therapeutic nanodisc, wherein the therapeutic nanodisc is (a) A lipid-binding polypeptide, which is encoded by SEQ ID NO: 3 or SEQ ID NO: 4, and (b) Lipid bilayer and (c) Therapeutic agents, including Therapeutic nanodiscs.

2. The therapeutic nanodisc according to claim 1, wherein the lipid bilayer is selected from diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside.

3. The lipid bilayer comprises dimyristoyl phosphatidylcholine (DMPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOP A therapeutic nanodisc according to claim 1 or 2, selected from E-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (transDOPE), and analogs thereof.

4. The lipid bilayer consists of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), cholesterol, 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoyloxypropylamine (PEG-C-DMA), 1,2-di A therapeutic nanodisc according to claim 1, 2, or 3, selected from linoleoyloxy-3-(N,N-dimethyl)aminopropane (DLinDMA), N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadiene-1-yl-1,3-dioxolan-4-ethanamine (DLin-KC2-DMA), 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadiene-1-yl-10,13-nonadecadiene-1-yl ester (DLin-MC3-DMA), and analogs thereof.

5. The therapeutic nanodisc according to any one of claims 1 to 3, wherein the lipid bilayer is a 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer.

6. The therapeutic nanodisc according to any one of claims 1 to 5, wherein the molar ratio of the lipid bilayer to the lipid-bound polypeptide is between 50:1 and 400:

1.

7. The therapeutic nanodisc according to any one of claims 1 to 6, wherein the molar ratio of the lipid bilayer to the lipid-bound polypeptide is between 80:1 and 120:

1.

8. A therapeutic nanodisc according to any one of claims 1 to 7, wherein the diameter measured by a transmission electron microscope (TEM) is between approximately 5 nm and approximately 100 nm.

9. The therapeutic nanodisc according to any one of claims 1 to 8, wherein the therapeutic agent is an antisense oligonucleotide (ASO), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), aptamer, ribozyme, mRNA, plasmid, ribonucleoprotein (RNP), or small molecule.

10. The therapeutic nanodisc according to any one of claims 1 to 9, wherein the therapeutic agent is ASO.

11. The therapeutic nanodisc according to claim 10, wherein the ASO targets mutant Huntington's syndrome (mHTT).

12. The therapeutic nanodisc according to any one of claims 1 to 11, wherein the therapeutic agent is selected from one or more of tominersen (CAS number: 1709886-74-7), lovanersen, WVE-120102, WVE-003, and AMT-130.

13. The therapeutic nanodisc according to any one of claims 1 to 12, wherein the therapeutic nanodisc is for delivering the therapeutic agent to the central nervous system.

14. The therapeutic nanodisc according to any one of claims 1 to 13, wherein the therapeutic nanodisc is for delivering the therapeutic agent to the brain.

15. The therapeutic nanodisc according to any one of claims 1 to 14, wherein the therapeutic nanodisc is for the treatment of neurological diseases or disorders.

16. The therapeutic nanodisc according to claim 15, wherein the neurological disease or disorder is selected from one or more of Huntington's disease (HD), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM), spinal muscular atrophy (SMA), spinocerebellar degeneration (SCA), spinal muscular atrophy (SMA), Parkinson's disease (PD), Alzheimer's disease (AD), frontotemporal dementia (FTD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), central nucleus myopathy, Alexander disease, cancer, stroke, meningitis, encephalitis, prion disease, polyneuropathy due to hereditary ATTR amyloidosis (hATTR-PN), and polyneuropathy of hereditary transthyretin-mediated amyloidosis (ATTR).

17. The therapeutic nanodisc according to claim 15 or 16, wherein the neurological disorder or impairment is HD.

18. The therapeutic nanodisc according to any one of claims 1 to 17, wherein the therapeutic agent is selected from one or more of tetrabenazine (TBZ), deutetrabenazine (DBZ), olanzapine, risperidone, citalopram, fluoxetine, sertraline, lamotrigine, and carbamazepine.

19. The aforementioned therapeutic agents are tominersen, WVE-120102 / 120101, WVE-003, (CUG)7, TTX-3360, AMT-130, and AAV. shHD2.1, VY-HTT01, Branapram, PTC518, TAK-686, ZF-KOX1, Pridopidine, Laquinimod, Fenofibrate, Neframapimod, Nilotinib, SRX246, Varenicline, SAGE-718, PBT2, Eteprirsen, SRP-5051, Casimersen, Renadylsen, Golodyrsen, Viltolarsen, WVE-N531, Nusinersen, ION306, ION859, ION464, IONIS-MAPTRx, ATL1102, Tofersen, Urefnersen, IONIS A therapeutic nanodisc according to any one of claims 1 to 18, selected from one or more of C9Rx, WVE-004, ION541, DYN101, zirganersen, ION717, aprontersen, inotersen, patisirane, butrisilane, giboshiran, lumasirane, inclisilane, and pegaptanib.

20. The therapeutic nanodisc according to any one of claims 1 to 19, wherein the therapeutic nanodisc is formulated as an aerosol.

21. The therapeutic nanodisc according to any one of claims 1 to 19, wherein the therapeutic nanodisc is formulated for intravenous or intranasal administration.

22. A method for treating neurological disorders or conditions, comprising administering a therapeutic nanodisc according to any one of claims 1 to 14, comprising an effective amount of therapeutic agent, to a person in need thereof. method.

23. The method according to claim 22, wherein the neurological disease or disorder is selected from one or more of HD, DMD, DM, SMA, SCA, SMA, PD, AD, FTD, MS, ALS, central nucleus myopathy, Alexander disease, cancer, stroke, meningitis, encephalitis, prion disease, hATTR-PN, and ATTR.

24. The method according to claim 22 or 23, wherein the administration is intravenous.

25. The method according to claim 22 or 23, wherein the administration is performed intranasally.

26. For the treatment of neurological disorders or conditions, Use of a therapeutic nanodisc according to any one of claims 1 to 14.

27. In the manufacture of pharmaceuticals for the treatment of neurological disorders or disabilities, Use of a therapeutic nanodisc according to any one of claims 1 to 14.

28. The use according to claim 26 or 27, wherein the neurological disorder or disability is selected from one or more of HD, DMD, DM, SMA, SCA, SMA, PD, AD, FTD, MS, ALS, central nucleus myopathy, Alexander disease, cancer, stroke, meningitis, encephalitis, prion disease, hATTR-PN, and ATTR.

29. The use according to claim 26, 27, or 28, wherein the neurological disorder or impairment is HD.

30. The use according to any one of claims 26 to 29, wherein the therapeutic agent is selected from one or more of tetrabenazine (TBZ), deutetrabenazine (DBZ), olanzapine, risperidone, citalopram, fluoxetine, sertraline, lamotrigine, and carbamazepine.

31. The aforementioned therapeutic agents are tominersen, WVE-120102 / 120101, WVE-003, (CUG)7, TTX-3360, AMT-130, and AAV. shHD2.1, VY-HTT01, Branapram, PTC518, TAK-686, ZF-KOX1, Pridopidine, Laquinimod, Fenofibrate, Neframapimod, Nilotinib, SRX246, Varenicline, SAGE-718, PBT2, Eteprirsen, SRP-5051, Casimersen, Renadylsen, Golodyrsen, Viltolarsen, WVE-N531, Nusinersen, ION306, ION859, ION464, IONIS-MAPTRx, ATL1102, Tofersen, Urefnersen, IONIS Use according to any one of claims 26 to 30, selected from one or more of C9Rx, WVE-004, ION541, DYN101, zirganersene, ION717, aprontersen, inotersen, patisirane, butricirane, giboshiran, lumasirane, inclisilane, and pegaptanib.

32. A composition, (a) A lipid-binding polypeptide, which is encoded by SEQ ID NO: 3 or SEQ ID NO: 4, and (b) A lipid bilayer comprising, composition.

33. The composition according to claim 32, wherein the lipid bilayer is selected from diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside.

34. The composition according to claim 32 or 33, wherein the lipid bilayer is selected from DMPC, DSPC, DOPC, DPPC, DOPE, POPPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, SOPE, and transDOPE.

35. The composition according to claim 32 or 33, wherein the lipid bilayer is selected from DOTMA, DOTAP, DODMA, DODAP, PEG-C-DMA, DLinDMA, DLin-KC2-DMA, DLin-MC3-DMA, and analogs thereof.

36. The composition according to any one of claims 32 to 35, wherein the lipid bilayer is a DMPC lipid bilayer.

37. The composition according to any one of claims 32 to 36, wherein the molar ratio of the lipid bilayer to the lipid-bound polypeptide is between 50:1 and 400:

1.

38. The composition according to any one of claims 32 to 37, wherein the molar ratio of the lipid bilayer to the lipid-bound polypeptide is between 80:1 and 120:

1.

39. The composition according to any one of claims 32 to 38, further comprising a therapeutic agent or a diagnostic agent.

Images