Apolipoprotein a-i nanodisks for central nervous system delivery

Abstract

The present disclosure provides a therapeutic nanodisk, the therapeutic nanodisk comprising: a lipid-binding polypeptide; a lipid bilayer and a therapeutic agent, wherein the therapeutic agent may be of use for treating, preventing a central nervous system disease, disorder, trauma or injury; or as a diagnostic agent for diagnosing a central nervous system disease, disorder, trauma or injury. The lipid bilayer may be 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the therapeutic agent may be a nucleic acid polymer. Further provided are methods for administration of the therapeutic nanodisk to treat, prevent or diagnose the central nervous system disease, disorder, trauma or injury and uses of such therapeutic nanodisks.

Description

[0001] APOLIPOPROTEINA-I NANODISKS FOR CENTRAL NERVOUS SYSTEM DELIVERY

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. US 63 / 472,886, filed 14 June 2023 and US 63 / 472,887, filed 14 June 2023.

[0004] TECHNICAL FIELD

[0005] The present disclosure relates to compositions and methods for treating, preventing or diagnosing a neurological disease or disorder. In particular, the compositions include Apolipoprotein A-I nanodisks for the delivery of a therapeutic agent to the central nervous system.

[0006] BACKGROUND

[0007] Huntington disease (HD) is an autosomal dominant, progressive neurodegenerative disorder characterized by the development of motor, cognitive, and psychiatric symptoms. HD is caused by a CAG trinucleotide repeat expansion in the HTT gene, which encodes the huntingtin protein [1], The resulting mutant huntingtin protein (mHTT), with an elongated polyglutamine tract, is associated with gain of toxic functions [2] that lead to progressive degeneration of neurons across the brain, the most prominent of which are cortical and striatal neurons [3],

[0008] Neuron loss also occurs in other brain regions, including the hippocampus, substantia nigra, and brainstem nuclei, albeit to a lower extent, and correlates with the extensive array of symptoms experienced by later stage HD patients [4], Current approved therapies for treating Huntington disease merely attempt to alleviate the symptoms of chorea without slowing down or arresting its progression.

[0009] One promising therapeutic approach is the use of gene-silencing techniques to lower mHTT levels in the brain. Previous studies using conditional knockout of mHTT in mice have suggested that down-regulating expression of mHTT improves pathological and behavioral outcomes of the disease [5], Further studies have demonstrated the therapeutic potential of mHTT-lowering using antisense oligonucleotides (ASOs) [6, 7], as well as other RNA interference therapies [8-12],

[0010] ASOs are a particularly promising approach for lowering mHTT levels, compared to other RNA- silencing therapies, due to chemical modifications that can attribute high water solubility, reduced toxicity, long tissue half-life, stability at room temperature, and cellular uptake via endocytosis [13, 14], However, the therapeutic potential of ASOs is limited by their inability to cross the blood-brain barrier (BBB) when administered into the systemic circulation. Thus, they are administered to the central nervous system (CNS) via direct surgical intervention into the cerebrospinal fluid (CSF) compartment. In mice, intracerebroventricular (ICV) injection of ASOs results in widespread distribution and dose-dependent suppression of HTT throughout the CNS

[0015] ,

[0011] However, in non-human primates, CSF administration via intrathecal infusion results in limited brain distribution, with higher ASO concentrations in superficial regions and lower concentrations in deeper regions, such as the basal ganglia [6], Since HD is a diffuse brain disorder, widespread delivery of HTT-lowering agents, with significant delivery to the deeper brain regions that are most affected, is necessary to realize the full therapeutic potential of huntingtin-lowering.

[0012] ASO drugs were recently administered via intrathecal (IT) infusion in clinical trials for several CNS disorders, including spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and HD [16-20], Success in terms of a therapeutic outcome for SMA and ALS occurs because these diseases primarily affects the spinal cord which is particularly amenable to intrathecal delivery. Despite providing a route for ASOs to enter the CNS, intrathecal delivery is invasive and requires repeated administration. As such, this route of administration carries a risk of infection, CSF leakage, and other serious surgical complications in highly-vulnerable patients

[0013]

[0021] .

[0014] Furthermore, based on previous non-human primate (NHP) studies, IT infusion of ASOs in patients is likely to result in limited brain distribution, and thus limited target engagement in areas of the brain most affected in HD, namely the deeper layers of the cortex and striatum [6], This medial-lateral concentration gradient is not unique to ASOs, and has also been observed around the catheter tip after intrathecal infusion of small molecule drugs, such as morphine, in patients

[0015]

[0022] ,

[0016] A phase III clinical trial in which HD patients received IT tominersen, an ASO targeting HTT, was stopped due to worsening of patient outcomes and the development of ventricular enlargement in one of the treatment arms with more frequent administration.

[0023] , It has been hypothesized that the increase in ventricular volume may be a result of increased CSF volume, impaired CSF flow and / or neuroinflammation, but a definitive explanation has yet to be determined

[0024] , Hydrocephalus has also been demonstrated to result after IT administration of dextran to Rhesus Macaques

[0025] , and has been reported in SMA patients treated intrathecally with the ASO nusinersen

[0026] , This raises the question of whether this ventricular enlargement could potentially be a consequence of IT delivery, with subsequent ventricular buildup of negatively charged moieties resulting in an inflammatory response and impaired CSF flow.

[0017] The intranasal route of administration offers a non-invasive approach for direct delivery of therapeutic macromolecules to the brain that do not otherwise cross the BBB. A growing number of proteins, siRNAs, viral and non-viral vectors, and even stem cells have been shown to rapidly enter the brain by the nasal route [27-32], several of which are being tested in human trials for CNS disorders [33, 34], Nasally delivered substances follow the trajectory of the olfactory and trigeminal nerve pathways, both of which originate in the nasal cavity, to gain uptake into the brain

[0035] , Once in the brain, intranasally administered biomolecules travel in perivascular spaces (surrounding blood vessels) and thus are rapidly distributed at the microvessel level, achieving deeper penetration into the brain parenchyma compared to intrathecal delivery

[0036] ,

[0018] IT infusion of ASOs into the cerebrospinal fluid is currently being used to deliver HTT lowering ASOs to the brain in clinical trials for HD. Recently, IT infusion of an ASO against human HTT, HTTRX, was shown to induce a dose-dependent reduction of mHTT concentrations in the CSF of HD patients, suggesting HTT target engagement in the brain

[0070] , In non-human primates, ASOs delivered by IT infusion distribute unevenly throughout the brain, resulting in higher HTT suppression in the superficial regions 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 since human brain volumes are several times larger than NHPs

[0071] , Moreover, since ASOs are degraded over time in vivo, repeated IT infusion is required to sustain HTT lowering effects making this route of administration quite invasive

[0014] , However, IT infusion of ASO would also bypass the peripheral tissues, which are also sites of significant pathology in HD

[0072] ,

[0019] An advantage of intranasal administration is that it bypasses the BBB, directly targets the brain, results in widespread brain distribution (particularly in deeper brain structures such as the basal ganglia), and limits peripheral exposure to the therapeutic agents. However, a potential limitation of intranasal delivery is that it is a low efficiency route of administration insofar as only a fraction of the nasally administered dose reaches the brain

[0035] , which could result in unwanted side effects. Unless, there is a benefit to treating peripheral tissues also.

[0020] One way to overcome this disadvantage is through the use of carriers or nanoparticle vehicles

[0037] that can potentially limit the clearance of macromolecules in the nasal cavity and / or promote their ability to penetrate the nasal mucosa and cross the olfactory epithelial layer - the rate-limiting steps of intranasal delivery

[0038] ,

[0021] SUMMARY

[0022] The present application is based in part on the fortuitous discovery that apolipoprotein A-I nanodisks (NDs) made with K133C or E146C ApoA-I lipid-binding polypeptides show much improved CNS / brain delivery of ASOs in comparison with wildtype ApoA-I.

[0023] Furthermore, in the present application, we demonstrate that NDs formed using apoA-I K133C or E146C administered intravenously show enhanced distribution to the brain compared to intravenous administration of WT apoA-I. We also show that a single intrnasal administration of apoA-I K133C NDs carrying a HTT-targeted ASO leads to significant reduction of mHTT in the cortex and striatum.

[0024] In one embodiment, there is provided a therapeutic nanodisk, the therapeutic nanodisk including: (a) a lipid-binding polypeptide, wherein the lipid-binding polypeptide encoded by SEQ ID NOs: 3 or 4; (b) a lipid bilayer; and (c) a therapeutic agent.

[0025] According to a further embodiment, there is provided a therapeutic nanodisk, the therapeutic nanodisk including: (a) a lipid-binding polypeptide, wherein the lipid-binding polypeptide encoded by SEQ ID NOs: 3 or 4; (b) a lipid bilayer; and (c) a nucleic acid polymer.

[0026] According to another embodiment, there is provided a composition, the composition including: (a) a lipid-binding polypeptide, wherein the lipid-binding polypeptide encoded by SEQ ID NOs: 3 or 4; and (b) a lipid bilayer. The composition may further include a therapeutic agent or diagnostic agent.

[0027] According to another embodiment, there is provided a method of treating a neurologic disease or disorder, the method including administration of a therapeutic nanodisk as described herein, containing an effective amount of the therapeutic agent, to a person in need thereof.

[0028] According to another embodiment, there is provided a method of treating or diagnosing a neurologic disease or disorder, the method including administration of a composition as described herein, containing an effective amount of a therapeutic agent, to a person in need thereof or a diagnostic agent to a person in need thereof.

[0029] According to another embodiment, there is provided a use of a therapeutic nanodisk as described herein, for treating a neurological disease or disorder.

[0030] According to another embodiment, there is provided a use of a therapeutic nanodisk as described herein, in the manufacture of a medicament for treating a neurological disease or disorder.

[0031] According to another embodiment, there is provided a use of a composition as described herein, for treating a neurological disease or disorder.

[0032] According to another embodiment, there is provided a use of a composition as described herein, in the manufacture of a medicament for treating a neurological disease or disorder.

[0033] According to another embodiment, there is provided a diagnostic method for diagnosing a neurologic disease or disorder, the method including administration of a composition as described herein, containing an effective amount of the diagnostic agent to a person in need thereof.

[0034] According to another embodiment, there is provided a method for administering a composition or a therapeutic nanodisk as described herein, in an effective amount to a person in need thereof.

[0035] The lipid bilayer may be selected from: diacylphosphatidylcholines; diacylphosphatidylethanolamines; ceramides; sphingomyelins; dihydro sphingomyelins; cephalins; and cerebrosides. The lipid bilayer may be selected from one or more of: dimyristoylphosphatidylcholine (DMPC); distearoylphosphatidylcholine (DSPC); dioleoylphosphatidylcholine (DOPC); dipalmitoylphosphatidylcholine (DPPC); dioleoylphosphatidylethanolamine (DOPE); palmitoyloleoylphosphatidylcholine (POPC); palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal); dipalmitoyl phosphatidyl ethanolamine (DPPE); dimyristoylphosphoethanolamine (DMPE); distearoylphosphatidylethanolamine (DSPE); 16-0-monomethyl PE; 16-O-dimethyl PE; 18-1-trans PE; 1- stearioyl-2-oleoyl-phosphatidy ethanol amine (SOPE); l,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE); and analogs thereof. The lipid bilayer may be selected from one or more of: N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);

[0036] 1.2-dioleoyl-3 -trimethylammonium propane (DOTAP); l,2-dioleyloxy-3 -dimethylaminopropane (DODMA); l,2-dioleoyl-3 -dimethylaminopropane (DODAP); cholesterol, 3-N-[(co- methoxypoly(ethylene glycol)2000)carbamoyl]-l,2-dimyristyloxy-propylamine (PEG-C-DMA);

[0037] 1.2-dilinoleyloxy-3-(N,N-dimethyl)aminopropane (DLinDMA); N,N-dimethyl-2,2-di-(9Z,12Z)- 9, 12-octadecadien-l-yl-l,3-dioxolane-4-ethanamine (DLin-KC2-DMA); 4-(dimethylamino)- butanoic acid, ( 10Z,13Z)-l-(9Z,12Z)-9,12-octadecadien-l-yl- 10, 13 -nonadecadi en-l-yl ester (DLin-MC3-DMA); and analogs thereof. The lipid bilayer may be a 1,2-Dimyristoyl-sn-glycero- 3 -phosphocholine (DMPC) lipid bilayer.

[0038] The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 400: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 350: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 300: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 250: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 200: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 190: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 180: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 170: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 160: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 150: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 140: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 130: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 120: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 110: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 105: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 100: 1 and 400: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 75: 1 and 167: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 61 : 1 and 124: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 80: 1 and 110: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 80: 1 and 120: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 70: 1 and 120: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 60: 1 and 120: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 50: 1 and 120: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 40: 1 and 120: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be between 100: 1 and 105: 1. The molar ratio of lipid bilayer to lipid-binding polypeptide may be 105:1.

[0039] The therapeutic nanodisk may be between about 5 nm to about 100 nm in diameter as determined by transmission electron microscopy (TEM). Alternatively, the therapeutic nanodisk may be between about 5 nm to about 40 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 5 nm to about 50 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 5 nm to about 60 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 5 nm to about 70 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 5 nm to about 80 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 5 nm to about 90 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 6 nm to about 40 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 7 nm to about 40 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 8 nm to about 40 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 9 nm to about 40 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 10 nm to about 40 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 10 nm to about 30 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 10 nm to about 20 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 5 nm to about 20 nm in diameter as determined by transmission electron microscopy (TEM). The therapeutic nanodisk may be between about 5 nm to about 30 nm in diameter as determined by transmission electron microscopy (TEM).

[0040] The therapeutic agent may be an antisense oligonucleotide (ASO); a short interfering RNA (siRNA); a short hairpin RNA (shRNA); a micro RNA (miRNA); an aptamer; a ribozyme; an mRNA; a plasmid; ribonucleoprotein (RNP); or a small molecule. The therapeutic agent may be an ASO. The ASO may target mutant huntingtin (mHTT). The therapeutic agent may be selected from one or more of: Tominersen (CAS No. : 1709886-74-7); Rovanersen; WVE-120102; WVE- 003; AMT-130; and SEQ ID NO:5. The therapeutic nanodisk may be for delivery of the therapeutic agent to the central nervous system. The therapeutic nanodisk may be for delivery of the therapeutic agent systemically. The therapeutic nanodisk may be for delivery of the therapeutic agent to the brain. The therapeutic nanodisk may be for delivery of the therapeutic agent to the spine. The therapeutic nanodisk may be for delivery of the therapeutic agent to a specific part or parts of the brain. The therapeutic nanodisk may be for delivery of the therapeutic agent to one or more of the olfactory bulbs and frontal cortex, the striatum, the thalamus and hypothalamus, and the brainstem and cerebellum.

[0041] The therapeutic nanodisk may be for the treatment of a neurological disease or disorder. The neurological disease or disorder may be selected from one or more of: Huntington disease (HD); Duchenne muscular dystrophy (DMD); Myotonic dystrophies (DMs); Spinal muscular atrophy (SMA); Spinocerebellar ataxias (SCAs); Spinal muscular atrophy (SMA); Parkinson's disease (PD); Alzheimer's disease (AD); Frontotemporal dementia (FTD); Multiple sclerosis (MS); amyotrophic lateral sclerosis (ALS); Centronuclear myopathies; Alexander Disease; cancer; stroke; meningitis; encephalitis; prion disease; polyneuropathy caused by hereditary ATTR amyloidosis (hATTR-PN); and polyneuropathy of hereditary transthyretin-mediated amyloidosis (ATTR). The CNS disease or disorder selected from one or more of: Huntington’s Disease; neurodegeneration; Alzheimer’s disease; Parkinson’s disease; amyotrophic lateral sclerosis; neuropathy; spinal muscular atrophy; dementia; frontotemporal dementia; cancer; multiple sclerosis; inherited metabolic diseases; autoimmune diseases or disorders. The neurological disease or disorder may be HD.

[0042] The neurological disease or disorder may be selected from one or more of: Huntington 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); Centronuclear myopathies; Alexander disease; Prion disease; Polyneuropathy caused by hereditary ATTR amyloidosis (hATTR-PN); Polyneuropathy of hereditary transthyretin-mediated amyloidosis (ATTR); CNS cancers. The therapeutic agent may be selected from one or more of: Tetrabenazine (TBZ); Deutetrabenazine (DBZ); Olanzapine; Risperidone; Citalopram; Fluoxetine; Sertraline; Lamotrigine; and Carbamazepine. The therapeutic agent may be selected from one or more of: Tominersen; WVE-120102 / 120101; WVE-003; (CUG)7; TTX-3360; AMT-130; AAV.shHD2.1; VY-HTT01; Branaplam; PTC518; TAK-686; ZF-KOX1; Pridopidine; Laquinimod; Fenofibrate; Neflamapimod; Nilotinib; SRX246; Varenicline; SAGE-718; PBT2; Eteplirsen; SRP-5051;

[0043] Casimersen; Renadirsen; Golodirsen; Viltolarsen; WVE-N531; Nusinersen; ION306; ION859; ION464; IONIS-MAPTRX; ATL1102; Tofersen; Ulefnersen; IONIS C9Rx; WVE-004; ION541; DYN101; Zilganersen; ION717; Eplontersen; Inotersen; Patisiran; Vutrisiran; Givosiran; Lumasiran; Inclisiran; and Pegaptanib. The therapeutic agent may also be SEQ ID NO:5.

[0044] The therapeutic nanodisk may formulated as an aerosol. The therapeutic nanodisk may be formulated for intravenous delivery or intranasal delivery. The therapeutic nanodisk may be formulated for intravenous delivery. The therapeutic nanodisk may be formulated for CNS delivery. The therapeutic nanodisk may be formulated for intravenous injection (IV), intranasal inhalation, intracerebroventricular (ICV) injection, lumbar intrathecal (IT) injection, intrathecal (IT) infusion, or cisterna magna injection delivery.

[0045] The composition as described herein may further include a therapeutic agent or diagnostic agent.

[0046] 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.

[0047] In some embodiments, the nucleic acid delivery particle comprises a molar ratio of about 30 moles lipid component to about 1 mole ApoA-I component. In some embodiments, the nucleic acid delivery particle comprises a molar ratio of about 55 moles DMPC to about 1 mole ApoA-I. In some embodiments, the nucleic acid delivery particle comprises a molar ratio of about 80 moles DMPC to about 1 mole ApoA-I. In some embodiments, the nucleic acid delivery particle comprises a molar ratio of about 105 moles DMPC to about 1 mole ApoA-I. In some embodiments, the nucleic acid delivery particle comprises a molar ratio of about 120 moles DMPC to about 1 mole ApoA-I.

[0048] 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 micro RNA (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 comprises a molar ratio of about 1 mole ApoA-I to about 5 moles ASO. In one embodiment, the nucleic acid delivery particle comprises a molar ratio of about 1 mole ApoA-I to about 3 moles ASO. In one embodiment, the nucleic acid delivery particle comprises a molar ratio of about 1 mole ApoA-I to about 1 mole ASO.

[0049] In a preferred embodiment, the nucleic acid delivery particle comprises a molar ratio of about 1 mole ApoA-I to about 3 moles ASO to about 105 moles DMPC.

[0050] In one embodiment, the nucleic acid polymer component associates with the lipid component and / or lipid-binding polypeptide component via electrostatic surface interactions. In one embodiment, the nucleic acid polymer component is conjugated or otherwise attached to the lipid component and / or lipid-binding polypeptide component. In one embodiment, the nucleic acid polymer may be attached to the lipid component and / or lipid-binding polypeptide component via a linker.

[0051] In one embodiment, there is provided a method of modulating the expression of a target gene, including administering a nucleic acid delivery particle for silencing the target gene expression or alternatively to induce expression of a target gene. In one embodiment, there is provided a method of modulating the expression of a target gene that is expressed in cells of the CNS or brain, including administering a nucleic acid delivery particle for silencing the target gene expression or alternatively to induce expression of a target gene. In one embodiment, the route of administration for the nucleic acid delivery particle is intravenous, parenteral or intranasal.

[0052] In one embodiment, there is provided a method of treating or preventing a CNS disease in a mammal, including administering an effective amount of the nucleic acid delivery particle is for modulating the target gene expression that is associated with a CNS disease or disorder wherein delivery of a therapeutic nucleic acid polymer to the brain or CNS is desirable. In a further aspect of the invention, the CNS disease or disorder may include but is not limited to, Huntington’s Disease, neurodegeneration, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, neuropathy, spinal muscular atrophy, dementia, frontotemporal dementia, cancer, multiple sclerosis, inherited metabolic diseases, autoimmune diseases or disorders. In one embodiment, there is provided a method of treating or preventing a musculoskeletal or neuromuscular disease or disorder in a mammal, including administering an effective amount of the nucleic acid delivery particle is for modulating the target gene expression that is associated with a musculoskeletal disease or disorder wherein delivery of a therapeutic nucleic acid polymer to the muscle tissue is desirable. 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-Dreifuss muscular dystrophy, distal myopathies, spinal muscular atrophy (SMA), muscular atrophy, or myotonia.

[0053] In one embodiment, there is provided a method of treating or preventing a liver disease or disorder in a mammal, including administering an effective amount of the nucleic acid delivery particle is for modulating the target gene expression that is associated with a liver disease or disorder wherein delivery of a therapeutic nucleic acid polymer to the liver is desirable. In a further aspect of the invention the liver disease or disorder may include, but is not limited to, cirrhosis, hepatocellular carcinoma, vviral hepatitis, non-viral hepatitis, cholestatic liver disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver fibrosis, and genetic liver diseases such as hemochromatosis, Wilson’s disease, alpha- 1 antitrypsin deficiency, hyperoxaluria and oxalosis.

[0054] In one embodiment, there is provided a method of treating or preventing a lung disease or disorder in a mammal, including administering an effective amount of the nucleic acid delivery particle is for modulating the target gene expression that is associated with a lung disease or disorder wherein delivery of a therapeutic nucleic acid polymer to the lung is desirable. 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.

[0055] In a further aspect, there is provided the use of a pharmaceutical composition described herein for the treatment, mitigation or prevention of a lung, a muscular or a liver, disease or disorder.

[0056] In a further aspect, there is provided the use of a pharmaceutical composition described herein for the treatment, mitigation or prevention of a CNS disease or disorder.

[0057] This summary of the invention does not necessarily describe all features of the invention. Other aspects, features and advantages of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention.

[0058] BRIEF DESCRIPTION OF THE FIGURES

[0059] FIGURE 1 shows ApoA-I NDs successfully form an electrostatic interaction with antisense oligonucleotides, (a) Electrophoretic mobility shift assay (EMSA) demonstrates that when ASO is incubated at 37°C for 1 hour with varying moles of apoA-I NDs (stoichiometry of ASO to apoA-I from 1 : 1 to 3 : 1), ASO was visualized at the level of the apoA-I NDs, suggesting a potential electrostatic interaction between the ASO and the apoA-I. (b) Size exclusion chromatography results demonstrating separation of nucleic acid, protein and phospholipid after ApoA-I NDs were incubated with ASO at 37°C for 1 hour (molar ratio of 2.5 moles of ASO : 1 mole of apoA-I : 105 moles of DMPC). ASO eluted in the same fractions as both the apoA-I and DMPC. (c) Size exclusion chromatography results from each individual component (nucleic acid, protein and phospholipid) run alone (as a negative control). The ASO, apoA-I and DMPC all exhibited different elution patterns, (d) Oligreen ASO association assay results indicate that after treatment with a detergent, Triton X-100, amount of ASO associated with both apoA-I WT and K133C NDs was similar to non-detergent treated NDs (two-way ANOVA demonstrates no significance by ND type, Triton X-100 treatment, or a significant interaction), (e) Dynamic light scattering demonstrate a minor shift in the size for NDs prepared using apoA-I WT or apoA-I K133C protein. ApoA-I WT NDs were larger in size (mean diameter ± SD = 11.95 ± 0.34 nm) compared to NDs formed with apoA-I K133C (n = 3; mean diameter ± SD = 10.76 ± 0.05 nm).

[0060] FIGURE 2 shows ApoA-I lipid-free protein and apoA-I NDs are successfully delivered to the brain following intranasal administration, (a) BACHD mice were treated intranasally with either 150 pg of apoA-I WT (lipid-free), apoA-I K133C (lipid-free), apoA-I WT NDs, apoA-I K133C NDs, or PBS and sacrificed two hours later. ApoA-I WT in nanodisk form resulted in higher whole brain delivery compared to lipid-free apoA- 1 WT protein. Lipid-free K133C achieved significantly higher levels in brain compared to both apoA-I WT and apoA-I WT NDs, while K133C apoA-I NDs achieved the highest apoA-I levels in whole brain. One-way ANOVA: PO.OOOl; Tukey’s multiple comparison test: *p<0.05, ***p<0.001, ****p<0.0001). (b) Across rostral-caudal brain slabs (A-D), apoA-I K133C NDs resulted in significantly higher levels of apoA-I in each region compared to apoA-I WT NDs except the most caudal brain region, D comprising of the brainstem and cerebellum. Two-way ANOVA indicates significance by brain region (P<0.0001), treatment (P<0.0001), and a significant interaction for brain region x treatment (P<0.01); Bonferroni’s multiple comparison test: **p<0.01, ****p<0.0001. (c) Ex vivo imaging of dorsal BACHD mouse brains 2 hr post-intranasal administration of PBS or apoA-I K133C NDs labelled with cf75O dye. (d) Ex vivo imaging of ventral BACHD mouse brains 2 hr post-intranasal administration of PBS or apoA-I K133C NDs labelled with cf75O dye. (e) A representative diagram demonstrating the rostral-caudal brain slabs used for ELISA. Section A comprises of the olfactory bulbs and frontal cortex, B the level of the striatum, C the level of the thalamus and hypothalamus, and D the level of the brainstem and cerebellum.

[0061] FIGURE 3 shows intranasal delivery of ApoA-I NDs is not affected by HD mutation at 2 and 6 months of age. WT and BACHD mice at 2 (a), 6 (b), and 12 (c) months of age were treated intranasally with K133C ApoA-I NDs and sacrificed 2 hours post-intranasal treatment, (a, b) At both 2 and 6 months, there were no significant differences in ApoA-I levels across the brain for WT vs BACHD mice. Two-way ANOVA indicates 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, A, comprising the olfactory bulbs and frontal cortex. Two-way ANOVA indicates significance by brain region, PO.OOOl; Bonferroni’s multiple comparison test: *p<0.05.

[0062] FIGURE 4 shows cellular localization of apoA-I after intranasal administration of apoA-I K133C NDs. After intranasal delivery of apoA-I K133C NDs, apoA-I deposits in in various brain cells across the brain including NeuN+ neurons (a-c), CD31+ capillary endothelial cells (d- f), Ibal+ microglia (g-i), GFAP+ astrocytes (j-1), lectin-labeled choroid plexus cells (m-o). Scale bar = 80 pm.

[0063] FIGURE 5 shows ApoA-I K133C NDs CNS rapidly enter the brain following intranasal administration and demonstrate a dose-dependent increase, (a-b) BACHD mice were administered 150 pg ApoA-I K133C NDs intranasally and sacrificed at 1, 2, 3, 6, and 24 hours post-treatment, (a) ApoA-I whole brain average was highest at 2 hours following intranasal administration. One-way ANOVA: P<0.0001; Tukey’s Multiple Comparison test: 2 and 24 hours vs 1 hr, ****p<0.0001. (b) ApoA-I levels were initially highest for the 1, 2, 3, and 6 hour time points in the most rostral brain region, A. Throughout all 4 rostral-caudal brain regions, ApoA-I levels were consistently significantly highest at 2 hrs post-intranasal treatment. Two-way ANOVA indicates significance by brain region (P<0.0001), treatment (P<0.0001) and interaction (PO.OOOl); Tukey’s Multiple Comparison test: **p<0.01, ***p<0.001, ****p<0.0001. (c-d) BACHD mice received either 75, 150, 300, or 500 pg apoA-I K133C NDs intranasally and were sacrificed at 2 hours post-intranasal administration, (c) Whole brain averages demonstrate a dose- dependent increase in apoA-I levels with the highest dose, 500 pg, achieving significantly higher levels than the lower doses (75 and 150 pg). 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, ****, pO.OOOl.

[0064] FIGURE 6 shows K133C apoA-I NDs are minimally distributed to peripheral tissues and rapidly cleared following intranasal administration. ApoA-I levels were measured in CSF (a), lung (b), liver (c), and plasma (d) of BACHD mice 1, 2, and 6 hrs post-intranasal treatment with K133C ApoA-I NDs. (a) ApoA-I levels in CSF were elevated at 1 hr and plateaued between 2-6 hours, (b) In lung, apoA-I levels were highest over the first 2 hours and apoA-I was almost completely eliminated from the lung by 6 hrs. (c) In liver, apoA-I levels remained minimal over the course of 6 hours, (d) In plasma, apoA-I levels were low at 1 hour and continued to rise steadily by 6 hours.

[0065] FIGURE 7 shows intranasal administration of apoA-I K133C NDs results in higher apoA-I levels in brain compared to intravenous administration of same dose, (a) BACHD mice received the same dose, 150 pg, of apoA K133C -I NDs either intranasally or intravenously. ApoA-I whole brain 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 apoA-I levels in the most rostral brain region (A) comprising of the olfactory bulbs and frontal cortex. Two-way ANOVA indicates significance by brain region (P<0.001), treatment (P<0.01), and interaction (P<0.001); Bonferroni’s Multiple Comparison test: ****p<0.0001.

[0066] FIGURE 8 shows single intranasal dose of K133C ASO-NDs results in significant mHTT lowering in brain, (a) BACHD mice received either PBS or apoA-I K133C ASO-NDs and were sacrificed 24 hours later. DL-IHC for ASO and NeuN demonstrate that ASO deposited in NeuN+ cells 24 hours post intranasal administration of ASO-NDs. (b-d) BACHD mice received either PBS, ASO alone, or apoA-I K133C ASO-NDs intranasally and were sacrificed 4 weeks later, (b) Whole brain averages of relative mHTT levels in brain lysates from these mice demonstrate a significant reduction in ASO-ND treated mice compared to PBS-and ASO-treated controls. Oneway ANOVA indicates significance by treatment, P<0.05; Tukey’s multiple comparison test, *p<0.05. (c) Representative western blots demonstrating mHTT and mouse HTT levels in microdissected brain regions after intranasal treatments, (d) An examination of relative mHTT levels in micro dissected brain regions shows significant mHTT lowering in the olfactory bulb, cortex, and striatum of ASO-ND treated mice. Two-way ANOVA indicates significance by treatment, PO.OOOl; Tukey’s multiple comparison test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0067] FIGURE 9 shows intranasal administration of 2 monthly doses of ApoA-I K133C ASO-NDs results in sustained mHTT lowering in deeper brain structures, (a) BACHD mice received either 2 monthly doses of PBS, ASO, or ASO-NDs, and 1 monthly dose of ASO-NDs, and were sacrificed 8 weeks after initial dose, (b) Whole brain averages demonstrate that both 1 and 2 doses of ASO-NDs resulted in significant mHTT-lowering. One-way ANOVA: P<0.0001; Tukey’s multiple comparison test: **p<0.01, ****p<0.0001 vs PBS-treated controls, (c) An examination of relative mHTT levels in micro dissected brain regions shows significant mHTT lowering in the olfactory bulb, striatum, and brainstem of mice that received 2 monthly doses of ASO-ND. Two- way ANOVA indicates significance by treatment, PO.OOOl; Tukey’s multiple comparison test: p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0068] FIGURE 10 shows in vitro characterization of ApoA-I nanodisks comprising varying ratios of the lipid component and lipid-binding protein component. (A) Native polyacrylamide gel electrophoresis of nanodisks comprising a ApoA-I:DMPC molar ratio ranging from 1 :30 up to 1 : 105. DPMC alone and ApoA-I alone are included as controls for comparison. (B) shows the % of ApoA-I that is incorporated into the nanodisks when combined at varying ratios of ApoA- I:DMPC.

[0069] FIGURE 11 shows antisense oligonucleotides associate with ApoA-I NDs. (A) shows the results of an electrophoretic mobility shift assay for a model antisense oligonucleotide (ASO alone), ApoA-I Nanodisks alone (NDs alone) or ApoA-I Nanodisks in the presence a model antisense oligonucleotide at defined ratios (1 :1, 1 :3, 1 :5 of ApoA-I:ASO). (B) Results of size exclusion chromatography showing the relative concentration of lipid (DMPC), antisense oligonucleotide (ASO), or the lipid-binding polypeptide (ApoA-1) within elution fractions.

[0070] FIGURE 12 shows ApoA-I NDs carrying ASO enhance suppression of HTT in HD patient- derived lymphoblast cells. (A) Western blot analysis of HTT protein expression in HD patient- derived lymphoblast cells exposed to either media alone, Antisense oligonucleotide (ASO) directed to the huntingtin gene, DMPC lipid alone, DMPC lipid and ASO, ApoA-I and ASO or the nanodisk nucleic acid delivery particle of the invention. (B) Graph showing the relative HTT protein levels for the wild type (wtHTT) and mutant HTT (mHTT) proteins following the treatment conditions shown in (A).

[0071] FIGURE 13 shows a model of ApoA-I “looped-belf ’ conformation on discoidal HDL.

[0072] FIGURE 14 shows ApoA-I amino acid sequence including the 24 amino acid signal peptide (SEQ ID NO: 1). The signal peptide sequence is shaded at the N-terminus of ApoA-I and mutation sites described in the invention are bolded and underlined.

[0073] FIGURE 15 shows ApoA-I mutants can form NDs with distinct properties. (A) Native PAGE protein gel showing the molecular weight of the complexes for nanodisks comprising either wild type ApoA-I (ApoA-I WT NDs), ApoA-I with a cysteine substitution at amino acid residue 133 (ApoA-I K133C NDs) or ApoA-I with a cysteine substitution at amino acid residue 146 (ApoA-I E146C NDs). (B) Electron microscopy of nanodisk nucleic acid delivery particles comprising either wild type ApoA-I (ApoA-I WT NDs), ApoA-I with a cysteine substitution at amino acid residue 133 (ApoA-I K133C NDs) or ApoA-I with a cysteine substitution at amino acid residue 146 (ApoA-I E146C NDs). (C) Graph showing the particle diameters in nanometers for nucleic acid delivery particles comprising either wild type ApoA-I (ApoA-I WT NDs), ApoA-I with a cysteine substitution at amino acid residue 133 (ApoA-I K133C NDs) or ApoA-I with a cysteine substitution at amino acid residue 146 (ApoA-I E146C NDs). (D) Dynamic light scattering analysis of the nucleic acid delivery particles comprising either wild type ApoA-I (ApoA-I WT NDs), ApoA-I with a cysteine substitution at amino acid residue 133 (ApoA-I K133C NDs) or ApoA-I with a cysteine substitution at amino acid residue 146 (ApoA-I E146C NDs).

[0074] FIGURE 16 shows ApoA-I in NDs can enter the brain more efficiently than lipid-free ApoA-I. ApoA-I levels as measured by ELISA in brain homogenate samples from mice that were administered intravenously with either ApoA-I wild type, nanodisks comprising wild type Apo- Al and lipid. FIGURE 17 shows ApoA-I K133C NDs enhance CNS entry following systemic administration. ApoA-I levels as measured by ELISA in various brain tissues from mice that were administered intravenously with either nanodisks comprising wild type Apo- Al and lipid (ApoA-I WT NDs), nanodisks comprising ApoA-I with a cysteine substitution at amino acid residue 133 and lipid (ApoA-I K133C NDs) or nanodisks comprising ApoA-I with a cysteine substitution at amino acid residue 146 and lipid (ApoA-I E146C NDs).

[0075] FIGURE 18 shows biodistribution of various ND formulations in the various regions of the brain (A) and in CSF (B) following IV injection. NP Fl : nanodisk comprising ApoA-I wild type; NP F2: nanodisk comprising ApoA-I K133C; NP F3: nanodisk comprising ApoA-I E146C; NP F4: nanodisk comprising ApoA-I K133C and E146C substitutions; NP F5: nanodisk comprising ApoA-I both R160V (valine substitution) and H162A (alanine substitution) substitutions.

[0076] FIGURE 19 shows (A) ex vivo imaging reveals increased CNS entry with ApoA-I K133C NDs as compared to Nanodisks comprising wild type ApoA-I. (B) shows a graphical quantification of the imaging results of relative fluorescence in the brain as a percent of fluorescence observed in the liver.

[0077] FIGURE 20 shows time-course of ApoA-I K133C ND or Nanodisk with wild type ApoA-I biodistribution shows rapid entry into the CNS following IV administration. (A) shows data for entry of nanodisks into the brain (B) shows data for entry of nandodisks into the CSF.

[0078] FIGURE 21 shows dose-dependent increase of ApoA-I K133C NDs in the brain (A) and CSF (B) following IV administration.

[0079] FIGURE 22 shows a dose-dependent increase of ApoA-I K133C NDs in all brain regions following IV administration.

[0080] FIGURE 23 shows HD mutation status does not significantly alter CNS entry of ApoA-I K133C NDs in wild type or BACHD animals at varying ages (2-12 months in age).

[0081] FIGURE 24 shows ASO carrying ApoA-I WT and ApoA-I K133C NDs can induce significant mutant HTT suppression in the CNS as compared to ASO alone (A: cortex, B: striatum, C: hippocampus, D: cerebellum).

[0082] FIGURE 25 shows ApoA-I K133C NDs increase peripheral exposure following systemic administration. ApoA-I levels as measured by ELISA in tissue homogenate samples (A: liver, B: Quad muscle, C: Kidney) from mice that were administered intravenously with either nanodisks containing wild type Apo- Al and lipid (ApoA-I WT NDs), nanodisks containing Apo- Al with a K133C substitution and lipid (ApoA-I K133C NDs) or nanodisks containing Apo- Al with a E146C substitution and lipid (ApoA-I E146C).

[0083] FIGURE 26 shows biodistribution of various ND formulations in the peripheral tissues following IV injection (A: Quadricep muscle, B: liver, C: Kidney). NP Fl: nanodisk comprising ApoA-I wild type; NP F2: nanodisk comprising ApoA-I K133C; NP F3: nanodisk comprising ApoA-I E146C; NP F4: nanodisk comprising ApoA-I K133C and E146C substitutions; NP F5: nanodisk comprising ApoA-I R160V (valine substitution) and H162A (alanine substitution) substitutions.

[0084] FIGURE 27 shows a time-course of ApoA-I K133C ND biodistribution (grey squares) or Nanodisk with wild type ApoA-I (black circles) into the liver (A) and quadriceps (B) tissues.

[0085] FIGURE 28 shows dose-dependent increase of ApoA-I K133C NDs in the liver (A) and quadriceps (B) following IV administration.

[0086] FIGURE 29 shows HD mutation status does not significantly alter peripheral distribution of ApoA-I K133C NDs in the liver (A) or Quadricep muscle (B) in wild type or BACHD animals at varying ages (2-12 months in age).

[0087] FIGURE 30 shows ASO carrying ApoA-I WT and K133C NDs can induce significant mutant HTT suppression in quadriceps (A), liver (B), lungs (C), and heart (D) as compared to ASO alone.

[0088] FIGURE 31 shows modification of ASO does not affect its HTT lowering activity. (A) HD patient-derived lymphoblast cells treated with either PBS, unmodified ASO, ASO modified with a 5’ end amine group (amino ASO) or ASO modified with a 5 ’end amine group and a 4- formylbenzamide moiety (4FB-ASO) and the protein HTT levels were detected by immunoblotting (A) and quantified in (B).

[0089] FIGURE 32 shows covalent conjugation of ASO to ApoA-I K133C does not affect formation of NDs. (A) Immunoblot and SDS-PAGE analysis of nanodisks comprising the ApoA-I K133C substitution (ApoA-I K133C) or modified ASO conjugated to the nanodisks comprising the ApoA-I K133C substitution (ASO: ApoA-I K133C). (B) Native PAGE analysis of increasing concentrations of the modified ASO conjugated to the nanodisks comprising the ApoA-I K133C substitution (ASO:ApoA-I K133C). (C) Electron microscopy analysis of the modified ASO conjugated to the nanodisks comprising the ApoA-I K133C substitution (ASO:ApoA-I K133C). (D) Graph depicting the diameter length in nanometers for the nanodisks comprising the ApoA-I K133C substitution (ApoA-I K133C) or modified ASO conjugated to the nanodisks comprising the ApoA-I K133C substitution (ASO:ApoA-I K133C).

[0090] FIGURE 33 shows conjugation of ASO to ApoA-I K133C NDs (ASO:ApoA-I K133C NC NDs) enhances HTT suppression in HD patient-derived lymphoblasts as compared to ASO alone. (A) shows the immunoblot analysis and (B) shows the graphical quantification.

[0091] FIGURE 34 shows ASO:ApoA-I K133C conjugate NDs (ASO:ApoA-I K133C NC NDs) enhance HTT lowering following intracerebroventricular injection in BACHD mice as compared to the PBS control or unmodified ASO in regions of the brain (A: cortex, B: striatum, C: hippocampus, D: cerebellum).

[0092] FIGURE 35 shows systemic delivery of ASO: ApoA-I K133C conjugate NDs (ASO: ApoA-I K133C NC NDs) results in potent suppression of mHTT in regions of the brain of BACHD mice (A: cortex, B: striatum, C: hippocampus, D: cerebellum).

[0093] FIGURE 36 shows ASO: ApoA-I K133C conjugate NDs (ASO: ApoA-I K133C NC NDs) enhance HTT lowering following intracerebroventricular injection in BACHD mice as compared to the PBS control or unmodified ASO in peripheral tissues (A: quadricep muscle, B: liver, C: lungs, D: heart).

[0094] FIGURE 37 shows systemic delivery of ASO: ApoA-I K133C conjugate NDs (ASO: ApoA-I K133C NC NDs) results in potent suppression of mHTT in peripheral tissues of BACHD mice (A: quadricep muscle, B: liver, C: lungs, D: heart).

[0095] DETAILED DESCRIPTION

[0096] The following detailed description will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, the figures demonstrate embodiments of the present invention. However, the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

[0097] Here, we explore the use of apolipoprotein A-I nanodisks as vehicles for intranasal delivery of ASOs to the brain. Nanodisks are biocompatible, discoidal complexes that are composed of phospholipids, and scaffolding proteins, such as apolipoprotein A-I (apoA-I). These nanodisks are spontaneously formed in the presence of apoA-I and phospholipids, with the phospholipids assembling into a bilayer that is circumscribed by two apoA-I molecules

[0039] , The apoA-I scaffold stabilizes the disk and shields the hydrophobic fatty acyl chains of the phospholipid bilayer from the hydrophilic environment

[0040] , One major advantage of these apoA-I nanodisks is their similarity to nascent high density lipoprotein (HDL) and thus lack of immunogenicity due to their ability to avoid recognition by macrophages

[0041] ,

[0098] ApoA-I nanodisks (NDs) have been used as in vivo drug delivery vehicles for hydrophobic drugs, including amphotericin B, all-trans retinoic acid, and curcumin [42-44], ApoA-I NDs have also been shown to bind siRNA and promote gene silencing in cultured cells

[0045] , 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 timedependent manner

[0046] , Here we present results from testing two ND formulations using two forms of the apoA-I protein, the wild-type protein and a K133C mutant. ApoA-I K133C was chosen because the insertion of the cysteine residue has been found to create a disulfide bond between the two anti-parallel apoA-I molecules, tethering the apoA-I tightly around the NDs, offering potentially greater cellular penetration and distribution

[0047] ,

[0099] ApoA-I NDs have multiple properties that make them ideal drug delivery vehicles, 1) they resemble nascent HDL particles and are thus biocompatible, 2) they form spontaneously in the presence of ApoA-I and lipids making them easy to generate, and 3) under the correct stoichiometry of ApoA-I:lipids they form monodisperse particles <50nm in diameter, which limits detection clearance by the immune and lymphatic systems

[0094] ,

[0100] This invention provides lipid NDs for non-invasive targeted delivery of nucleic acid cargo to the CNS for treatment of HD and other CNS-related diseases or disorders. Lipid NDs of the present invention comprise (a) a lipid, (b) a lipid-binding polypeptide, and (c) a nucleic acid polymer.

[0101] Lipid NanoDisks

[0102] A therapeutic nanodisk (ND) as used herein refers to a lipid-binding polypeptide, a lipid bilayer, and a therapeutic agent, wherein the lipid-binding polypeptide encoded by SEQ ID NOs: 3 or 4. NDs are discoidal particles composed of phospholipid bilayers encircled by recombinant ApoA- I, which acts as a membrane scaffolding protein to organize lipids and shield hydrophobic fatty acyl chains from the hydrophilic environment [84, 85], Notably, NDs can self-assemble into particles with a controlled size depending on the lipid composition and molar ratio of ApoA- Llipid. In a structural context, NDs resemble nascent HDL particles prior to loading of esterified cholesterol and transitioning to a spherical particle

[0086] , NDs have been used extensively to study the structure and function of integral membrane proteins [87-90] and have also been used as a delivery platform for bioactive compounds, such as all-trans retinoic acid and amphotericin B [42, 44, 91], Lipid NDs as described herein are composed of lipid-binding polypeptide and a lipid bilayer, with an optional therapeutic agent, or diagnostic agent. The therapeutic agent is optionally selected from: an antisense oligonucleotide (ASO); a short interfering RNA (siRNA); a short hairpin RNA (shRNA); a micro RNA (miRNA); an aptamer; a ribozyme; an mRNA; a plasmid; ribonucleoprotein (RNP); or a small molecule. The therapeutic agent may 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 micro RNA (miRNA); an aptamer; a ribozyme; an mRNA; or a plasmid. The nucleic acid may be an antisense oligonucleotide (ASO); a short interfering RNA (siRNA); a short hairpin RNA (shRNA); a micro RNA (miRNA); an aptamer; a ribozyme; an mRNA; a plasmid; or form part of a ribonucleoprotein (RNP).

[0103] The lipid bilayer may be selected from: dimyristoylphosphatidylcholine (DMPC); distearoylphosphatidylcholine (DSPC); dioleoylphosphatidylcholine (DOPC); dipalmitoylphosphatidylcholine (DPPC); dioleoyl-phosphatidylethanolamine (DOPE); palmitoyloleoylphosphatidylcholine (POPC); palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal); dipalmitoyl phosphatidyl ethanolamine (DPPE); dimyristoylphosphoethanolamine (DMPE); distearoyl-phosphatidylethanolamine (DSPE); 16-0-monom ethyl PE; 16-O-dimethyl PE; 18-1 -trans PE; l-stearioyl-2-oleoyl-phosphatidy ethanol amine (SOPE); 1,2-dielaidoyl-sn- glycero-3-phophoethanolamine (transDOPE); and analogs thereof.

[0104] Alternatively, the lipid bilayer may be selected from: N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA); l,2-dioleoyl-3 -trimethylammonium propane (DOTAP); l,2-dioleyloxy-3 -dimethylaminopropane (DODMA); l,2-dioleoyl-3- dimethylaminopropane (DODAP); cholesterol, 3-N-[(co-methoxypoly(ethylene glycol)2000)carbamoyl]-l,2-dimyristyloxy-propylamine (PEG-C-DMA); l,2-dilinoleyloxy-3- (N,N-dimethyl)aminopropane (DLinDMA); N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-l- yl-l,3-dioxolane-4-ethanamine (DLin-KC2-DMA); 4-(dimethylamino)-butanoic acid, ( 1 OZ, 13Z)- 1 -(9Z, 12Z)-9, 12-octadecadien- 1 -yl- 10, 13 -nonadecadi en- 1 -yl ester (DLin-MC3 - DMA); and analogs thereof.

[0105] In certain embodiments, the lipid NDs include a neutral lipid. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides. Exemplary lipids include, for example, dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1- stearioyl-2-oleoyl-phosphatidy ethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE). In one embodiment, the neutral lipid is dimyristoylphosphatidylcholine (DMPC).

[0106] In certain embodiments, the lipid NDs include apolipoprotein as the lipid-binding polypeptide component. Apolioproteins are proteins that bind lipids (oil-soluble substances such as fat and cholesterol) to form lipoproteins. They transport lipids through the lymphatic and circulatory systems. Apolipoprotein Al (ApoA-I) is encoded by the APOA1 gene and plays a role in lipid metabolism. Apolipoprotein A-I is the main protein component of high-density lipoprotein (HDL) particles in the plasma. HDL is involved in the reverse cholesterol transport (RCT) of cholesterol from peripheral tissues to the liver for excretion or recycling

[0073] , ApoA-I is one of the most abundant apolipoproteins in the CSF [74-76] and the brain [77, 78], However, APOA-I mRNA levels in the brain are very low during human development and adulthood

[0079] , which suggests that the source of ApoA-I present in the CNS originates from the plasma where it is primarily produced by the liver and intestines

[0080] , There is also evidence that brain microvascular endothelial cells may also produce and secrete ApoA-I

[0081] , In one embodiment, ApoA-I has been modified to promote disulfide bridge formation between ApoA-I molecules of the 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 position 133 (K) or 146 (E). The lipid-binding polypeptide may be encoded by SEQ ID NOs: 3 or 4 or a sequence having 90% sequence identity to either SEQ ID NOs: 3 or 4. Preferably, the lipid-binding polypeptide is encoded by SEQ ID NOs: 3 or 4. More preferably, the lipid-binding polypeptide is encoded by SEQ ID NO: 3. Furthermore, the lipid-binding polypeptides as used herein may encompasses both modified and unmodified amino acids. The modifications may be beneficial to one or more of: loading of the therapeutic agent (including a nucleic acid polymer), or the diagnostic agent; delivery of the therapeutic agent or the diagnostic agent; targeting of the therapeutic agent or the diagnostic agent; bioavailability of the therapeutic agent or the diagnostic agent; half-life of the therapeutic agent or diagnostic agent; or the minimization of unwanted effects of the therapeutic agent or the diagnostic agent.

[0107] Nucleic Acid Polymers

[0108] The lipid NDs 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 meant to include any oligonucleotide or polynucleotide. Fragments containing up to 50 nucleotides are generally termed oligonucleotides, and longer fragments are called polynucleotides. In particular embodiments, oligonucleotides of the present invention are 8-50 nucleotides in length. In particular embodiments, oligonucleotides of the present invention are 8-150 nucleotides in length. In particular embodiments, oligonucleotides of the present invention are 8-500 nucleotides in length. Oligonucleotides are classified as deoxyribooligonucleotides or ribooligonucleotides. A deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5' and 3' carbons of this sugar to form an alternating, unbranched polymer. A ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose. The nucleic acid that is present in a lipid nanoparticle according to this invention includes any form of nucleic acid that is known. The nucleic acids used herein can 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.

[0109] Although various embodiments of the 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 this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. The following examples are provided for the purpose of illustrating, not limiting, the claimed invention.

[0110] The present disclosure provides compositions for treating, preventing or diagnosing a central nervous system condition, disease, disorder, trauma or injury using therapeutic nanodisk, wherein the therapeutic nanodisk comprises: a lipid-binding polypeptide; a lipid bilayer; and a therapeutic agent. Alternatively, the therapeutic nanodisk comprises: a lipid-binding polypeptide; a lipid bilayer; and a nucleic acid polymer.

[0111] Non-limiting examples of neurologic diseases and disorders, and other disorders, and the nucleic acid polymers that may be used to treat them are provided in TABLE 1 below.

[0112] TABLE 1: Central Nervous System Diseases or Disorders and Nucleic Acid Polymers Used in the Treatment Thereof

[0113] Disease / Disorder Nucleic Acid Polymer

[0114] Huntington disease (HD) Tominersen; Rovanersen; WVE-120102; WVE-003; and

[0115] AMT- 130

[0116] Duchenne muscular dystrophy (DMD) Eteplirsen; SRP-5051; Casimersen; Renadirsen;

[0117] Golodirsen; Viltolarsen; and WVE-N531

[0118] Spinal muscular atrophy (SMA) Nusinersen; and ION306

[0119] Alzheimer’s disease (AD) IONIS-MAPTRX

[0120] Parkinson’s disease (PD) ION859; and ION464

[0121] Multiple sclerosis (MS) ATL1102

[0122] Amyotrophic lateral sclerosis (ALS) Tofersen; Ulefhersen; IGNIS C9Rx; WVE-004; and

[0123] ION541

[0124] Centronuclear myopathies DYN 101

[0125] Alexander disease Zilganersen

[0126] Prion disease ION717

[0127] Polyneuropathy caused by hereditary ATTR Eplontersen; Vutrisiran; and Patisiran amyloidosis (hATTR-PN)

[0128] Polyneuropathy of hereditary transthyretin- Inotersen mediated amyloidosis (ATTR)

[0129] CNS cancers Aprinocarsen (ISIS 3521); Trabedersen (AP12009); CpG- ODN; IMV-001; Imetelstat; Oblimersen; OGX-Ol l;

[0130] LErafAON; TASO-OOl; WGI-0301; AZD8701; anti-miR- 221 / 222; anti-miR-lOb; and Pegaptanib

[0131] Other Givosiran; Pegaptanib; Inclisiran; and Lumasiran

[0132] As used herein, therapeutic agent is meant to include: an antisense oligonucleotide (ASO); a short interfering RNA (siRNA); a short hairpin RNA (shRNA); a micro RNA (miRNA); an aptamer, a ribozyme, an mRNA, a plasmid, or a small molecule.

[0133] As used herein, nucleic acid polymer, is meant to include: an antisense oligonucleotide (ASO); a short interfering RNA (siRNA); a short hairpin RNA (shRNA); a micro RNA (miRNA); an aptamer, a ribozyme, an mRNA, or a plasmid. The nucleic acid polymer may be selected from one or more of the nucleic acid polymers listed in TABLE 1.

[0134] As used herein a diagnostic agent is meant to include a substance used to examine the body in order to detect impairment of its normal functions. Diagnostic agents as described herein may include radiopharmaceuticals and contrast agents for use in imaging techniques (including x-rays, magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), computed tomography (CT) (including Volumetric Quantitative CT (vQCT); High-Resolution CT (hrCT); and Micro-CT (pCT)), positron emission tomography (PET), single-photon emission computed tomography (SPECT), thermography, electrical source imaging (ESI), digital mammography, tactile imaging, magnetic source imaging (MSI), medical optical imaging, ultrasonic and electrical impedance tomography (EIT), diagnostic sonography, PET / CT hybrid, three-dimensional ultrasound computed tomography (3D USCT), and simultaneous PET / MRI)).

[0135] TABLE 2: Informal Sequence Listing

[0136] ( Signal Sequence ) + = LNA modi fications and * = phosphorothioate modi fications

[0137] - C in sequences 3 and 4 represent positions 133 and 146 , respectively

[0138] As used herein, the term “messenger RNA” or “mRNA”, refers to a polynucleotide that encodes and expresses the secretory protein.

[0139] The nucleic acid polymers as used herein encompasses both modified and unmodified. In one embodiment, the nucleic acid polymers comprise one or more coding and non-coding regions. The nucleic acid polymers can be purified from natural sources, produced using recombinant expression systems and optionally purified, or may be chemically synthesized. Modifications to the nucleic acid polymers can improve immunogenicity, stability, and translational efficiency and fidelity of the nucleic acid polymers and thus improve desired activity.

[0140] The nucleic acid polymers may include nucleotide analogues. The nucleic acid polymers may include a modified internucleoside linkage. The modified internucleoside linkage may be a peptide-nucleic acid linkage, a morpholino linkage, a N3’ to P5’ phosphoramidate linkage, a methylphosphonate linkage or a phosphorothioate linkage. The nucleic acid polymers may have one or more modified sugar moieties. The modified sugar moiety may be 2 ' -O-alkyl oligoribonucleotide. The nucleic acid polymers may be a gapmer. The nucleic acid polymers may have a 2'MOE gapmer modification. The nucleic acid polymers may have a modified nucleobase. The modified nucleobase may be a 5-methyl pyrimidine or a 5- propynyl pyrimidine. The one or more nucleotide analogues may include a locked nucleic acid (LNA). The LNA units include beta- D- oxy -LNA monomers. The nucleic acid analogues may be selected from one or more of: locked nucleic acids (LNA); MOE, 2,-O-(2-methoxyethyl); BNA, 2’,4’-bridged nucleic acid; N-Me- aminooxy BNA, 2’-N-(methyl)-4’-C-aminooxymethylene 2’,4’-bridged nucleic acid; N-Me- aminooxy 2’,4’-bridged nucleic acid, 2’-N-(methyl)-4’-C-aminooxymethylene 2’,4’-bridged nucleic acid; 2’,4’-BNANC[NMe], 2’-O,4’-C-(N-methyl) aminomethylene 2’,4’-bridged nucleic acid; 2’-O,4’-C-aminomethylene 2’,4’-bridged nucleic acid (2’,4’-BNA); and 2’,4’-BNANC. AS used herein and non-nucleotide analogue nucleic acid may be a standard nucleic acid, but the nucleic acid polymers may still have a linkage is modified or not modified.

[0141] The nucleic acid polymers may further include a modified internucleoside linkage. The modified intemucleoside linkage may be a peptide-nucleic acid linkage, a morpholino linkage, a N3’ to P5’ phosphoramidate linkage, a methylphosphonate linkage or a phosphorothioate linkage. The nucleic acid polymers may further include a modified sugar moiety. The modified sugar moiety may be a 2 ' -O-alkyl oligoribonucleotide. The nucleic acid polymers may further have a 2'MOE gapmer modification. The ASO may further have a 2'0Me gapmer modification. The nucleic acid polymers may further include a modified nucleobase. The modified nucleobase may be a 5-methyl pyrimidine or a 5- propynyl pyrimidine.

[0142] In those embodiments in which a nucleic acid polymer is chemically synthesized, the nucleic acid polymer can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and / or backbone modifications. In some embodiments, a nucleic acid polymer is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl- cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5- iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, 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-phosphoramidite linkages).

[0143] The mRNAs of the disclosure may be synthesized according to any of a variety of known methods. For example, mRNAs in certain embodiments may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. While mRNA provided from in vitro transcription reactions may be desirable in certain embodiments, other sources of mRNA are contemplated, such as mRNA produced from bacteria, fungi, plants, and / or animals.

[0144] In some embodiments, a nucleic acid polymer include a 5' and / or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect a nucleic acid polymer's stability or translation, for example, an iron responsive element. In some embodiments, a nucleic acid polymer may be between about 15 and 500 nucleotides in length or longer.

[0145] Routes of administration

[0146] In some embodiments, the therapeutic nanodisk or the composition described herein may be part of a pharmaceutical composition. The pharmaceutical composition may provide a prophylactic (preventative), ameliorative or a therapeutic benefit. The pharmaceutical composition may be administered at any suitable dosage. The type of administration used to introduce a therapeutic agent or a diagnostic agent includes any route that delivers the therapeutic nanodisk or the composition to a central nervous system (CNS) site or systemically. Alternatively, the delivery may be to the extracellular space comprises the interstitial and / or cerebrospinal fluid cerebrospinal fluid. The site includes the brain and / or spinal cord. The administration methods include but are not limited to intravenous injection (IV), intranasal inhalation, intracerebroventricular (ICV) injection, lumbar intrathecal (IT) injection, intrathecal (IT) infusion, cistema magna injection, and the use of a catheter (e.g., intrathecal catheter) to introduce the therapeutic nanodisk or the composition to the brain or spinal cord. The therapeutic nanodisks or the compositions as described herein may be administered to a subject. As used herein, a “subject” may be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected of having or at risk for having a central nervous system disease, disorder, trauma or injury, are known to those of ordinary skill in the art. Some examples are listed in TABLE 1.

[0147] Methods & Materials

[0148] ApoA-I expression and purification

[0149] ApoA-I wt, K133C, and E146C plasmids were a gift from Michael Oda were optimized for bacterial expression. Plasmid expression and purification were performed as described by Ryan et al.

[0048] and Oda et al.

[0049] , In summary, BL21(DE3)pLysS cells transformed with pNFXex- apoA-I WT and K133C plasmids (Martin et al.

[0039] ) were cultured in NCZYM™ media (Sigma™, catalog # N3643) containing 50 pg / ml ampicillin at 37°C. Expression was induced by addition of isopropyl thiogalactoside (IPTG™) to a final concentration of 0.5 mM when the culture reached an OD600 of 0.6. After 3 hours of incubation, the bacteria were centrifuged at 10,000 g for 15 min and lysed in B-PER (Thermo Fisher™ #78248) with addition of lysozyme (final concentration of 1 mg / ml) and DNase I (1 pl per 10 ml) followed by sonication on ice and then centrifugation at 20,000 g for 30 min at 4°C. The supernatant fraction was mixed with an equal volume of 2x column loading buffer (40 mM NaJLPC , 1 M NaCl, 6 M guanidine HC1, pH 7.4) and frozen at - 20°C for 1-3 days before purification.

[0150] For purification, the lysate was then thawed and centrifuged at 5,000 g for 10 min at 4°C, passed through a 0.22 pm filter, and then added to a Ni-NTA agarose pre-prepared column and flow- through was passed through the column 2 additional times. Column was washed with 25mL lx column loading buffer, then washed with wash buffer (20 mM NaH2PC>4, 0.5 M NaCl, 20mM imidazole, 0.05% Tween-20, pH 7.4) until A280 reached 0. Finally, column was eluted with 25 mL elution buffer (20 mM NaH2PC>4, 0.5 M NaCl and 0.5 M imidazole, pH 7.4) in 1 mL fractions. Protein was detected by A280 (corrected for imidazole) and sample was dialyzed in PBS with 1 mM benzamidine and 1 mM EDTA at 4°C. Alternatively, fractions containing protein were pooled, dialyzed in PBS at 4°C for 24 h, filter sterilized using a 0.2 pm syringe filter (Pall™, catalog # 4612) and stored at -80°C until use.

[0151] Nanodisk preparation

[0152] For nanodisk preparation, apoA-I protein (i.e. wt; K133C; and E146C) was thawed slowly on ice to reduce precipitation, then filter sterilized using a 0.2 pm filter and concentrations were measured using a DC protein absorbance assay (Bio-Rad™, catalog # 5000113 and 5000114). For in vitro applications, apoA-I was diluted to the desired concentration in Dulbecco’s phosphate buffered saline (PBS; Sigma-Aldrich™, catalog # D8537). For in vivo applications, apoA-I protein was concentrated using centrifugal filter units with a 10 kDa nominal molecular weight cutoff (MWCO. Millipore™, catalog # MRCPRT010), filter sterilized using a 0.2 pm syringe filter and adjusted to the desired concentrations with PBS. l,2-Dimyristoyl-sn-glycero-3- phosphocholine (DMPC) suspended in chloroform at 25 mg / mL (Avanti Polar Lipids™, catalog # 850345C or NC0845353) was brought to room temperature before use and transferred to a v-bottom glass vial and dried under an argon stream then placed at 37°C for 2 min to equilibrate temperature in a biosafety cabinet. Dried lipids were then resuspended with the apoA-I solution at a molar ratio of 105: 1, gently triturated for 1 min and then incubated to spontaneously form nanodisks (NDs) at 22°C for 16 h.

[0153] For ASO-ND preparation, nanodisks were first prepared as described above. The next day, ASO was thawed slowly on ice and then the desired volume was mixed with the nanodisks. ASO nanodisk mixture was incubated at 37°C for 1 hour, and gently mixed before use. Antisense oligonucleotide (ASO) targeting human HTT

[0154] In this study, we used an ASO gapmer designed to target intron 22 of the the human HTT transcript with a fully modified phosphorothioate backbone and locked nucleic acid (LNA) wing modifications (+T*+A*+A*+T*+A*C*G*T*A*A*G*T*G*T*+C*+A*+C*+A*+A; + = LNA modifications and * = phosphorothioate modifications - MW = 6416 g / mol), as previously described [22, 50, 98], The same HTT ASO was also synthesized with a reactive amine group modification attached to the 5' end nucleotide base with a 6-carbon linker (amino-HTT ASO, MW = 6596 g / mol) to allow for conjugation to apoA-I K133C. The ASO was commercially synthesized (Qiagen™) and HPLC-purified and resuspended in Dulbecco’s phosphate buffered saline (PBS; Sigma-Aldrich™, catalog #D8537). Sterile PBS was used to re-suspend the ASO followed by incubation at 37°C for 2 h. The following equation was used to calculate ASO concentrations: Concentration (M) = OD260 x dilution factor / s260 in which s260 = extinction coefficient of oligo at 260 nm. For ASO treatments, the ASO was diluted to the indicated concentration in sterile PBS or prepared in ApoA-I nanodisks and administered as described below.

[0155] HTT ASO conjugation to apoA-I KI 33C

[0156] For modification of apoA-I K133C, aliquoted protein was thawed on ice, quantified by DC assay, then diluted to 5 pg / pL in l x modification buffer (10x modification buffer: 1.0 M NasPO4, 1.5 M NaCl, pH 8.0). Zeba Spin™ desalting columns with a 7 kDa MWCO (Thermo Scientific™, catalog # 89892) were used to complete the buffer exchange of apoA-I K133C into 1 x modification buffer. Following buffer exchange, apoA-I K133C protein concentration was re-quantified by DC assay, and this value was used to calculate the quantity of succinimidyl-6-hydrazinonicotinamide (S-HyNic™. Vector Laboratories™, catalog # S- 1002-010) needed to modify the protein with a molar substitution ratio (MSR) of 3, according to the manufacturer’s protocol. For each reaction, fresh S-HyNic™ was resuspended in the calculated volume of anhydrous N, N- dimethylformamide (DMF. Sigma- Aldrich™, catalog # 227056- 100ML), added to the desalted protein, vortexed and incubated for 3 h at room temperature. After incubation, HyNic-modified apoA-I K133C protein was desalted using Zeba Spin desalting columns into 1 x conjugation buffer (10x conjugation buffer: 1.0 M NasPCL, 1.5 M NaCl, pH 6.0). Modified protein was then stored frozen at -80°C until further use.

[0157] For modification of amino-HTT ASO, the molecule was resuspended in nuclease-free dH2O, then diluted to 0.5 OD / pL by adding 10x modification buffer to a final concentration of l x. Succinimidyl-4- formylbenzamide (s-4FB. Vector Laboratories™, catalog # S- 1004- 105) was resuspended fresh for each use in anhydrous DMF and modification of amino-HTT ASO was done for 3 h at room temperature according to the manufacturer’s protocol. Following incubation, the 4FB-modified HTT ASO (4FB-HTT ASO) was diluted 10-fold in l x conjugation buffer, and loaded in 500 pL aliquots onto Amicon Ultra™ 0.5 mL centrifugal filter unit columns with a 3 kDa MWCO (Amicon™, catalog # UFC500324) and concentrated for 20-30 min at 14,000 xg at 4°C. Following concentration, the 4FB-HTT ASO was washed 3x with l x conjugation buffer, centrifuged for 20-30 min at 14,000 xg at 4°C for each wash, and stored at -80°C until use.

[0158] For conjugation of HyNic-modified apoA-I K133C and 4FB-HTT ASO, reagents were thawed on ice, centrifuged briefly to remove precipitate, and quantified by DC assay and Nanodrop™, respectively. The MSR was measured for both modified apoA-I K133C and HTT ASO according to the manufacturer’s protocol. In a glass vial, calculated volumes of HyNic-apoA-I K133C and 4FB-HTT ASO were combined with l x TurboLink™ catalyst buffer (10x TurboLink™ catalyst buffer: 100 mM aniline, 100 mM Na3PO4, 150 mM NaCl, pH 6.0), and incubated at room temperature for 2 h, then desalted into PBS using Zeba Spin Desalting Columns™. The MSR of the HTT ASO + apoA-I K133C conjugate was quantified according to the manufacturer’ s protocol. Conjugation was confirmed by immunoblot and electrophoretic mobility shift assay (EMSA) as described below. NDs were generated with the HTT ASO + apoA-I K133C conjugate and DMPC at a molar ratio of 1 : 105 based on the concentration of apoA-I as described above.

[0159] Electrophoretic mobility shift assay (EMSA)

[0160] ApoA-I NDs were generated as described above using 37.37 nmol (25.33pg) DMPC and 355.87pmol (lOpg) apoA-I, then incubated at 37°C for 1 hour with 355.36 pmol (2.28 pg), 1.01 nmol (6.8pg) or 1.78nmol (11.4pg) ASO (MW = 6417.12 g / mol) corresponding to stoichiometries ranging from 1 : 1 to 1 :5 apoA-I: ASO. ND formulations were run on a 2% agarose gel at 100V for ~45 minutes and the gel was stained with OliGreen™ (Invitrogen™ #07582) to visualize ASO. For additional validation of the HTT ASO + apoA-I K133C conjugate, 5 pg (0.78 nmol) of HTT ASO and the molar equivalent of HTT ASO + apoA-I K133C conjugate were resolved on a 2% agarose gel containing SyberSafe™ at 130 V for 30 min. The gel was imaged using a GelDoc system (BioRad™). For comparing apoA-I ND migration in plasma, samples from animals injected with either PBS, apoA-I WT NDs or apoA-I K133C NDs were diluted 1 :25 in a buffer containing 50 mM Tris pH 8, 150 mM NaCl, 1% IGEPAL™ and protease inhibitors (Roche™; catalog # 11836145001). Samples were then diluted with 2* Native Tris-Glycine Sample Buffer (Invitrogen™, LC2673) and run in duplicate on 4-20% Tris- Glycine gels (Invitrogen, XP04200BOX) in Tris-Glycine Native Running Buffer (Invitrogen™, LC2672) at 130 V for 2 h. NativeMark™ protein standard (Invitrogen™, LC0725) was used as a size standard. The gel was then cut in half to visualize total protein and apoA-I. One half was stained with Coomassie R- 250™ (Bio-Rad™, catalog # 161-0400) according to the manufacturer’s protocol. The other half was transferred onto 0.2 pm nitrocellulose in NuPAGE™ transfer buffer (Invitrogen™, catalog # NP00061) at 18 V for 2 h and probed for apoA-I as described above. Both the gel and blot were imaged using a LI-COR Odyssey Imaging System™.

[0161] Size exclusion chromatography assay

[0162] Sephadex™ G-50 Medium beads (Pharmacia™, 17-0043-01) were re-hydrated according to manufacturer’ s instructions, loaded into a 15 ml column, and washed 3 times with PBS. ASO ND formulations were generated using 37.37 nmol (25.33pg) DMPC, 355.87 pmol (10pg) apoA- I, and 1.01 nmol (6.8 pg) ASO in a 200pL total volume, and were slowly added to the top of the bead bed and allowed to settle for 2 minutes before 3mL of PBS was added. Approximately 60- 65 50pL fractions were collected in a 96-well plate for measurement of DNA, protein and lipid concentrations in each fraction. DNA concentrations were measured using a NanoDrop™ spectrophotometer (ssDNA), protein concentrations were measured using a DC protein absorbance assay and lipid concentrations were measured using a phospholipid detection assay (Sigma™, catalog # MAK 122).

[0163] Oligreen ASO association assay

[0164] ApoA-I NDs were prepared as described above and incubated with ASO (37°C for 1 hour) then dialyzed to remove free, unbound ASO. Dialyzed formulations were then treated with Triton X- 100™ (final concentration 1%) and incubated for 15 min at 37°C. Oligreen™ working stock was prepared by diluting Oligreen™ reagent in IxTE buffer (pH 7.5) and kept in the dark until use. Triton X-100™ treated NDs were then diluted to 1 :20 in IxTE buffer. 100 pl of each sample and standard were pipetted onto a 96 well-plate and 100 pl of Oligreen™ working stock was added to each well and incubated for 3 min at room temperature in the dark. Absorbance was measured at 520 nm. Optical density (OD) measurements

[0165] DMPC alone formulations were generated using 25.3 pg (37.4 nmol) DMPC resuspended in 10 pL PBS and incubated at 22°C for 16 h. ApoA-I NDs were generated as described above using 25.3 pg (37.4 nmol) DMPC and 10 pg (356 pmol) apoA-I in 10 pL PBS total volume. OD measurements were done in triplicate at 590 nm using a NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific™).

[0166] Dynamic light scattering analysis (DLS)

[0167] ApoA-I NDs were generated as described above using 25.3 pg (37.4 nmol) DMPC and 10 pg (356 pmol) apoA-I in 100 pL PBS total volume. Samples were diluted 5* in PBS immediately prior to loading into cuvettes and DLS size measurements were performed using the Malvern Zetasizer™ (Malvern Panalytical™) according to the manufacturer instructions. Mean particle size and poly dispersity index (PDI) from 5 readings on each sample or the 1stpeak are shown.

[0168] Native polyacrylamide gel electrophoresis (PAGE)

[0169] Nanodiscs were prepared as described earlier and mixed with 15% glycerol at the desired concentrations before loading into Novex Tris-Glycine 4-20% Wedge We ll™ gels. Gels were run at 120V for 120 min at RT with IX Tris-Glycine native running buffer. Gel was removed and incubated in the dark for 60 min in Syber Safe™ (1 :4000 in IxTBE), then rinsed 3 times with IxTBE and scanned using Bio-Rad™ XR+ imager. Gel was then rinsed 3 times with IxPBS and stained with Coomassie blue ((0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid) for 30 min at RT before rinsing 3 times in IxTBE and scanning the gel using Odyssey Infrared Imaging™ system (LI-COR Biosciences™).

[0170] Alternatively, ND formulations were generated as described above using molar ratios of DMPC and apoA-I calculated from published molecular weights (MW; DMPC = 677.93 g / mol, apoA-I = 28,100 g / mol). For DMPC: apoA-I stoichiometry studies, the molar ratio of DMPC used for each formulation was calculated based on 10 pg (356 pmol) apoA-I. For comparing electrophoretic mobility of apoA-I WT and K133C NDs, 25.3 pg (37.4 nmol) DMPC and 10 pg (356 pmol) apoA- I (corresponding to 105: 1 DMPC: apoA-I molar ratio) in 10 pL total volume were used. Samples were diluted with 2* Native Tris-Glycine Sample Buffer (Invitrogen™, LC2673), loaded on 4- 20% Tris-Glycine gels (Invitrogen™, XP04200BOX) and run in Tris-Glycine Native Running Buffer (Invitrogen™, LC2672) at 130 V for 2 h. High MW ladder (GE™, GE17-0445-01) was loaded on the same gels and used as a size standard. Gels were then stained with Coomassie R- 250 (Bio-Rad™, catalog # 161-0400) according to the manufacturer’s protocol and imaged using a LI-COR Odyssey Imaging System™ (LI-COR Biosciences™).

[0171] Transmission electron microscopy (TEM)

[0172] Negative-staining TEM of apoA-I ND formulations was performed as previously described

[0095] , Briefly, apoA-I NDs were generated as described above using 38 pg (56 nmol) DMPC and 15 pg (534 pmol) apoA-I in 200 pL PBS total volume. 10 pL of each formulation was added onto a carbon-coated grid and after 20 seconds excess liquid was wicked away. A small droplet of 2% potassium phosphotungstate (pH 6.5) was then added to the grid and incubated for 10 seconds. Excess stain was then removed and grid was allowed to air dry. Samples were imaged using the Hitachi H7600™ TEM (Hitachi™) at 80 kV, with 500, 000-600, 000* direct magnification, and acquired with the XR51 camera with exposure = 800 msec, gain = 1, and bin =1. The diameter of apoA-I NDs were analyzed using Fiji™ (Image!™) and sizes in nm are presented.

[0173] Brain microvascular endothelial-like cell (BMEC) differentiation

[0174] GM03621 fibroblast cells were obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research. Fibroblasts were reprogrammed to induced pluripotent stem cells (iPSCs) using an episomal, non-integrating reprogramming protocol using OCT4, SOX2, KLF4, L-MYC, LIN28 and p53 shRNA described in

[0096] , pCXLE-hOCT3 / 4-shp53 (Addgene™ plasmid # 27077), pCXLE-hSK (Addgene™ plasmid # 27078), and pCXLE-hUL (Addgene™ plasmid # 27080) were gifts from Shinya Yamanaka

[0096] , Markers of pluripotency were validated using qPCR and immunofluorescence, and normal karyotype was confirmed. BMECs were differentiated from GM03621 iPSCs as described

[0097] with the following modifications. GM03621 iPSCs were grown on Matrigel™ (Corning™, catalog # 354277) coated 6-well plates with daily mTESRl (StemCell™, catalog # 85850) media changes until cells reached 50% confluency. Cells were then switched into DMEM / F12 media containing 20% Knockout Serum Replacement™ (Gibco™, catalog # 10828010), 1% MEM non-essential amino acids (Gibco™, catalog # 11140050), 1% GlutaMax™ (Gibco™, catalog # 35050061), 1% Pen / Strep (Gibco™, catalog # 15140122) and P -mercaptoethanol for 5 days with daily media changes. On day 6 of differentiation, cells were switched into complete endothelial serum-free media (EM. Gibco™, catalog # 11111044) containing 1% platelet poor serum (Sigma-Aldrich™, catalog # P2918) and human basic FGF (Prospec™, catalog # CYT-218) for 2 days with daily media changes.

[0175] Immunofluorescence (IF)

[0176] BMECs were seeded on coverslips coated with 400 pg / mL collagen IV (Sigma- Aldrich™, catalog # C5533) and 100 pg / mL fibronectin (Sigma-Aldrich™, catalog # F4759) at a density of 25,000 cells / cm2in complete EM, and media was changed daily until confluency was reached. Culture media was then removed, cells were washed 2* with PBS, then fixed with 4% paraformaldehyde in PBS for 12 min at room temperature. Cells were washed 2* with PBS, then permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature. BMECs were then washed 2* with PBS, blocked with blocking buffer containing 5% FBS (Gibco™, catalog # 12483-020) + 0.1% Triton X-100 in PBS for 2 h and probed with primary antibody combinations of either rabbit anti- PECAM-1 (1 :50. Developmental Studies Hybridoma Bank™, catalog # P2B1) and mouse anticlaudin-5 (1 :500. Invitrogen™, catalog # 35-2500) or rabbit anti-occludin (1 :250. Invitrogen™, catalog # 71-1500) and mouse anti-GLUT-1 (1 : 1000. Abeam™, catalog # ab40084) in antibody dilution buffer containing 1% FBS + 0.1% Triton X-100 in PBS for 2 h at room temperature. Cells were washed 3* with blocking buffer and primary antibodies were detected using secondary antibody combinations of goat anti-rabbit Alexa Fluor 488™ (1 :500. Invitrogen™, catalog # A32731) and goat anti-mouse Alexa Fluor 594™ (1 :500. Invitrogen™, catalog # A-11005) for 1 hour at room temperature in antibody dilution buffer. Coverslips were then washed 3* with blocking buffer and mounted on slides using ProLong Gold Antifade™ with DAPI (Thermo Fisher Scientific™, catalog # P36931).

[0177] Transmigration experiments

[0178] BMECs were seeded into 1.12 cm2 Transwell™ permeable inserts with a 0.4 pm pore size (Coming™, catalog #3401) coated with 400 pg / mL collagen IV and 100 pg / mL fibronectin at a minimum density of 25,000 cells / cm2in complete EM, and media was changed daily until confluency was reached. Prior to each trans-migration experiment, the trans-endothelial electrical resistance (TEER) for each Transwell™ was measured in triplicate using an epithelial volt / Q meter (World Precision Instruments™, catalog # EV0M2). ApoA-I ND formulations composed of 38 pg (56 nmol) DMPC and 15 pg (534 pmol) apoA-I in 100 pL PBS were generated as described above. An equivalent amount (534 pmol) of lipid-free apoA-I WT and apoA-I K133C were prepared in 100 pL PBS. Complete EM media from the insert (apical chamber) and basolateral chamber were replaced with transport buffer composed of Hank’s Balanced Salt Solution™ (Gibco™, catalog # 14025092) with 10 mM HEPES and 1% FBS. At time = 0 min, 100 pL of lipid-free apoA-I or apoA-I NDs were added to the insert and 200 pL aliquots were taken at 15 min intervals and replaced with fresh transport buffer to maintain a constant volume in the basolateral chamber. ApoA-I concentrations we measured using a human apoA-I ELISA as described below.

[0179] Animal treatments

[0180] Studies were carried out in BACHD mice

[0051] in strict compliance with protocols approved by the University of British Columbia Animal Care Committee (A16-0130, A20-0107).

[0181] Approximately equal numbers of male and female mice were used for all experiments. Animals were maintained under a 12 h light: 12 h dark cycle in a clean barrier facility and given ad lib access to food and water. For intranasal treatments, mice were anesthetized with ketamine and xylazine (100 / 10 mg / kg, i.p.) and then placed in a supine position with their noses upright and heads flat on the surface. A 10 pl Hamilton™ syringe with a blunted 30 gauge needle used to administer treatments in 2.5 pl increments per nare every 2 min alternating sides until the total volume (60- 90 pl) was given.

[0182] Mice remained supine for 30 min after intranasal administration.

[0183] For intravenous treatments, the animals received the same doses / volumes of treatments via tail vein injection.

[0184] Animal collection and tissue processing

[0185] Animals allocated for biochemical analyses were terminally anesthetized using 2,2,2-Tribro- moethanol (Avertin™, Sigma-Aldrich™, catalog # T48402). For selected experiments, terminal CSF was collected from the cisterna magna and whole blood was then collected via cardiac puncture for isolation of plasma as previously described

[0098] , Alternatively, CSF was collected as a previously described [22, 50], In summary, a 50 pL Hamilton™ syringe with 12° bevel was inserted into the cistema magna using an adapted stereotaxic frame and an UltraMicroPump™ with Micro4™ controller was used to withdraw CSF.

[0186] For apoA-I ELISA studies only, animals were perfused with PBS for 4 min prior to collection to flush out any loosely-associated apoA-I. Brains were then either harvested whole or micro- dissected into separate brain regions and peripheral tissues were also collected. Samples were snap frozen in liquid N2 and stored at -80°C until use.

[0187] Animals allocated for ex vivo imaging, histology and immunohisto-chemistry (IHC) were terminally anesthetized using 2,2,2-Tribromoe-thanol and perfused transcardially with ice cold PBS for 4 min followed by 4% paraformaldehyde (PF A) in PBS for 4 min. Brains were removed and post-fixed in 4% PFA in PBS for 24 h at 4°C then either transferred into PBS for ex vivo imaging, 70% ethanol for histology, or 30% sucrose containing 0.01% NaNs until equilibrated for IHC.

[0188] Longitudinal plasma collection was conducted on mice by bleeding from the saphenous vein using EDTA-coated microvette tubes (Sarstedt™, catalog # 16.444.100) and kept on ice. Blood was then centrifuged at 4000 *g at 4°C for 10 min, plasma was isolated and transferred to a clean micro-centrifuge tube and snap frozen in liquid N2.

[0189] Plasma from animals that received apoA-I NDs was collected longitudinally from the saphenous vein as described above. Samples were diluted 2-fold with PBS and sent for analysis using the Mouse Cytokine 44-Plex Discovery Assay™ (Eve Technologies Corporation™). Sample values that fell below the limit of detection were designated at 0 pg / mL. Levels of LIF fell below the limit of detection for all samples and were not reported. Statistical outliers identified using the ROUT method with a cutoff Q value set to 1% were excluded from analysis.

[0190] ApoA-I concentrations in plasma were measured by ELISA as described above and plasma PK properties were modeled using the Phoenix WinNonlin™ software (Certara™).

[0191] IV tail vein injections

[0192] Animals were weighed then restrained in a mouse tail illuminator (Bioseb™, catalog # MTI). The tail was warmed for 2-3 min on the illuminator to highlight the tail vein, sterilized with 70% ethanol, and an insulin syringe with a 31 gauge needle (BD™, catalog # 320440) was used to deliver formulations through the tail vein as a bolus injection with a volume up to 10 mL / kg.

[0193] For biodistribution and tolerability studies, dosing of apoA-I NDs was calculated based on mg apoA-I / kg animal body weight and were delivered at doses ranging from 5-60 mg / kg (178-2136 nmol / kg). For mHTT target engagement experiments, dosing of HTT ASO or the molar equivalent of HTT ASO as HTT ASO NDs was calculated based on mg ASO / kg animal body weight and delivered at a range of 1.67-10 mg / kg (260-1559 nmol / kg). ICV injections

[0194] Animals received unilateral ICV bolus injections as previously described [71,72], For mHTT target engagement studies, 5 pg (0.78 nmol) or 15 pg (2.34 nmol) of HTT ASO or the molar equivalent of HTT ASO as HTT ASO NDs was delivered by ICV injection.

[0195] Tissue preparation

[0196] For immunohistochemistry, the animals were sacrificed by transcardial perfusion under 2,2,2- Tribromoethanol (Avertin™) anesthesia with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PF A) in PBS. Brains were removed and post-fixed in 4% PFA for 24 hr and then placed in 30% sucrose for 2-4 days. The brains were then cryo-sectioned at 25 pm and sections were stored at 4°C in PBS with 0.01% NaN3.

[0197] For biochemical analysis, mice were anesthetized using 2,2,2-Tribromoethanol (Avertin) and CSF was collected. Blood was then collected via cardiac puncture for plasma isolation. Brains were removed and placed on ice for ~1 min to increase tissue rigidity. A mouse brain matrix was used to divide the brains into left and right hemispheres, and the right hemispheres were then microdissected into different regions (ex. striatum, cortex, hippocampus, cerebellum), while the left hemispheres were dissected into 4 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.

[0198] For ELISA and immunoblotting, samples were homogenized in SDP lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1% IGEPAL, 40 mM [3- glycerophosphate, and 10 mM NaF) with protease inhibitors (IX cOmplete protease inhibitor cocktail, 1 mM phenylmethanesulfonyl fluroride in isopropanol, 1 mM sodium orthovanadate, and 5 pM Z-VAD-FMK) and 0.1% sodium dodecyl sulfate (SDS) as previously described

[0052] ,

[0199] HD lymphoblast culture and treatments

[0200] GM04724 lymphoblast cells were obtained from the NIGMS Human Genetic Cell Repository at the Cori ell Institute for Medical Research™. Lymphoblasts were grown in RPMI-1640 media (Gibco™, catalog # 11875093) supplemented with 10% FBS. For all treatment experiments, GM04724 lymphoblasts were triturated to a single cell suspension, counted using a hemocytometer, and 5 x 105 cells in 1 mL RPMI-1640 were seeded per well of a 12-well plate. For activity studies, the HTT ASO, amino-HTT ASO, 4FB- HTT ASO and HTT ASO NDs were diluted to 3 pM in pre-warmed RPMI-1640 media. Treatments were then added to wells containing lymphoblasts to a final concentration of 1 pM, cells were triturated to a single cell suspension and incubated for 120 h prior to collection

[0201] Histology

[0202] For evaluation of tolerability, fixed tissues were rinsed 3* with PBS before being placed in 70% ethanol overnight. Tissues were then processed, embedded in paraffin, sectioned (5 pm), and stained with Hematoxylin and Eosin (H&E) (BC Children’s Hospital Histology Core Laboratory). H&E slides were tile-scanned using the Olympus BX61™ microscope (Olympus Scientific Solutions™) using a 10x air objective.

[0203] Immunohisto-chemistry (IHC)

[0204] For evaluation of cellular tropism, brains equilibrated in 30% sucrose were frozen on dry ice, mounted in Tissue-TEK O.C.T. embed-ding compound (Sakura™, catalog # 4583), and cut via cryostat (Leica™ CM3050S) into 25 pm coronal sections free-floating in PBS + 0.01% NaNs. A series of sections spaced 200 pm apart and spanning the striatum were co-stained with the rabbit anti-apoA-I antibody (1 : 1000, Abeam™, catalog # ab52945) and either mouse anti-NeuN (1 :1000. Sigma-Aldrich™, catalog # MAB377) or tomato lectin DyLight 649™ (1 : 1000. Vector Laboratories™, catalog # DL-1178-1). Primary antibodies were detected with goat anti-rabbit AlexaFluor-488™ (1 :500, Invitrogen™, catalog # A-11008) and goat anti-mouse AlexaFluor- 568™ (1 :500, Invitrogen™, catalog # A-11004) secondary antibodies. Sections were then mounted using ProLong Gold Antifade™ with DAPI.

[0205] ApoA-I enzyme-linked immunosorbent assay (ELISA)

[0206] Human apoA-I quantification was carried out using Elisa Pro: Human ApoA-I kit (Mabtech™, 3710-1HP-10) according to manufacturer’s instructions. A final volume of 100 pl / well was maintained throughout assays. Pre-coated plates were washed 5 times in wash buffer before use and between all steps. All subsequent steps were carried out at room temperature with continuous shaking. Samples were diluted in Apo ELISA buffer, and samples, standards, and background control were added to wells. Plates were then incubated for 2 hours. Next, the detection antibody was added to each well and incubated for 1 hour. Streptavidin-HRP was then added to each well and plates were incubated for 1 hour. TMB substrate was added to the wells and incubated for 10- 15 min until full color development occurred, protected from direct light. The reaction was stopped by addition of Stop Solution to each well. Absorbance was measured at 450 nm and apoA-I concentration was normalized to total mg protein in samples. Whole brain averages were calculated by averaging the normalized ApoA-I levels across all 4 rostral-caudal brain slabs indicated in FIGURE 2e.

[0207] Alternatively, ApoA-I concentrations in culture media, tissue lysates and biofluids were quantified using the human apoA-I ELISA kit (Mabtech™, catalog # 3710-1HP-2) according to the manufacturer’s protocol. Absorbance measurements were taken at 450 nm and 600 nm using a POLARstar Omega™ plate reader (BMG LabTech™). Dilutions for each sample type were tested to ensure measurements fell in the linear range of the assay. A standard curve generated using a purified human recombinant apoA-I standard (provided with the ELISA kit) at concentrations ranging from 0.6 to 40 ng / mL was included on every experimental plate to calculate absolute apoA-I concentrations (not shown). A comparison of detection using equal concentrations of purified recombinant apoA-I WT and apoA-I K133C was performed prior to running experimental samples. Detection of both apoA-I proteins was approximately equal with absorbance values falling in the linear range of the assay (not shown). Tissues and biofluids from PBS treated mice were included on every experimental plate, and mean absorbance values for the same tissue / biofluid were subtracted from experimental sample values (with the exception of not shown) to provide apoA-I concentrations above background. ApoA-I concentrations in culture media (ng / mL), tissues (ng of apoA-I / mg of lysate) or biofluids (ng of apoA-I / pL of biofluid) are presented.

[0208] Fluorescent dye labeling of apoA-I

[0209] Labelling of apoA-I WT and apoA-I K133C proteins with the near infrared CF750 fluorescent dye (Sigma-Aldrich™, catalog # SCJ4600059) for ex vivo imaging studies was performed according to the manufacturer’s protocol under the following conditions. Briefly, apoA-I WT or apoA-I K133C proteins were diluted to 5 mg / mL (356 nmol / mL) in PBS and 0.66 mL of each protein (3.3 mg) was adjusted to pH 8.3 in a final concentration of 0.1 M NaHCCh for labeling. For each apoA- I protein, 1 pmol CF750 succinimidyl ester was dissolved in 100 pL dH20 to a concentration of 10 mM and dye was added in a dropwise fashion to the apoA-I solution. Labelling reactions were incubated for 1 h at room temperature under constant agitation. Excess dye was then removed using centrifugal filter units with a 10 kDa MWCO (Amicon™, catalog # UFC5010). Ex vivo imaging

[0210] Two month old BACHD mice allocated for ex vivo imaging received 250 pg intravenous injections of CF750-labelled apoA-I NDs and animals were perfused 2 h post-injection as described above. Fixed whole or coronally sectioned brains were imaged using the AMI HTX Imaging System™ (Spectral Instruments Imaging™) using the 745 / 790 nm excitation / emission filters, power = 10%, exposure = 2 s, and bin = 1. Mean fluorescence intensity values (photons / s / cm2 / sr) were determined using the aura imaging software (Version 2.0.1, Spectral Instruments Imaging™) by drawing individual regions of interest for each brain and intensity values from PBS injected brains were subtracted to provide fluorescence signal above background. A fire lookup table (LUT) was applied using ImageJ™ to highlight fluorescence intensity.

[0211] Immunoblotting

[0212] For validation of the HTT ASO + apoA-I K133C conjugate, 4* NuPAGE LDS Sample Buffer™ (Invitrogen™, catalog # NP0007) and 50 mM dithiothreitol (DTT) were added to 10 pg (356 pmol) of apoA-I K133C and the molar equivalent of ASO + apoA-I K133C conjugate, and samples were denatured for 10 min at 70°C. Samples were run in duplicate on pre-cast NuPAGE™ 12%, bisTris gels (Invitrogen™, catalog # NP0341) in NuPAGE™ MOPS SDS running buffer (Invitrogen™, catalog # NP0001) at 175 V for 45-50 min. The gel was then cut in half for imaging of both ASO and apoA-I. One half was incubated in Quant-iT OliGreen™ ssDNA Reagent (1 : 10,000. Invitrogen™, catalog # 07582) for 30 min at room temperature and imaged using a GelDoc. The other half was transferred onto 0.2 pm nitrocellulose in NuPAGE™ transfer buffer (Invitrogen™, catalog # NP00061) at 18 V for 2 h. The membrane was blocked for 30 min in PBS with 0.05% Tween-20™ (PBST™) containing 5% skim milk at room temperature on a shaker, washed 3* with PBST and incubated with the rabbit anti-apoA-I anti-body (1 :1000, Abeam™, catalog # ab52945) in 5% BSA in PBST with 0.01% NaN3 overnight on a rocker at 4°C. After incubation, the membrane was washed 3* with PBST, incubated with the goat anti- rabbit IgG (H + L) Alexa Fluor 680™ (1 :5000. Invitrogen™, catalog # A- 21076) in 5% skim milk / PBST for 1 h at room temperature. Finally, membranes were washed for 4* PBST and imaged using a LI- COR Odyssey Imaging System™. Quantitative immunoblotting to measure levels of HTT in cell and tissue lysates was performed as previously described

[0052] , Double-label immunohistochemistry

[0213] Free-floating coronal sections of mouse brain (25 gm thick) were blocked in a solution containing either 10% normal goat serum (NGS), 10% normal donkey serum (NDS), or both in PBS for 1 hr. Sections were then incubated overnight at 4°C with primary antibodies to both ApoA-1 (rabbit anti-human ApoA-I; Abeam™ ab52945; 1 :250) and either GFAP (rat anti-GFAP, Invitrogen™ 13-0300; 1 : 1000), NeuN (mouse anti-NeuN; Millipore™ MAB377; 1 : 1000), ionized calcium- binding adapter molecule 1 (Ibal; goat anti-Ibal, Abeam™ AB107159; 1 : 1000), tomato lectin- conjugated Dylight 649™ (Vector Laboratories™, #DL-1178-1; 1 : 1000), or rabbit anti-ASO antibody (gift from lonis Therapeutics™; 1 :2000) in PBS containing 5% NDS, 5% NGS, or both. Sections were then incubated for 1 hr with the corresponding secondary antibodies diluted 1 :500 in PBS.

[0214] The secondary antibodies were goat anti-rabbit Alexa Fluor 488™ (Invitrogen™, Al 1008) or donkey anti-rabbit Alexa Fluor 488™ for detection of ApoA-I, goat anti-rat Alexa Fluor 568™ (Invitrogen™, Al 1077) for detection of GFAP, goat-anti mouse Alexa Fluor 633™ (Invitrogen™, A- 21052) for detection of NeuN, and donkey anti-goat Alexa Fluor 594™ (Invitrogen™, Al 1058) for detection of Ibal.

[0215] Sections were mounted on slides and cover-slipped using Fluoromount-G (Southern Biotech™).

[0216] Huntingtin low-bis western blots

[0217] Resolution of full length HTT alleles was carried out using allelic separation immunoblotting as previously described

[0046] , In summary, 50 pg of total protein was loaded on 10% bis:acrylamide (1 :200 bis:acrylamide) gels. Proteins were transferred to 0.45 pm nitrocellulose membranes and incubated with mouse 1CU-4C8 anti -HTT antibody (Fisher Scientific™, # MAB2166) and rabbit anti-calnexin antibody (Sigma™, # C4731). IR dye 800CW goat anti-mouse (Rockland™, # 610- 131-007) and Al exaFluor™ 680 goat anti-rabbit (Molecular Probes™, # A2107) secondary antibodies were used. The Odyssey Infrared Imaging™ system (LI-COR) was used to scan blots and Image Studio Lite™ v5.2 was used to perform densitometry to quantify band intensity. IOD values for HTT bands were normalized to the IOD for the loading control, calnexin, and then normalized to the same allele for PBS-treated mice on the same membrane.

[0218] Microscopy

[0219] Leica SP8 STED confocal microscope with a 40* objective was used to image stained sections. Image format was 2048 x 2048 pixels at a scan speed of 600 Hz. All images for each set of markers were taken with the same laser and gain settings to allow for comparison. A z-stack was imaged (257 nm) and Hygens Professional Deconvolution™ software was used to deconvolute images. HD BMECs and brain sections for tropism studies were imaged using the Leica TCS SP5™ laser scanning confocal inverted microscope (Leica Microsystems™) with 40 and 63 x oil immersion objectives, and the Leica Application Suite Advanced Fluorescence™ software.

[0220] Experimental design and statistical analyses

[0221] All statistical analyses were performed using GraphPad Prism™ (version 8 or 9 GraphPad™). Groupwise comparisons of normally distributed data with similar variance were analyzed using either the two-tailed Student’s t-test (two groups) or ANOVA (three or more groups). Post hoc analyses for ANOVA were performed using the Tukey or the Sidak tests to correct for multiple comparisons. Linear regressions were performed to assess the dose- response. Bar graphs are presented as scatter dot plots to demonstrate data complexity and variability. Group data from averages of multiple animals are represented as means ± standard error of the mean. Experiments involving animals included approximately the same number of males and females. The number of animals (biological replicates; N) used for each experiment is reported in the figure legends. Alpha values of <0.05 were considered significant for all analyses, p values for each statistical test are presented in the text and corresponding figure legends where * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

[0222] EXAMPLES

[0223] Example 1: ApoA-I NDs Form an Electrostatic Interaction with Antisense Oligonucleotides

[0224] We first sought to determine if apoA-I nanodisks were capable of binding an antisense oligonucleotide (ASO) designed to promote degradation of the human HTT transcript

[0050] . The secondary structure of apoA-I contains an amino-terminus globular domain spanning residues 1- 43 and 10 amphipathic a-helices encompassing residues 44-243 with helices 5, 6 and 7 containing polar faces with positively charged residues[53-55]. We hypothesized that the positively charged faces within the secondary structure of apoA-I could form an electrostatic interaction with the negatively charged ASO. To test this, we performed an electrophoretic mobility shift assay (EMSA) whereby a constant amount of ASO was incubated with decreasing moles of apoA-I NDs (stoichiometry of apoA-I to ASO from 1 : 1 to 1 :3) at 37°C for 1 hour. We visualized ASO at the level of the apoA-I NDs, suggesting a potential electrostatic interaction between the ASO and the apoA-I (FIGURE la)

[0225] We further validated the interaction between apoA-I NDs and the ASO using size exclusion chromatography (SEC). ApoA-I NDs were incubated with increasing moles of ASO at 37°C for 1 hour and formulations were applied to a column. We observed that at a molar ratio of 2 moles of ASO : 1 mole of apoA-I : 105 moles of DMPC, the ASO consistently eluted in the same fractions as both the apoA-I and DMPC, suggesting an interaction between the ASO and NDs (FIGURE lb). As a negative control, each individual component was through the column alone and fractions were collected for analysis. We observed that the ASO, apoA-I and DMPC eluted with different patterns. (FIGURE 1c).

[0226] To quantify the percent of ASO incorporated into apoA-I NDs, we optimized an assay using the OliGreen™ fluorescent dye, used for quantifying oligonucleotides and single stranded DNA in solution. Both ApoA-I WT and K133C NDs were incubated with ASO (37°C for 1 hour) then dialyzed to remove free, unbound ASO. Dialyzed formulations were then treated with a detergent, Triton X-100™, and incubated with OliGreen™ to quantify the amount of oligonucleotide associated with apoA-I NDs. We rationalized that if the oligonucleotides were located / associating along the inside of the apoA-I NDs then the OliGreen™ signal would be higher after Triton X- 100™ treatment compared to apoA-I NDs that were not treated with Triton X-100, due to disruption of the NDs by the detergent freeing ASO from the inside of the disks. Our results demonstrated that, in the absence of Triton X-100™, approximately 83-84% of the ASO was associated with both apoA-I WT and K133C NDs. Notably, treatment with Triton X-100 did not dramatically change the percent of ASO associated with either form of apoA-I NDs (FIGURE Id). Thus, these data suggest that the ASO may associate with apoA-I along the exterior of apoA- I NDs.

[0227] Using dynamic light scattering (DLS; FIGURE le), we found that apoA-I WT NDs were larger in size (mean diameter ± SD = 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 ± SD = 10.76 ± 0.05 nm, PDI = 0.0.067). Both apoA-I WT and K133C NDs fall well within the ideal size range for intranasal delivery to the brain and subsequent perivascular distribution.

[0228] To determine whether there might be a difference between apoA-I WT and K133C ND association with ASO, native PAGE was carried out and gels were stained with SYBR safe to visualize ASO followed by a Coomassie stain for total protein to visualize the NDs (not shown). Signals for both stains demonstrated a similar pattern at the level of the NDs confirming ASO association with the NDs. Signal intensity was quantified and was found to be similar for both apoA-I WT NDs and apoA-I K133C NDs (not shown).

[0229] Example 2: ApoA-I K133C NDs Exhibit Enhanced CNS Entry Following Intranasal Administration

[0230] Our first in vivo goal was to determine whether NDs formed with either apoA-I WT or apoA-I K133C could enter the CNS after intranasal administration. BACHD mice were treated with a 150 pg dose of either lipid-free apoA-I WT, lipid-free apoA-I K133C, apoA-I WT NDs, or apoA-I K133C NDs (in 30 pl PBS) intranasally. ApoA-I levels in the brain were quantified 2 hours after intranasal administration using an ELISA specific for human apoA-I, as previously described

[0046] , Levels of apoA-I were higher in the brain after intranasal administration of apoA- I WT NDs compared to lipid-free apoA-I WT, suggesting that the ND formulation may improve CNS entry. Whole brain levels of apoA-I were higher after intranasal delivery of lipid-free apoA-I K133C compared to lipid-free apoA-I WT (FIGURE 2a). One-way ANOVA p<0.001; Tukey’s multiple comparison ****p<0.0001), and for apoA-I K133C NDs compared to apoA-I WT NDs (****p<0.0001).

[0231] To assess CNS distribution of the mutant apoA-I K133C after intranasal administration, mice were sacrificed 2 hours after intranasal delivery and brains were micro-dissected into 4 rostral- caudal brain slabs (A-D; FIGURE 2e). ApoA-I K133C NDs achieved significantly higher CNSentry across the 3 most rostral brain regions A-C (FIGURE 2b). Two-way ANOVA indicates 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 NDs- A: ***p<0.001, B: ****p<0.0001, C **p<0.01, D: n.s.; WT NDs vs K133C NDs- A: ****p<0.0001, B: ****p<0.0001, C: *p<0.05). We also assessed gross brain distribution of apoA-1 labeled with cf75O dye after intranasal administration of apoA-I K133C NDs by carrying out ex vivo imaging of brains 2 hours post- intranasal administration. When examining the dorsal brain (FIGURE 2c), high concentrations were achieved in the whole brain, particularly in the olfactory bulbs, cortex, and cerebellum. On the ventral side (FIGURE 2d), high concentrations were also observed in the olfactory bulbs, the central layers of the cortex, around the circle of Willis and hypothalamus, the pons, and medulla oblongata. Example 3: HD Mutation Does Not Significantly Alter CNS Entry and Distribution of ApoA-I K133C NDs

[0232] Our next aim was to determine whether the HD mutation in BACHD mice was affecting the amount of apoA-I NDs delivered to the brain after intranasal administration, since the integrity of the blood-brain barrier has been shown to be affected in both HD mice[56, 57] and patients

[0058] . Separate groups of BACHD mice and their wild-type littermates at 2, 6, and 12 months of age received the same dose of apoA-I K133C NDs intranasally (150 pg of apoA-I in 30 pl PBS). Animals were sacrificed 2 hours after intranasal delivery and brains were dissected into 4 rostral- caudal slabs, A-D as indicated in FIGURE 2e. For both the 2 and 6 month groups, there was no difference in delivery efficacy of apoA-I across all 4 brain regions (FIGURE 3a and 3b, Two-way ANOVA indicates significance by brain region, p<0.0001, but no significance by genotype, p=0.4482 and p=0.8395 respectively, or interaction, p=0.7308 and p=0.9755 respectively). However, for the 12 month group, the A region (comprising of the olfactory bulb and frontal cortex) had significantly higher levels of apoA-I in the BACHD mice compared to their wildtype littermates. There were no significant differences between two groups across the other 3 brain regions. (FIGURE 3c, Two-way ANOVA indicates significance by brain region, p<0.0001, but no overall significance by genotype, p=0.3135, or interaction, p=0.0522; Bonferroni’s multiple comparisons test, *p<0.05 for region A). These results provide important evidence that the delivery of apoA-I to the brain after intranasal delivery of apoA-I K133C NDs is not a result of anatomical changes due to the HD mutation (at 2 and 6 months). For the remainder of our studies, we continued to use 2 month old BACHD mice to eliminate any potential variability with older mice.

[0233] Example 4: After Intranasal Delivery of ApoA-I K133C NDs, ApoA-I Localizes in Multiple Cell Types Across the Brain

[0234] To determine the cellular distribution of our NDs, we delivered apoA-I K133C NDs intranasally to 2-month-old BACHD mice and sacrificed the animals 2 hours later. Immunohistochemical staining for human apoA-I and different cellular markers was carried out on sagittal sections to determine the cell types in which apoA-I was localized at this time point. We found extensive colocalization of apoA-I with both CD31+ capillary endothelial cells, as well as NeuN+ neurons across the brain. Less frequently, apoA-I also colocalized with GFAP+ astrocytes and Ibal+ microglia (FIGURE 4). These localizations were not specific to any brain regions, but rather occurred across the whole sagittal axis of sections. Additionally, apoA-I colocalized extensively with lectin-stained choroid plexus cells.

[0235] Example 5: ApoA-I K133C NDs CNS Rapidly Enter the Brain and CSF Following Intranasal Administration

[0236] To determine the optimal dosing parameters of apoA-I K133C NDs, we carried out studies to evaluate the time-course of CNS distribution of apoA-I in the brain and CSF after intranasal delivery. ApoA-I K133C NDs were delivered intranasally to 5 groups of mice and harvested 1, 2, 3, 6, and 24 hours post-intranasal delivery (FIGURES 5a and 5b). The highest apoA-I levels were found across all 4 brain regions, A-D, at 2 hours post intranasal delivery. At the 1 hour time point, levels were highest in the A region (comprising of the olfactory bulb and frontal cortex), but decreased over time. Simultaneously, levels were lowest in the D region (comprised of the cerebellum and the brainstem) at the 1 hour time point but continued to rise over time (FIGURE 5b, Two-way ANOVA indicates significance by time point p<0.0001, brain region p<0.0001, and a significant interaction p<0.0001; Tukey’s multiple comparison test **p<0.01, ***p<0.001, ****p<0.0001). Furthermore, apoA-I levels were measurable (above background) across all 4 brain regions 24 hours after intranasal delivery. Whole brain averages demonstrate the same trend, with levels of apoA-I peaking at 2 hours post-intranasal delivery and falling to about 17% of peak levels at 24 hours (FIGURE 5a, One-way ANOVA indicates significance by time point, p<0.0001; Tukey’s multiple comparison test compared to 1 hr ****p<0.0001).

[0237] Example 6: ApoA-I K133C NDs Undergo Dose-dependent Increase in Brain Following Intranasal Administration

[0238] We next sought to determine the relationship between dose of apoA-I K133C NDs administered intranasally and the levels of apoA-I detected in the brain. To answer this question, groups of mice received ascending doses of apoA-I K133C NDs (75, 150, 300, or 500 pg apoA-I). Since apoA-I levels were found to peak in the brain 2 hours post-intranasal administration, animals for this study were harvested 2 hours after intranasal administration of apoA-I K133C NDs and tissues were analyzed by ELISA for human apoA-I (FIGURES 5c and 5d). When comparing whole brain averages of apoA-I levels for each of the treatment groups, there was a linear trend between the doses of 150-500 pg of apoA-I K133C NDs in which the average apoA-I levels in whole brain doubled with doubling dose (r2= 0.9843). The 500 pg dose was significantly higher than both the 75 pg and 150 jug doses (FIGURE 5c, One-way ANOVA indicates significance by dose (p<0.01); Tukey’s multiple comparison test, **p<0.01 for 500 pg vs 75 and 150 pg).

[0239] Dose-dependent increases in apoA-I levels were observed along the rostral-caudal axis of the brain after intranasal delivery of the NDs (FIGURE 5d, Two-way ANOVA indicates significance by dose (p<0.01), brain region (p<0.0001) and interaction (p<0.01). In the most rostral brain region, A, comprising of the olfactory bulbs and frontal cortex, where apoA-I levels were highest at 2 hours post-intranasal administration, this dose-dependent increase is clearly evident, with significantly highest levels after the 500 pg dose, followed by the 300 pg dose (FIGURE 5d, Tukey’s multiple comparison test, **p<0.01,**p<0.001, ****, pO.OOOl). A similar trend was also observed in the medial brain region B, which encompasses striatum, with higher levels of apoA-I measured after the 500 pg dose of NDs. Simple linear regression confirmed that in regions A and B, apoA-I levels significantly increased with increased dose (non-zero slope, p<0.05). In the more caudal brain regions, C and D, no significant trend was observed at this time point.

[0240] Example 7: ApoA-I K133C NDs are Minimally Distributed to Peripheral Tissues and Rapidly Cleared Following Intranasal Administration

[0241] 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 pg dose of apoA-I K133C NDs. ApoA-I levels were measured in the CSF, lung, liver, and plasma at 1, 2, and 6 hours post-intranasal delivery. At 1 hour post-intranasal administration, apoA-I had already accessed the CSF compartment and remained steady between 2 and 6 hours after treatment (FIGURE 6a). In the lung, apoA-I levels were elevated at 1 and 2 hours post-intranasal delivery, but were completely eliminated from the lung at 6 hours (FIGURE 6b). This suggests that some of the intranasally delivered dose of NDs is inhaled into the lungs where it is eliminated over the course of 6 hours. One hour after intranasal administration, apoA-I had not yet reached the liver, but levels were elevated over the next hour to 10 ng apoA-I / mg protein and there was a trend towards elimination by the 6 hour time point (FIGURE 6c) These results demonstrate that intranasal delivery primarily bypasses the first pass effect, with minimal amounts of apoA-I being distributed to the liver over a 6 hour time period.

[0242] In plasma, apoA-I levels were extremely low at 1 hour post-intranasal administration, and continued to rise over the next 5 hours (FIGURE 6d). This suggests that there is a slow exchange occurring with apoA-I being redistributed from the lungs and CSF to plasma over the course of 6 hours.

[0243] Example 8: Intranasal Administration of ApoA-I K133C ND Results in Enhanced CNS Levels in Brain Compared to IV Administration of Equal Dose

[0244] Since we have previously demonstrated that our apoA-I K133C NDs can access the brain after intravenous administration, we sought to compare efficiency of brain delivery after both intranasal and intravenous administration. Animals for this study received the same dose (300 pg) of ApoA- I K133C NDs either intravenously or intranasally and were harvested 2 hours after administration. ELISA results indicated that the whole brain average of ApoA-I levels was significantly higher for mice that received the NDs intranasally compared to those that received intravenous NDs. In fact, apoA-I whole brain averages were almost 4 times higher in the intranasal cohort compared to the intravenously-treated group (FIGURE 7a, individual t-test, *p<0.05). In the most rostral brain region examined, A, comprising of the olfactory bulbs and frontal cortex, ApoA-I levels were significantly higher for mice that received apoA-I K133C NDs intranasally compared to those that underwent intravenous administration. There were no signficiant differences between treatment groups in the more caudal brain regions examined (B- D) (FIGURE 7b, Two-way ANOVA indicates significance by route of administration (P<0.01), brain region (P<0.001) and interaction (P<0.001); A region: Bonferroni’s multiple comparison test ****p<0.0001).

[0245] Example 9: A Single Intranasal Dose of ASO K133C NDs Results in Potent Suppression of mHTT in the Cortex and Striatum of BACHD Mice

[0246] To visually confirm whether apoA-I K33C NDs could effectively deliver ASO to the brain we delivered either PBS or ASO-loaded apoA-I K133C NDs (or ASO-NDs) intranasally to 2 month- old BACHD mice and perfused the animals after 2 hours. Double-label IHC for ASO and NeuN demonstrates that at 24 hours post-intranasal administration, ASO was deposited in neuronal cells throughout the brain (FIGURE 8a).

[0247] To determine whether apoA-I K33C NDs could effectively deliver ASO to the brain in concentrations sufficient for achieving target engagement, we delivered a single dose of either PBS, ASO alone (300 pg ASO), or ASO-loaded apoA-I K133C NDs (or ASO-NDs; 300 pg ASO: 660 pg apoA-I) intranasally to 2 month-old BACHD mice and harvested the animals after 4 weeks. HTT western blots results demonstrated that relative mHTT was significantly reduced by -30% across the whole brain of ASO-ND treated mice compared to PBS-treated controls (FIGURE 8b; One-way ANOVA indicates significance by treatment, P<0.05; Tukey’s multiple comparison test, *p<0.05). Relative mHTT levels were significantly lower in the olfactory bulbs, cortex, and striatum of the ASO-ND treated mice compared to the PBS-treated controls. In all other brain regions examined, -20-25% reduction in relative mHTT levels was observed (FIGURE 8d; Two-way ANOVA indicates significance by treatment, PO.OOOl; Tukey’s multiple comparison test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0248] Next, we wanted to determine whether this mHTT lowering lasted over the course of 8 weeks and whether a second dose after 4 weeks would help maintain mHTT suppression. To assess these questions, BACHD mice received either PBS, 2 doses of ASO (300 pg ASO given 4 weeks apart), 1 dose of ASO-NDs (300 pg ASO : 660 pg apoA-I), or 2 doses of ASO-NDs (300 pg ASO : 660 pg apoA-I given 4 weeks apart) and were sacrificed 8 weeks after the initial treatment (FIGURE 9a). The whole brain average of relative mHTT levels for mice treated intranasally with two doses of ASO-ND was significantly lower than for those treated with just one dose of ASO-ND dose by about 10% (FIGURE 9b; One-way ANOVA indicates significance by brain region, P<0.0001; Tukey’s multiple comparison test, **p<0.01, ****p<0.0001).

[0249] In micro-dissected brain regions, a second ASO dose 4 weeks after the first did not achieve significant lowering of mHTT in any brain region. However, two ASO-ND doses achieved significant mHTT lowering in olfactory bulbs, striatum, and brainstem (compared to PBS-treated controls). mHTT lowering was similar in all brain regions after intranasal administration of either one or two doses of ASO-NDs, except in the brainstem where two ASO-ND doses resulted in significant mHTT lowering compared to a single ASO-ND dose. (FIGURE 9c; Two-way ANOVA indicates significance by brain region, P<0.05, and by treatment, P<0.0001; Tukey’s multiple comparison test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0250] Example 10: Characterization of ApoA-I NanoDisks in Vitro

[0251] Recently, it was demonstrated that ApoA-I can efficiently enter the CNS via the blood-CSF barrier (BCSFB)

[0046] as well as the blood brain barrier (BBB)

[0082] following intravenous administration in mice. It was shown that ApoA-I undergoes transcytosis across brain microvascular endothelial cells of the BBB by receptor-mediated endocytosis via scavenger receptor Bl (SR-B1)

[0083] , Multiple studies have demonstrated that apolipoprotein A-I can interact with bilayer vesicles of charged and / or neutral lipids to form discoidal complexes known as nanodisks (NDs) [84, 85], Optimization of ApoA-I wild type (WT) concentrations and a neutral lipid, l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), was carried out to identify a formulation where the majority of ApoA-I was incorporated into NDs. NDs were generated with constant moles of ApoA-I and increasing moles of DMPC and these formulations by native polyacrylamide electrophoresis (PAGE) (FIGURE 10A). A ratio of 105 moles of DMPC : 1 mole of ApoA-I resulted in >99% of free ApoA-I incorporated into NDs (FIGURE 10B). This ratio of DMPC lipid to ApoA-I was used for formation of NDs for all subsequent studies.

[0252] Example 11: Antisense Oligonucleotides Associate with ApoA-I NanoDisks

[0253] Recently, it was demonstrated that ApoA-I NDs could be used to deliver chemotherapeutic agents to glioblastoma multiforme and induce tumor regression

[0092] , Moreover, ApoA-I NDs can bind siRNAs to promote gene silencing in cultured cells

[0093] , To determine if ApoA-I NDs are capable of binding an antisense oligonucleotide (ASO) designed to target HTT mRNA for RNase Hl -mediated degradation, an ASO that was previously shown to potently suppress HTT in a mouse model of HD was used

[0050] , This synthetic ASO contains a phosphorothioate- modified backbone with locked nucleic acid (LNA)-modified nucleotides wings flanking an unmodified gap (gapmer), giving the molecule a net negative charge. The secondary structure of ApoA-I contains an amino-terminus globular domain spanning residue 1-43 and 10 amphipathic a-helices encompassing residues 44-243 with helices 5, 6 and 7 containing polar faces with positively charged residues [53-55], To test whether the positively charged faces within the secondary structure of ApoA-I could form an electrostatic interaction with the negatively charged ASO, an electrophoretic mobility shift assay (EMSA) was performed.

[0254] ApoA-I NDs were generated (as described above) and incubated with increasing moles of ASO (molar ratios of ApoA-I: ASO from 1 : 1 to 1 : 5) at 37°C for 1 hour. A shift in the size of ApoA-I ND formulations incubated with ASO was observed, and the intensity and size of the formulations increased with increasing levels of ASO, which suggests an interaction between the ASO and ApoA-I NDs (FIGURE HA).

[0255] Since the EMSA requires formulations to be exposed to an electric field the integrity of the electrostatic interaction between the negatively charged ASO and the positively charged ApoA-I residues on the NDs may be altered. To validate the interaction between ApoA-I NDs and the ASO a complementary method, size exclusion chromatography (SEC), was utilized.

[0256] ApoA-I NDs were generated and incubated with increasing moles of ASO at 37°C for 1 hour and formulations were applied to a column containing G-50 Sephadex beads (data not shown). Multiple fractions were collected and quantified for levels of nucleic acid, protein and lipid. The ASO consistently eluted in the same fractions as the ApoA-I and DMPC suggesting an interaction between the ASO and NDs. As a negative control, each component was mixed together just before the SEC assay and each component eluted in different fractions. Notably, a molar ratio of 3 moles of ASO : 1 moles of ApoA-I : 105 moles of DMPC was the optimal ratio for this complex under these conditions (FIGURE 11B).

[0257] Example 12: ApoA-I NanoDisks Carrying ASO Enhance Suppression of HTT in HD Patient-derived Cells

[0258] To evaluate if ApoA-I NDs could improve delivery of ASOs into cells, different ND formulations were compared to ASO alone in HD patient lymphoblasts using levels of HTT as a readout for delivery efficacy. Previous studies have shown that ASOs are able to enter lymphoblasts in the absence of a carrier following bath application. ASO ApoA-I WT NDs (53%) significantly increased HTT suppression compared to ASO alone (25%) after a 96 hour treatment (FIGURES 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 huntingtin. Consistent with non-selective HTT lowering, no significant difference in magnitude of wild type HTT (wtHTT) compared to mHTT suppression following treatment was observed (FIGURE 12B).

[0259] Example 13: ApoA-I Mutants form NanoDisks

[0260] Based on amino acid scanning and electron paramagnetic resonance, it was previously proposed that ApoA-I adapts a “looped belt” conformation on reconstituted discoidal HDL particles, with the loop spanning amino acids 133-146

[0039] , This loop structure partially overlaps with the lecithin cholesterol acyltransferase (LCAT) activation site, which spans amino acids 143-165 of ApoA-I

[0095] , LCAT is required for the esterification of free cholesterol. Once esterified, cholesterol moves from the surface to the hydrophobic core of nascent discoidal HDL leading to the formation of spherical HDL. Within each discoidal HDL particle, there are 2 ApoA-I molecules in a head to tail (anti-parallel) orientation with ApoA-I helices lying perpendicular to the phospholipid acyl chains [96, 97], Substitution mutations of the amino acids along the loop region (133-146), and neighboring amino acids including the LCAT site (123-165), with a cysteine could promote disulfide bridge formation between the two ApoA-I molecules, across the loop, thereby locking them into position and preventing conversion of discoidal to spherical HDL (FIGURE 13 modified with permission from

[0039] ). ApoA-I bearing cysteine mutations at selected sites may limit loading of cholesterol into NDs, thus reducing their involvement in the RCT pathway, and may promote increased biodistribution to the CNS. To test this, a series of ApoA-I mutants with amino acid substitutions at particular sites within 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 made with substitutions at other amino acid residues R160V (valine substitutions) and H162A (alanine substitution) (FIGURE 14).

[0261] It was previously shown that cysteine substitutions at selected residues in ApoA-I does not impair its ability to form NDs

[0039] , Using the optimized molar ratio of 105 moles of DMPC : 1 mole of ApoA-I we found that both ApoA-I K133C and ApoA-I E146C nanodisks formed higher order complexes by native PAGE (FIGURE 15A). To further characterize the morphology and size of mutant NDs, electron microscopy (EM) on ApoA-I WT, ApoA-I K133C and ApoA-I E146C NDs was performed. Each of the ApoA-I WT, ApoA-I K133C mutant and ApoA-I E146C mutant formed distinct disc shaped particles (FIGURE 15B). Quantification of these particles revealed that ApoA-I WT NDs were significantly smaller in diameter than NDs formed by ApoA-I K133C (FIGURE 15C One-way ANOVA p<0.0001. Mean diameter: 15.67nm vs 18.32nm. Tukey’s multiple comparison test p<0.0001) and ApoA-I E146C (FIGURE 15C Mean diameter: 15.67nm vs 19.10nm. Tukey’s multiple comparison test p<0.0001). Additionally, dynamic light scattering was performed to further evaluate the particle size and dispersity of ApoA-I WT, K133C and E146C NDs. Consistent with the native PAGE and EM data, ApoA-I WT NDs were found to be smaller in size than NDs formed with the ApoA-1 mutants, K133C and E146C (FIGURE 15D).

[0262] Example 14: ApoA-I K133C NDs Enhance CNS Entry Following Systemic Administration

[0263] It has been shown that lipid-free ApoA-I can enter the CNS following intravenous administration [46, 82], To evaluate whether NDs formed with ApoA-I WT could also enter the CNS, we compared levels of ApoA-I in the brain 2 hours after IV administration of either lipid-free ApoA- I WT or ApoA-I WT NDs at 30mg / kg. Levels of ApoA-I WT NDs were significantly higher in the brain compared to lipid-free ApoA-I, suggesting that NDs improves CNS entry (FIGURE 16 Unpaired t-test *p=0.0282). ApoA-I levels were quantified using an ELISA specific for human ApoA-I, as previously described

[0046] , To determine whether NDs formed with ApoA-I mutants could improve CNS entry compared to ApoA-I WT NDs, BACHD mice received a bolus intravenous injection of either ApoA-I WT NDs, ApoA-I K133C NDs, and ApoA-I E146C NDs (FIGURE 17) at 20mg / kg. Animals were sacrificed at 2 hours post-injection and brains were microdissected into cortex, striatum, hippocampus, cerebellum and brainstem. A significant effect of ApoA-I mutants as well as brain region was observed (FIGURE 17 Two-way ANOVA brain region p=0.0271, ApoA-I p<0.0001 and interaction p=0.1221) with K133C showing a significant increase compared to WT in all brain regions (striatum: *p=0.03, hippocampus: ****p<0.0001, cortex: *p=0.0361, cerebellum: ***p=0.0001 and brainstem: **p=0.0057) and compared to E146C in all brain regions but the striatum (hippocampus: ####p<0.0001, cortex: ##p=0.0064, cerebellum: ####p<0.0001 and brainstem: ###p=0.0001). To determine whether K133C and E146C NDs could improve CNS entry compared to other ApoA-I mutant NDs, the amount of various ApoA-I ND formulations in the brain and CSF was determined (FIGURE 18 A and B) Based on these findings, ApoA-I K133C was identified as the lead mutant for subsequent studies.

[0264] Ex vivo imaging was performed to validate the finding that ApoA-I K133C NDs are able to enter the brain more efficiently compared to ApoA-I WT NDs. ApoA-I WT and ApoA-I K133C proteins were labelled with the cf75O near-infrared dye and it was confirmed that this conjugation did not alter the ability of ApoA-I to form NDs (data not shown). The cf75O succinimidyl ester labels primary amines such as those found at the amino terminus of the protein or on lysine residues. Since the K133C mutant has one less lysine than ApoA-I WT, the degree of cf75O labelling would be different for each protein making it difficult to compare them directly. Although absolute fluorescence was found to be higher with cf75O-labelled ApoA-I K133C NDs compared to ApoA-I WT NDs (FIGURE 19A), relative fluorescence in the brain was quantified as a percent of fluorescence in the liver. Consistent with the ELISA data, we measured significantly higher levels of ApoA-I K133C NDs fluorescence in the brain compared to ApoA-I WT NDs (FIGURE 19B 10% vs 5% of liver fluorescence, Unpaired t-test p=0.0051).

[0265] Example 15: Time-course of ApoA-I K133C NanoDisk Biodistribution Shows Rapid Entry into the CNS Following IV Administration

[0266] To evaluate the biodistribution profile of NDs over time, BACHD mice received a bolus intravenous injection with 20mg / kg of either ApoA-I WT NDs or ApoA-I K133C NDs. At different time intervals post-injection, brain and CSF were harvested. In the brain, significantly higher levels of ApoA-I K133C NDs were measured compared to WT NDs at 2 and 4 hours postinjection (FIGURE 20A Two-way ANOVA ApoA-I pO.OOOl, timepoint p=0.1523 and interaction p=0.2058. Sidak’s multiple comparison 2 hours: ***p=0.0004, 4 hours: *p=0.0151). No significant effect of time interval post-injection on levels of ApoA-I was found in the brain. Levels of ApoA-I K133C NDs peaked at 2 hours post-injection whereas levels of ApoA-I WT NDs peaked at 1 hour post-injection. In the CSF, significantly higher levels of ApoA-I K133C NDs were detected compared to ApoA-I WT NDs at 1 and 2 hours post-injection (FIGURE 20B Two-way ANOVA ApoA-I pO.OOOl, timepoint p<0.0001 and interaction pO.OOOl. Sidak’s multiple comparison 1 hour: ***p=0.0004, 2 hours: ****p<0.0001). There was also a significant effect of time interval post-injection on levels of ApoA-I in the CSF with levels of both ApoA-I WT and ApoA-I K133C NDs peaking at 2 hours post-injection.

[0267] Example 16: Dose-dependent Increases of ApoA-I K133C NanoDisks in the Brain Following IV Administration

[0268] To evaluate whether increasing the injected dose of ApoA-I K133C NDs would result in increased CNS entry, a dose-response experiment was performed where BACHD mice received a single IV injection with 10-60mg / kg of ApoA-I K133C NDs. Since levels of ApoA-I K133C NDs were found to peak in the brain at 2 hours post-injection, tissues were harvested at this timepoint for quantification using the human ApoA-I ELISA. A significant dose-dependent increase of ApoA- I K133C in the brain was observed (FIGURE 21A One-way ANOVA pO.OOOl . Tukey’s multiple comparison test ***pO.05 compared to lOmg / kg, ##pO.05 compared to 20mg / kg. Linear regression R2 =0.7594, slope non-zero pO.OOOl). No dose-dependent increase of ApoA-I in the CSF was observed at 2 hours post-injection (FIGURE 21B One-way ANOVA p=0.1082. Linear regression R2 =0.2172, slope non-zero p=0.1745).

[0269] To gain resolution into the regional distribution of NDs in the brain at a range of doses, BACHD mice received a single IV injection with 5-30mg / kg of ApoA-I K133C NDs (FIGURE 22). Animals were sacrificed at 2 hours post-injection and brains were microdissected into brain regions (striatum, cortex, hippocampus, cerebellum and brainstem) for quantification of ApoA-I by ELISA. A significant dose-response in every brain region was observed (Linear regression striatum R2 =0.6402, slope non-zero pO.OOOl; cortex R2 =04971, slope non-zero p=0.0011; hippocampus R2 =0.5155, slope non-zero p=0.0005; cerebellum R2 =0.5773, slope non-zero p=0.0002; brainstem R2 =0.5492, slope non-zero p=0.0003). There was no significant difference in ApoA-I levels between brain regions (Two way ANOVA brain region 0.5176, dose pO.OOOl, interaction 0.4233).

[0270] Example 17: HD Mutation does not Significantly Alter CNS Entry of ApoA-I K133C NanoDisks

[0271] The blood brain barrier has been shown to be impaired in HD mice and patients [57, 58], To investigate whether the HD mutation could impact the concentration of ApoA-I NDs getting into the brain following IV injection, an experiment where BACHD and wild type littermate controls receiving a single IV injection with lOmg / kg of ApoA-I K133C NDs at 2, 6 and 12 months of age was performed. Although no BBB impairment has been reported in the striatum of BACHD mice up to 12 months of age

[0098] , this experiment served as an important control to exclude the possibility that the HD mutation was responsible for the brain exposure levels seen with ApoA-I K133C NDs. 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 levels of ApoA-I in the brain at any age was observed (FIGURE 23 Two-way ANOVA genotype 0.8201, age p=0.3699, interaction p=0.3699).

[0272] Example 18: ASO Carrying ApoA-I WT and K133C NanoDisks Can Induce Modest Mutant HTT Suppression in the CNS

[0273] To evaluate the potential of ApoA-I NDs as ASO carriers in vivo, 2-month-old BACHD mice received a single IV injection with either PBS or 300pg of ASO, ASO-ApoA-I WT NDs or ASO- ApoA-I K133C NDs and HTT target engagement in the CNS was measured 4 weeks later. In the CNS, a significant reduction of mHTT levels was observed in the cortex (FIGURE 24A One-way ANOVA p=0.0195), striatum (FIGURE 24B One-way ANOVA p=0.0309. Tukey’s multiple comparison *p<0.05 compared to PBS) and hippocampus (FIGURE 24C One- way ANOVA p=0.0166. Tukey’s multiple comparison **p<0.01 compared to PBS), but not in the cerebellum (FIGURE 24D One-way ANOVA p=0.8781).

[0274] Example 19: ApoA-I K133C NDs Increase Peripheral Exposure Following Systemic Administration

[0275] It has been shown that lipid-free ApoA-I can enter the CNS following intravenous administration [46, 82], To evaluate whether NDs formed with ApoA-I mutants could alter biodistribution in peripheral tissues compared to ApoA-I WT NDs, BACHD mice received a bolus intravenous injection of ApoA-I WT, ApoA-I K133C NDs and ApoA-I E146C NDs at 20mg / kg. Animals were sacrificed at 2 hours post-injection and liver, quadriceps, 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 pO.OOOl. Tukey’s multiple comparison **p=0.0022 K133C vs WT, ###p<0.0001 K133C vs E146C). Similarly, in the quadriceps 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 in ApoA-I was measured, however, there was a strong trend towards an increase with both ApoA-I K133C and ApoA-I E146C compared to 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 NDs (NP F2) and ApoA-I E146C (NP F3) NDs could improve distribution to peripheral tissues compared to other ApoA-I mutant NDs, the amounts of various ApoA-I ND formulations in the quadriceps, liver and kidney were determined (FIGURE 26 A- C). Based on these findings, ApoA-I K133C (NP F2) was identified as the lead mutant for subsequent study.

[0276] Example 20: Time-course of ApoA-I K133C NanoDisk Biodistribution Following IV Administration

[0277] To evaluate the biodistribution profile of NDs over time, BACHD mice received a bolus intravenous injection with 20mg / kg of either ApoA-I WT NDs or ApoA-I K133C NDs. At different time intervals post-injection, liver and quadricep were harvested. In the liver, significantly higher levels of ApoA-I K133C NDs were detected compared to WT NDs at 0.5, 1, 2 and 4 hours post-injection (FIGURE 27A Two-way ANOVA apoA-I p<0.0001, timepoint p=0.0010 and interaction p=0.0103. Sidak’s multiple comparison 0.5 hours: ****p<0.0001, 1 hour: **p=0.0020, 2 hours: ***p=0.0001, 4 hours: ***p=0.0005). There was also a significant effect of time interval post-injection on levels of ApoA-I in the liver with levels peaking at 30 minutes post-injection for ApoA-I K133C NDs but peaking at 2 hours post-injection for ApoA-I WT NDs.

[0278] Additionally, there was a significant increase of ApoA-I K133C NDs compared to WT NDs at 1, 2 and 4 hours post-injection in the quadriceps (FIGURE 27B Two-way ANOVA apoA-I pO.OOOl, timepoint p=0.6290 and interaction p=0.6958. Sidak’s multiple comparison 1 hour: *p=0.0125, 2 hours: ***p=0.0001, 4 hours: ***p=0.0002). There was no significant effect of time interval post-injection on levels of ApoA-I in the quadriceps. The levels of apoA-I K133C NDs peaked at 4 hours post-injection, whereas levels of ApoA-I WT NDs peaked at 2 hours postinjection. The levels of ApoA-I K133C remained high (>6000ng / mg of ApoA-I) in the quadriceps up to 24 hours post-injection.

[0279] Example 21: Dose-dependent Increases of ApoA-I K133C NanoDisks in the Quadriceps Following IV Administration

[0280] To evaluate whether increasing the injected dose of ApoA-I K133C NDs would result in increased levels in peripheral tissues, a dose-response experiment was performed where BACHD mice received a single IV injection with 10-60mg / kg of ApoA-I K133C NDs. Tissues were harvested at 2 hours post-injection for quantification using the human ApoA-I ELISA. A significant dosedependent increase of apoA-I K133C was observed quadriceps (FIGURE 28B One-way ANOVA pO.OOOl. Tukey’s multiple comparison test ***p<0.05 compared to lOmg / kg, ###p<0.05 compared to 20mg / kg, &p<0.05 compared to 40mg / kg. Linear regression R2 =0.9308, slope nonzero p<0.0001). In the liver, a strong trend towards a 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).

[0281] Example 22: HD Mutation does not Significantly Alter Peripheral Distribution of ApoA-I K133C NanoDisks

[0282] To determine whether the HD mutation could impact the distribution of ApoA-I NDs getting to the peripheral tissues, relative ApoA-I levels in the liver and quadriceps were determined (FIGURE 29 A and B). BACHD and wild type littermate controls received a single IV injection with lOmg / kg of ApoA-I K133C NDs at 2, 6 and 12 months of age. Relative ApoA-I levels are presented rather than absolute values because different cohorts were collected and analyzed at different times.

[0283] Example 23: ASO Carrying ApoA-I WT and K133C NanoDisks Can Induce Modest Mutant HTT Suppression in Peripheral Tissue

[0284] To evaluate the potential of ApoA-I NDs as ASO carriers in vivo, 2-month-old BACHD mice received a single IV injection with either PBS or 300pg of ASO, ASO-ApoA-I WT NDs or ASO- ApoA-I K133C NDs and HTT target engagement was measured 4 weeks later. In the periphery, a significant reduction of mHTT in the quadriceps of mice treated with ASO, ASO-ApoA-I WT NDs and ASO-ApoA-I K133C NDs was observed compared to PBS (FIGURE 30A pO.OOOl. Tukey’s multiple comparison ****p<0.0001 compared to PBS). There was also a significant reduction of mHTT in the liver with ASO-ApoA-I WT NDs and ASO-ApoA-I K133C NDs compared to PBS and ASO treatment (FIGURE 30B p<0.0001. Tukey’s multiple comparison ****p0.0001 compared to PBS, ##p<0.01 compared to ASO). In the lungs, there was a significant reduction of mHTT with all treatment conditions compared to PBS (FIGURE 30C pO.OOOl. Tukey’s multiple comparison ****p<0.0001 compared to PBS). A significant reduction of mHTT in the heart with ASO-ApoA-I WT ND treatment was observed compared to PBS and ASO alone treatment (FIGURE 30D. p<0.0001. Tukey’s multiple comparison ****p0.0001 compared to PBS, #pO.05 compared to ASO).

[0285] Example 24: Covalent Conjugation of ASO to ApoA-I K133C does not Affect Formation of NanoDisks

[0286] Optimization of the conditions for covalent conjugation of ApoA-I K133C to an ASO designed to target human huntingtin (HTT), could increase the potential for ASO to remain attached to the NDs in vivo and exploit the enhanced brain entry seen with the ApoA-I K133C mutant. To conjugate the ASO to ApoA-I, a 6-carbon linker with an amine group was first added to the 5’ end of the ASO. For conjugation of the ASO and ApoA-I, the amine group of the ASO was modified with a 4-formylbenzamide (4FB) moiety and the primary amines of the ApoA-I K133C protein were modified with a 6-hydrazinonicotinamide (HyNic) moiety. The resulting conjugate with covalent linker was stable at a range of pHs and temperatures. Prior to optimizing conjugation conditions, addition of the amino group to the ASO and the modification of the ASO with the 4FB moiety were tested to determine whether the overall activity of the ASO would be altered compared to unmodified ASO alone. HD patient-derived lymphoblasts (Coriell NIGMS GM04724) were treated for 120 hours with either PBS, unmodified ASO, amino ASO or 4FB- ASO at 1 pM in the culture medium and HTT target engagement was measured by quantitative immunoblotting (FIGURE 31A). Bath application of ASO in the culture medium (in the absence of transfection reagents) has previously resulted in suppression of HTT in lymphoblasts. In this example, all treatment conditions resulted in significant reduction of HTT (FIGURE 31B One- way ANOVA pO.OOOl. Tukey’s multiple comparison test ****p<0.0001) whereas there was no significant difference in magnitude of HTT lowering between formulations, suggesting that the modification of the ASO did not affect its activity.

[0287] To validate the conjugation of ASO to ApoA-I K133C, ApoA-I K133C and the conjugate were run by SDS-PAGE and stained with Coomassie to detect protein and Sybr Safe™ to detect nucleic acids (FIGURE 32A). FIGURE 32A shows that the ASO was conjugated to the ApoA-I K133C protein. Since there can be a range of HyNic™ modifications per ApoA-I K133C molecule, multiple conjugate bands corresponding to different molar ratios of ASO to ApoA-I K133C were observed. Formation of ASO:ApoA-I K133C conjugate NDs was validated using native PAGE (FIGURE 32B) and EM (FIGURE 32C). Notably, ASO: ApoA-I K133C non-cleavable conjugate (NC) NDs formed NDs with morphology similar to ApoA-I K133C NDs, but were significantly larger in diameter (FIGURE 32D).

[0288] Example 25: ASO: ApoA-I K133C Conjugate NanoDisks Enhance HTT Suppression in HD Patient-derived Lymphoblasts and Following ICV Injection in BACHD Mice

[0289] ASO:ApoA-I K133C NC NDs were evaluated to determine whether they could enhance HTT suppression compared to ASO alone in HD patient-derived lymphoblasts. Cells were treated for 120 hours with either PBS, ASO alone or ASO: ApoA-I K133C NC NDs at a concentration of 1 pM in the culture medium and HTT suppression was measured by quantitative immunoblotting (FIGURE 33A). ASO alone and ASO:ApoA-I K133C NC NDs resulted in significant reduction of HTT levels (FIGURE 33B ASO = 60% reduction, ASO: ApoA-I K133C NDs = 72% reduction. One-way ANOVA pO.OOOl). Treatment with ASO:ApoA-I K133C NC NDs resulted in a significant increase in the magnitude of HTT suppression compared to ASO alone (t-test p=0.0277). These data suggest that ASO:K133C NDs may enhance cellular entry compared to ASO alone.

[0290] HTT suppression in the brain was compared following ICV injection of either PBS, ASO or ASO:ApoA-I K133C NC NDs. A dose of 15pg of ASO or molar equivalent ASO:ApoA-I K133C NC NDs were administered in a single ICV injection and animals were harvested 4 weeks later for quantification of HTT target engagement. Significant reduction with both ASO and ASO: ApoA- I K133C NC NDs in all brain regions was observed (FIGURES 37A-D. One-way ANOVA pO.OOOl for all brain regions. Tukey’s multiple comparison ****p0.0001 compared to PBS). Notably, ASO: ApoA-I K133C NC NDs resulted in a significant increase in the magnitude of HTT suppression compared to ASO alone in both the cortex (FIGURE 34A Tukey’s multiple comparison test #p=0.0136) and the striatum (FIGURE 34B Tukey’s multiple comparison test #p=0.0313).

[0291] HTT suppression in the periphery was evaluated following ICV administration of either PBS, ASO or ASO: ApoA-I K133C NC NDs. A significant reduction of HTT in the quadriceps was observed with ASO: ApoA-I K133C NC ND treatment but not with ASO (FIGURE 35A One-way ANOVA p=0.0101. Tukey’s multiple comparison test: compared to PBS *p=0.0141, compared to ASO #p=0.0426). Furthermore, there was a significant reduction of HTT with ASO:ApoA-I K133C NC ND treatment compared to ASO alone but not PBS in the liver (FIGURE 35B One-way ANOVA p=0.0233. Tukey’s multiple comparison: compared to PBS p=0.1575, compared to ASO #p=0.0190) and the lungs (FIGURE 35C One-way ANOVA p=0.0043. Tukey’s multiple comparison test: compared to PBS p=0.2018, compared to ASO #p=0.0030). No significant reduction of HTT was observed with either ASO or ASO:apoA-I K133C NC NDs in the heart (FIGURE 35D One-way ANOVA p=0.1500).

[0292] Example 26: Systemic Delivery of ASO: ApoA-I K133C Conjugate NanoDisks Results in Potent Suppression of mHTT in the Striatum and Peripheral Tissues of BACHD Mice

[0293] To evaluate the potential of ASO:ApoA-I K133C NC NDs to suppress HTT in the CNS, 2 month old BACHD mice received a single IV injection with either PBS or 300pg of ASO: ApoA-I K133C NC NDs and HTT target engagement was assessed in the CNS 4 weeks post-injection. In the brain, a significant reduction of mHTT 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).

[0294] To evaluate the potential of ASO: ApoA-I K133C NC NDs to suppress HTT in the periphery, 2- month old BACHD mice received a single IV injection with either PBS or 300pg of ASO: ApoA- I K133C NC NDs and HTT target engagement was assessed 4 weeks post-injection. In the periphery, treatment with ASO: ApoA-I K133C NC NDs resulted in significant reduction of mHTT in the quadriceps (FIGURE 37A. Unpaired t-test ***p=0.0001), liver (FIGURE 37B Unpaired t-test ****p<0.0001), lungs (FIGURE 37C Unpaired t-test ***p=0.0006) and heart (FIGURE 37D Unpaired t-test ***p=0.0001). Discussion

[0295] These studies demonstrate that apoA-I nanodisks or therapeutic nanodisks can enhance intranasal or intravenous delivery of a therapeutic agent to the CNS. In particular, the delivery of mHTT- lowering antisense oligonucleotides (ASOs) to the brain, and improve their distribution and target engagement. Intranasal overcomes some of the key limitations associated with ASO delivery to the brain, including bypassing the blood brain barrier and the liver, avoiding invasiveness of intrathecal delivery, limiting systemic exposure, while ensuring target engagement in the deeper brain structures most affected in HD. Similarly, systemic delivery of ASOs show potent suppression of mHTT in striatum and peripheral tissues and organs.

[0296] ApoA-I NDs assembled with mutant K133C and E146C were able to enter the brain more effectively than both apoA-I WT NDs or lipid-free apoA-I protein, demonstrating that this mutation in the apoA-I protein results in improved CNS delivery. Since both the apoA-I WT and K133C proteins formed similarly sized NDs

[0059] , it is unlikely that the improved brain penetration is a result of ND size. The enhanced delivery of the K133C mutant is potentially due to the natural helical registry of apoA-1 bringing the cysteine resides into close opposition, allowing for the formation of disulfide bonds tightly tethering the apoA-I molecules around the nanodisk

[0047] . This may result in the mutant K133C form of apoA-I having a marked 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 facilitates HDL uptake into cells

[0060] ,

[0297] Furthermore, it has been reported that SR-B1 is expressed in the nasal mucosa of mice

[0061] , Tsuzuki et al. have confirmed mRNA and protein expression of the SR-B1 receptor in the nasal mucosa tissue, and have demonstrated immune-histochemical expression of SR-B1 receptor in the olfactory epithelium

[0061] , It has been demonstrated that differences in apoA-I tertiary conformation markedly influence its recognition by this receptor

[0062] , Further characterization of the tertiary structure and conformation of ApoA-I K133C NDs would help to elucidate the characteristics that enhance their delivery to the brain, especially after intranasal delivery.

[0298] The widespread distribution across the brain after intranasal delivery of apoA-I K133C NDs was confirmed using ex vivo imaging. This was characteristic of the intranasal route of administration’ s reliance on the olfactory and trigeminal nerve endings for entry into the brain and perivascular distribution with high levels around the arteries of the Circle of Willis

[0063] ,

[0299] Since the mutant HTT transgene has been known to cause anatomical changes in the brains of several transgenic mouse models of HD including BBB damage in both R6 / 2 mice and YAC128 mice

[0056] , we sought to determine whether the enhanced entry of apoA-I K133C NDs into the brain could be a result of impaired BBB in the BACHD mice. Our results indicated that at both 2 and 6 months of age, there was no significant difference in the amount of apoA-I K133C that was measured in the brains of BACHD mice after intranasal delivery of NDs compared to WT mice. Thus, the apoA-I K133C NDs are capable of penetrating the brain after intranasal delivery, regardless of any anatomical changes in the animals as a result of their phenotype.

[0300] However, in the 12-month-old BACHD mice, there were higher levels of apoA-I in the A region (comprising of the olfactory bulbs and frontal cortex) compared to the WT mice. This increase could potentially be attributed to changes in olfactory mucosa in the nasal cavity of the BACHD mice (thus improved entry into the brain along the trajectory of the olfactory nerve terminals) or even anatomical changes in the olfactory bulbs themselves (the latter of which has been observed in HD patients [64, 65]). Regardless of the cause, improved CNS delivery could prove beneficial for older patients in later stages of the disease.

[0301] Since the intranasal route of administration results in distribution of substances in perivascular spaces across the brain, multiple studies involving intranasal delivery of therapeutics have demonstrated predominantly perivascular deposition of the agents [36, 37, 66] in the brain. Thus, the physicochemical properties of the therapeutic agent and / or its delivery vehicle are important factors in ensuring delivery to the intended cellular targets. We initially hypothesized that apoA- I NDs would primarily deposit in capillary endothelial cells, neurons, and astrocytes due to the expression of the SR-B1 receptor on the surface of these cells

[0060] since apoA-I is a known ligand of this receptor. Indeed, the results of our immunohistochemical analysis demonstrated that after intranasal delivery of apoA-INDs, apoA-I extensively deposited in both capillary endothelial cells and neurons across the brain. Although we did observe apoA-I colocalization in astrocytes, it was much lower than in the aforementioned cells despite their expression of the SR-B1 receptor.

[0302] Similarly, we observed apoA-I deposition in microglia to a much lower extent as well. At two hours post-intranasal delivery, there was a great deal of apoA-I co-localization with leptin- stained choroid plexus cells. This is consistent with findings by Stukas et al.

[0046] which demonstrated that intravenously administered apoA-I enters the CNS via the choroid plexus. This cellular distribution pattern was similar for both WT and K133C apoA-I NDs, although there significantly more apoA-I positive cells observed across the brain after intranasal treatment with the apoA-I K133C NDs compared to apoA-I WT NDs. Since we only carried out IHC staining at 2 hours post-intranasal treatment, the question of whether some apoA-I from lung-plasma exchange was still accessing the brain via the choroid plexus or whether the apoA-I from intranasal delivery to perivascular spaces was entering the brain parenchyma via the choroid plexus remains to be answered. It is our hypothesis that the latter is in fact the case, since we observed higher apoA-I levels in CSF compared to plasma at both 1 hour and 2 hours post-intranasal treatment, indicating that exchange from CSF and lung to plasma is the more likely direction.

[0303] Consistent with most intranasal delivery studies, apoA-I was already delivered to the brain within 1 hour after intranasal delivery. Since brain distribution after intranasal delivery is a dynamic process, we carried out a time-course study to determine when apoA-I levels were highest in the brain and what the change in regional distribution looked like at different timepoints after treatment. Our results indicated that delivery was highest at 2 hours post-intranasal delivery across all 4 rostral-caudal brain regions. Additionally, levels remained highest in the most rostral brain region A (comprising olfactory bulbs and frontal cortex), but began to shift towards the most caudal brain regions over the course of 24 hours. These results indicate two important findings; the first is that if this rapid entry into the CNS translates to human administration, patients would not need to be immobile or anesthetized (as is the case with mice due to their limited nasal cavity volume and being obligate nose breathers), and a nasal spray or instillation would be sufficient for humans. The second important finding is that apoA-I was still detectable in the brain after 24 hours and thus rapid clearance would not be a limiting factor for delivery of ASOs via this modality.

[0304] The dose-dependent increase in apoA-I levels observed is similarly promising for translation to HD patients, and would potentially allow for patient-tailored doses by increasing frequency of dosing for certain patients based on disease progression and severity. The plateau reached at the highest dose we measured is most likely due to the limited size and volume of the rodent nasal cavity, which not only is significantly larger in humans, but also associated with a longer residence time

[0067] ,

[0305] One of the main advantages of intranasal administration is its ability to delivery therapeutics to the brain whilst bypassing the first pass effect. Indeed, our results confirmed this to be the case for apoA-I NDs, where minimal liver exposure was detected over the course of 6 hours post- treatment with a trend towards complete clearance. In the lungs, higher levels of apoA-I were detected at 1 hour post-intranasal delivery but were completely cleared by 6 hours. This is most likely a result of the mice inhaling some of the dose due to the limited volume of their nasal cavity. The rapid clearance of apoA-I from the lung and liver over the course of several hours and the fact that the empty WT apoA-I NDs have been demonstrated to not illicit a cytotoxic effect in vitro

[0042] , and neither the apoA-I WT or K133C NDs has been shown to illicit an immune response in vivo (data not shown) suggests that they would not have long-term negative effects as a delivery modality. In this regard, it will be of importance to examine the immunogenicity and / or off-target effects of apoA-I NDs after repeated long-term intranasal treatments in vivo.

[0306] Generally, minimal peripheral exposure for a gene therapy intended for the CNS results in fewer side effects. Our results effectively demonstrated the improved delivery of apoA-I NDs to the CNS with intranasal administration compared to intravenous treatment with the same dose. At the two-hour time point, the difference in whole brain apoA-I levels in intranasally-treated animals compared to intravenously-treated mice can primarily be attributed to the increase in the olfactory bulbs and frontal cortex. However, since brain distribution after intranasal administration is a dynamic process (as demonstrated in FIGURE 5b), it stands to reason that the large amount of apoA-I measured in the most rostral brain region (A) will be distributed along the rostral-caudal brain axis over the course of the next several hours. In the case of HD, where mHTT is expressed throughout the body including the periphery, different doses could potentially be tailored to the brain via intranasal administration and to the periphery via intravenous treatment if necessary, allowing for more comprehensive target engagement throughout the body.

[0307] We successfully demonstrated that intranasal delivery of a single dose of apoA-I K133C ASO- NDs significantly reduced mHTT levels in the striatum, as well as the cortex and olfactory bulbs (which were sustained over the course of 8 weeks). With a second dose, four weeks after the first intranasal dose, we achieved over 25% lowering of mHTT in all brain regions except for the cerebellum. In contrast, the current preferred method for ASO delivery to the CNS, the intrathecal route, results in a concentration gradient with higher levels of the ASO being delivered to the outer layers of the cortex and minimal distribution to deeper brain regions such as the striatum [6], Our results demonstrate the therapeutic potential of intranasal apoA-I NDs as a delivery modality for ASOs to the brain. The only other study demonstrating successful ASO delivery after intranasal administration in mice required daily doses of 100 pg ASO (of a alpha-synuclein lowering ASO conjugated to indatraline to target monoamine neurons) over the course of 28 days to achieve significant target engagement

[0068] and did not have sustained lowering with a shorter treatment paradigm

[0069] , The fact that our apoA-I delivery modality is not only completely non-invasive, but that it can also result in sustained and significant target engagement in deeper brain structures is a major advantage and improvement over current ASO delivery methods.

[0308] In conclusion, these studies successfully demonstrate the therapeutic potential of intranasally administered apoA-I K133C NDs for delivery of antisense oligonucleotides to the brain. We have shown that this approach can successfully deliver ASO to the brain and significantly reduce mHTT expression in the regions most affected in HD, the cortex and striatum. This delivery modality overcomes the obstacles that have thus far limited the clinical effectiveness of mHTT-lowering ASOs and would allow for easier and more accessible dosing for patients. Not only is this modality completely non-invasive, but its ability to bypass the BBB and deliver and distribute ASO to deeper brain tissues make it an ideal approach for treating Huntington disease, as well as other neurologic diseases and disorders. Most importantly, these studies demonstrate both the utility and potential of apoA-I nanodisks for delivering macromolecules (i.e. an antisense oligonucleotide (ASO); a short interfering RNA (siRNA); a short hairpin RNA (shRNA); a micro RNA (miRNA); an aptamer, a ribozyme, an mRNA, a plasmid, or a small molecule) to the brain via the intranasal route or via systemic injection. Combining the non-invasive intranasal route with non- immunogenic apoA-I ND delivery vehicles has widespread applications for delivery of ASOs, as well as other macromolecules, for treatment of various CNS diseases and disorders.

[0309] Although various embodiments of the 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 this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word “comprising” is used herein as an open-ended term, substantially equivalent to the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a thing” includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings. References

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Claims

CLAIMS:

1. A therapeutic nanodisk, the therapeutic nanodisk comprising:(a) a lipid-binding polypeptide, wherein the lipid-binding polypeptide encoded by SEQ ID NOs: 3 or 4;(b) a lipid bilayer; and(c) a therapeutic agent.2 The therapeutic nanodisk of claim 1, wherein the lipid bilayer is selected from: diacylphosphatidylcholines; diacylphosphatidylethanolamines; ceramides; sphingomyelins; dihydro sphingomyelins; cephalins; and cerebrosides.3 The therapeutic nanodisk of claim 1 or 2, wherein the lipid bilayer is selected from: dimyristoylphosphatidylcholine (DMPC); distearoylphosphatidylcholine (DSPC); dioleoylphosphatidylcholine (DOPC); dipalmitoylphosphatidylcholine (DPPC); dioleoylphosphatidylethanolamine (DOPE); palmitoyloleoylphosphatidylcholine (POPC); palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal); dipalmitoyl phosphatidyl ethanolamine (DPPE); dimyristoylphosphoethanolamine (DMPE); distearoylphosphatidylethanolamine (DSPE); 16-0-monomethyl PE; 16-O-dimethyl PE; 18-1-trans PE; 1- stearioyl-2-oleoyl-phosphatidy ethanol amine (SOPE); l,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE); and analogs thereof.4 The therapeutic nanodisk of claim 1, 2, or 3, wherein the lipid bilayer is selected from: N- [l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); l,2-dioleoyl-3- trimethylammonium propane (DOTAP); l,2-dioleyloxy-3 -dimethylaminopropane (DODMA); 1,2- dioleoyl-3 -dimethylaminopropane (DODAP); cholesterol, 3-N-[(co-methoxypoly(ethylene glycol)2000)carbamoyl]-l,2-dimyristyloxy-propylamine (PEG-C-DMA); l,2-dilinoleyloxy-3- (N N-dimethyl)aminopropane (DLinDMA); N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-l- yl-l 3-dioxolane-4-ethanamine (DLin-KC2-DMA); 4-(dimethylamino)-butanoic acid, (10Z,13Z)-l- (9Z 12Z)-9,12-octadecadien-l-yl- 10, 13 -nonadecadi en-l-yl ester (DLin-MC3 -DMA); and analogs thereof.5 The therapeutic nanodisk of any one of claims 1-3, wherein the lipid bilayer is a 1,2- Dimyristoyl-sn-glycero-3 -phosphocholine (DMPC) lipid bilayer.6 The therapeutic nanodisk of any one of claims 1-5, wherein the molar ratio of lipid bilayer to lipid-binding polypeptide is between 50: 1 and 400: 1.

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

8. The therapeutic nanodisk of any one of claims 1-7, wherein the nanodisk is between about 5 nm to about 100 nm in diameter as determined by transmission electron microscopy (TEM).

9. The therapeutic nanodisk of any one of claims 1-8, wherein the therapeutic agent is: an antisense oligonucleotide (ASO); a short interfering RNA (siRNA); a short hairpin RNA (shRNA); a micro RNA (miRNA); an aptamer; a ribozyme; an mRNA; a plasmid; ribonucleoprotein (RNP); or a small molecule.

10. The therapeutic nanodisk of any one of claims 1-9, wherein the therapeutic agent is an ASO.

11. The therapeutic nanodisk of claim 10, wherein the ASO targets mutant huntingtin (mHTT).

12. The therapeutic nanodisk of any one of claims 1-11, wherein the therapeutic agent is selected from one or more of: Tominersen (CAS No. : 1709886-74-7); Rovanersen; WVE-120102; WVE-003; and AMT-130.

13. The therapeutic nanodisk of any one of claims 1-12, wherein the therapeutic nanodisk is for delivery of the therapeutic agent to the central nervous system.

14. The therapeutic nanodisk of any one of claims 1-13, wherein the therapeutic nanodisk is for delivery of the therapeutic agent to the brain.

15. The therapeutic nanodisk of any one of claims 1-14, wherein the therapeutic nanodisk is for the treatment of a neurological disease or disorder.

16. The therapeutic nanodisk of claim 15, wherein the neurological disease or disorder is selected from one or more of: Huntington disease (HD); Duchenne muscular dystrophy (DMD); Myotonic dystrophies (DMs); Spinal muscular atrophy (SMA); Spinocerebellar ataxias (SCAs); Spinal muscular atrophy (SMA); Parkinson's disease (PD); Alzheimer's disease (AD);Frontotemporal dementia (FTD); Multiple sclerosis (MS); amyotrophic lateral sclerosis (ALS); Centronuclear myopathies; Alexander Disease; cancer; stroke; meningitis; encephalitis; prion disease; polyneuropathy caused by hereditary ATTR amyloidosis (hATTR-PN); and polyneuropathy of hereditary transthyretin-mediated amyloidosis (ATTR).

17. The therapeutic nanodisk of claim 15 or 16, wherein the neurological disease or disorder is HD.

18. The therapeutic nanodisk of any one of claims 1-17, 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 therapeutic nanodisk of any one of claims 1-18, wherein the therapeutic agent is selected from one or more of: Tominersen; WVE-120102 / 120101; WVE-003; (CUG)7; TTX-3360; AMT-130; AAV.shHD2.1; VY-HTT01; Branaplam; PTC518; TAK-686; ZF-KOX1; Pridopidine; Laquinimod; Fenofibrate; Neflamapimod; Nilotinib; SRX246; Varenicline; SAGE-718; PBT2; Eteplirsen; SRP-5051; Casimersen; Renadirsen; Golodirsen; Viltolarsen; WVE-N531; Nusinersen; ION306; ION859; ION464; IONIS-MAPTRX; ATL1102; Tofersen; Ulefnersen; IONIS C9Rx;WVE-004; ION541; DYN101; Zilganersen; ION717; Eplontersen; Inotersen; Patisiran; Vutrisiran; Givosiran; Lumasiran; Inclisiran; and Pegaptanib.

20. The therapeutic nanodisk of any one of claims 1-19, wherein the therapeutic nanodisk is formulated as an aerosol.

21. The therapeutic nanodisk of any one of claims 1-19, wherein the therapeutic nanodisk is formulated for intravenous delivery or intranasal delivery.

22. A method of treating a neurologic disease or disorder, the method comprising administration of a therapeutic nanodisk of any one of claims 1-14, containing an effective amount of the therapeutic agent, to a person in need thereof.

23. The method of claim 22, wherein the neurological disease or disorder is selected from one or more of: HD; DMD; DMs; SMA; SC As; SMA; PD; AD; FTD; MS; ALS; Centronuclear myopathies; Alexander Disease; cancer; stroke; meningitis; encephalitis; prion disease; hATTR- PN; and ATTR.

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

25. The method of claim 22 or 23, wherein the administration is intranasal.

26. Use of a therapeutic nanodisk of any one of claims 1-14, for treating a neurological disease or disorder.

27. Use of a therapeutic nanodisk of any one of claims 1-14, in the manufacture of a medicament for treating a neurological disease or disorder.

28. The use of claim 26 or 27, wherein the neurological disease or disorder is selected from one or more of: HD; DMD; DMs; SMA; SC As; SMA; PD; AD; FTD; MS; ALS; Centronuclear myopathies; Alexander Disease; cancer; stroke; meningitis; encephalitis; prion disease; hATTR- PN; and ATTR.

29. The use of claim 26, 27, or 28, wherein the neurological disease or disorder is HD.

30. The use of any one of claims 26-29, wherein the therapeutic agent is selected from one ormore of: Tetrabenazine (TBZ); Deutetrabenazine (DBZ); Olanzapine; Risperidone; Citalopram; Fluoxetine; Sertraline; Lamotrigine; and Carbamazepine.

31. The use of any one of claims 26-30, wherein the therapeutic agent is selected from one or more of: Tominersen; WVE-120102 / 120101; WVE-003; (CUG)7; TTX-3360; AMT-130;AAV.shHD2.1; VY-HTT01; Branaplam; PTC518; TAK-686; ZF-KOX1; Pridopidine;Laquinimod; Fenofibrate; Neflamapimod; Nilotinib; SRX246; Varenicline; SAGE-718; PBT2;Eteplirsen; SRP-5051; Casimersen; Renadirsen; Golodirsen; Viltolarsen; WVE-N531; Nusinersen;ION306; ION859; ION464; IONIS-MAPTRX; ATL1102; Tofersen; Ulefnersen; IONIS C9Rx;WVE-004; ION541; DYN101; Zilganersen; ION717; Eplontersen; Inotersen; Patisiran; Vutrisiran; Givosiran; Lumasiran; Inclisiran; and Pegaptanib.

32. A composition, the composition comprising:(a) a lipid-binding polypeptide, wherein the lipid-binding polypeptide encoded by SEQ ID NOs: 3 or 4; and(b) a lipid bilayer.

33. The composition of claim 32, wherein the lipid bilayer is selected from: diacylphosphatidylcholines; diacylphosphatidylethanolamines; ceramides; sphingomyelins; dihydro sphingomyelins; cephalins; and cerebrosides.

34. The composition of claim 32 or 33, wherein the lipid bilayer is selected from: DMPC;DSPC; DOPC; DPPC; DOPE; POPC; POPE; DOPE-mal; DPPE; DMPE; DSPE; 16-0- monomethyl PE; 16-O-dimethyl PE; 18-1-trans PE; SOPE; and transDOPE.

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

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

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

39. The composition of any one of claims 32-38, wherein the composition further comprises a therapeutic agent or diagnostic agent.

Images