Nucleic Acid Delivery to the Central Nervous System

Abstract

The present invention features polymeric nanocarriers (e.g., PLGA nanoparticles) with encapsulated nucleic acids (e.g., antisense oligonucleotides) for delivery to the central nervous system (e.g., intrathecally). These polymeric nanocarriers are useful for treating central nervous system disorders. They can deliver their cargo (e.g., antisense oligonucleotides) in higher amounts, for longer periods, and to deeper regions of the brain than free or unformulated antisense oligonucleotides. Efficient delivery and distribution of antisense oligonucleotides results in reduced dosing frequency and patient compliance, and improves patient experience.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 169,539, filed April 1, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on Mar. 25, 2022, is named 13751-0301WO1_SL.txt and is 10,722 bytes in size.

[0003] The present disclosure relates generally to compositions for delivering therapeutic agents to the central nervous system and methods of using same in the treatment of neurological disorders. [Background technology]

[0004] Delivery of drugs to the central nervous system (CNS) has been a challenge in the treatment of neurological diseases (such as Alzheimer's and Parkinson's diseases). For drugs to reach the nervous system, they must first penetrate the blood-brain barrier (BBB), which is a major challenge due to the selectivity of the BBB. The BBB acts as a semipermeable membrane, preventing most molecules from entering the nervous system from the blood, allowing only low molecular weight (less than 400 Da) and lipophilic compounds to pass through. Most small and large molecules (such as monoclonal antibodies and antisense oligonucleotides) cannot pass this barrier. Due to this difficult process of drug penetration across the BBB, less than 10% of therapeutic agents for neurological diseases advance to clinical trials.

[0005] One way to directly access the CNS is through the use of intrathecal (IT) injection. By injecting directly into the cerebrospinal fluid (CSF), therapeutic agents can directly access the CNS. However, because the CSF turns over several times a day, the residence time of therapeutic agents can be limited. There is a need in the art for improved methods to deliver therapeutic agents to the CNS. Summary of the Invention

[0006] This application relates in part to compositions for delivering therapeutic agents (e.g., nucleic acids such as antisense oligonucleotides) to the central nervous system (CNS). It also features methods of treating neurological disorders using such compositions. Additionally, methods are provided for increasing the amount and / or residence time of a therapeutic agent delivered to the brain of a human subject. Furthermore, the disclosure relates to methods for delivering a therapeutic agent deeper into the brain of a human subject in need thereof.

[0007] In one aspect, the disclosure features a central nervous system (CNS) delivery composition. The composition includes a polymeric nanocarrier and an antisense oligonucleotide. The antisense oligonucleotide is encapsulated within the polymeric nanocarrier and directly precomplexed with a counteragent (a cationic molecule) prior to encapsulation.

[0008] In some cases, the polymeric nanocarrier is selected from the group consisting of poly(l-lactide), poly(glycolide), poly(d,l-lactide) (PLA), poly(dioxanone), poly(d,l-lactide-co-l-lactide), poly(d,l-lactide-co-glycolide), poly(glycolide-co-trimethylene carbonate), poly(caprolactone) ("polycaprolactone"), poly(d,l-lactide-co-glycolide) (PLGA), poly(dioxanone) poly(glycolide-co-trimethylene carbonate), and mixtures thereof. In some cases, the polymeric nanocarrier is PLGA. When the CNS delivery composition is a PLGA nanoparticle, the PLGA nanoparticle comprises a ratio of lactic acid:glycolic acid ranging from 2:98 to 98:2. In certain cases, the PLGA nanoparticles comprise lactic acid:glycolic acid in a ratio selected from the group consisting of: 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 11:89, 12:88, 13:87, 14:86, 15:85, 16:84, 17:83, 18:82, 19:81, 20:8 0, 21:79, 22:78, 23:77, 24:76, 25:75, 26:74, 27:73, 28:72, 29:71, 30:70, 31:69, 32:68, 33:67, 34:66, 35:65, 36:64, 37:63, 38:62, 39:61, 40:60, 41:59, 42:58, 43:57, 44:56, 45:55, 46:54 , 47:53, 48:52, 49:51, 50:50, 51:49, 52:48, 53:47, 54:46, 55:45, 56:44, 57:43, 58:42, 59:41, 60:40, 61:39, 62:38, 63:37, 64:36, 65:35, 66:34, 67:33, 68:32, 69:31, 70:30, 71:29, 72:28 , 73:27, 74:26, 75:25, 76:24, 77:23, 78:22, 79:21, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:4, 98:2, and 100:0.

[0009] The counteragent is a cationic molecule that complexes with the antisense oligonucleotide. In some cases, the cationic molecule is a cationic peptide. In other cases, the cationic molecule is chitosan. In some cases, the cationic molecule is hexadecylamine. In some cases, the cationic molecule is lauric arginate. In still other cases, the cationic molecule is polyethyleneimine (PEI). In some cases, the PEI is linear PEI. In other cases, the PEI is cross-linked PEI.

[0010] In some cases, the CNS delivery composition further comprises a therapeutic agent. In certain cases, the therapeutic agent is selected from the group consisting of a small molecule, a cDNA, an mRNA, an siRNA, an miRNA, an aptamer, a ribozyme, and a different antisense oligonucleotide.

[0011] In certain cases, the CNS delivery composition is formulated for intrathecal delivery to a human subject.

[0012] In some cases, the antisense oligonucleotide is a gapmer or splice switching antisense oligonucleotide. In some cases, the antisense oligonucleotide is useful for treating neurodegenerative diseases (e.g., tauopathy, synucleinopathy). In certain cases, the antisense oligonucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 1. In some cases, the antisense oligonucleotide consists of 18 linked nucleosides, and the oligonucleotide has the nucleobase sequence Me U Me CA Me C Me U Me U Me U Me CA Me UAA Me U.G. Me C MeA nucleic acid sequence consisting of UGG (SEQ ID NO: 1), each internucleoside bond of the oligonucleotide is a phosphorothioate bond, and each nucleoside of the oligonucleotide is a 2'-methoxyethyl nucleoside; Me U is 5-methyl-uracil, Me C is 5-methylcytosine.

[0013] In another aspect, the disclosure relates to a method of treating a CNS disorder in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the CNS delivery composition described above.

[0014] In some cases, administration is by intrathecal injection. In certain cases, intrathecal injection is bolus injection. In some cases, the CNS disorder is synucleinopathy or tauopathy. In certain cases, the CNS disorder is spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, Angelman syndrome, frontotemporal dementia (FTD), Creutzfeldt-Jakob disease, spinocerebellar ataxia type 3 (SCA3) or Menks' disease.

[0015] In another aspect, the disclosure features a method of treating SMA in a human subject in need of such treatment, increasing the inclusion of exon 7 in SMN2 messenger ribonucleic acid (mRNA) transcripts in human subjects having a loss of both functional copies of the SMN1 gene, or increasing the inclusion of exon 7 in SMN2 messenger ribonucleic acid (mRNA) transcripts in human subjects having a mutation in the SMN1 gene that causes functional SMN protein deficiency. The method includes administering a CNS delivery composition by injection into the intrathecal space of the human subject, wherein the antisense oligonucleotide consists of 18 linked nucleosides, and the oligonucleotide has the nucleobase sequence Me U Me CA Me C Me U Me U Me UMe CA Me UAA Me U.G. Me C Me A nucleic acid sequence consisting of UGG (SEQ ID NO: 1), each internucleoside bond of the oligonucleotide is a phosphorothioate bond, and each nucleoside of the oligonucleotide is a 2'-methoxyethyl nucleoside; Me U is 5-methyl-uracil, Me C is 5-methylcytosine.

[0016] In some embodiments, the injection is a bolus injection.

[0017] In another aspect, the disclosure relates to a method for delivering antisense oligonucleotides to the CNS of a human subject, the method comprising administering, by intrathecal injection, an antisense oligonucleotide encapsulated within PLGA nanoparticles, the PLGA nanoparticles having a lactic acid:glycolic acid ratio ranging from 2:98 to 100:0, and the antisense oligonucleotide complexed with PEI or another cationic molecule (e.g., cationic peptides, chitosan, hexadecylamine, lauric arginate).

[0018] In certain cases, the PLGA nanoparticles comprise lactic acid:glycolic acid in a ratio selected from the group consisting of: 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 11:89, 12:88, 13:87, 14:86, 15:85, 16:84, 17:83, 18:82, 19:81, 20:8 0, 21:79, 22:78, 23:77, 24:76, 25:75, 26:74, 27:73, 28:72, 29:71, 30:70, 31:69, 32:68, 33:67, 34:66, 35:65, 36:64, 37:63, 38:62, 39:61, 40:60, 41:59, 42:58, 43:57, 44:56, 45:55, 46:54 , 47:53, 48:52, 49:51, 50:50, 51:49, 52:48, 53:47, 54:46, 55:45, 56:44, 57:43, 58:42, 59:41, 60:40, 61:39, 62:38, 63:37, 64:36, 65:35, 66:34, 67:33, 68:32, 69:31, 70:30, 71:29, 72:28 , 73:27, 74:26, 75:25, 76:24, 77:23, 78:22, 79:21, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:4, 98:2, and 100:0.

[0019] In certain cases, the human subject has CNS disorder.In some cases, the CNS disorder is synucleinopathy or tauopathy.In some cases, the CNS disorder is SMA, ALS, Parkinson's disease, Alzheimer's disease, Huntington's disease, Angelman syndrome, frontotemporal dementia (FTD), Creutzfeldt-Jakob disease, spinocerebellar ataxia type 3 (SCA3) or Menkes disease.

[0020] In some cases, the antisense oligonucleotide is delivered to the CNS of the human subject (e.g., cortex, striatum, thalamus, substantia nigra, cerebellum) about 0.1 hours to about 1 week after administration. In some cases, the antisense oligonucleotide is delivered to the CNS of the human subject (e.g., cortex, striatum, thalamus, substantia nigra, cerebellum) 1 day to 7 days after administration. In some cases, the antisense oligonucleotide is delivered to the CNS of the human subject (e.g., cortex, striatum, thalamus, substantia nigra, cerebellum) 1 day to 6 days after administration. In some cases, the antisense oligonucleotide is delivered to the CNS of the human subject (e.g., cortex, striatum, thalamus, substantia nigra, cerebellum) 1 day to 5 days after administration. In some cases, the antisense oligonucleotide is delivered to the CNS of the human subject (e.g., cortex, striatum, thalamus, substantia nigra, cerebellum) 1 day to 4 days after administration. In some cases, the antisense oligonucleotide is delivered to the CNS of the human subject (e.g., cortex, striatum, thalamus, substantia nigra, cerebellum) 1 to 3 days after administration. In some cases, the antisense oligonucleotide is delivered to the CNS of the human subject (e.g., cortex, striatum, thalamus, substantia nigra, cerebellum) 1 to 2 days after administration. In some cases, the antisense oligonucleotide is delivered to the CNS of the human subject (e.g., cortex, striatum, thalamus, substantia nigra, cerebellum) 1 day after administration. In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, cerebellum of the human subject within 0.1 hours to 48 hours after administration. In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, cerebellum of the human subject within 0.1 hours to 36 hours after administration. In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, or cerebellum of a human subject within 0.1 to 24 hours after administration. In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, or cerebellum of a human subject within 0.1 to 12 hours after administration. In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, or cerebellum of a human subject within 0.1 to 6 hours after administration.In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, or cerebellum of a human subject within 0.1 to 3 hours after administration. In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, or cerebellum of a human subject within 0.1 to 2 hours after administration. In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, or cerebellum of a human subject within 24 hours after administration. In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, or cerebellum of a human subject within 12 hours after administration. In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, or cerebellum of a human subject within 6 hours after administration. In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, or cerebellum of a human subject within 3 hours after administration. In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, or cerebellum of a human subject within 2 hours after administration. In some cases, the antisense oligonucleotide is delivered to the cortex, striatum, thalamus, substantia nigra, or cerebellum of a human subject within 1 hour after administration.

[0021] In another aspect, the disclosure provides a method for increasing the amount of antisense oligonucleotide delivered to the spinal cord and / or brain of a human subject in need thereof, as compared to delivery of a solution of the antisense oligonucleotide in an aqueous buffer. The method includes intrathecal injection of PLGA nanoparticles encapsulating the antisense oligonucleotide, the antisense oligonucleotide being precomplexed with PEI or another cationic molecule (e.g., cationic peptide, chitosan, hexadecylamine, lauric arginate), and the lactic acid:glycolic acid ratio of the PLGA nanoparticles ranges from 2:98 to 100:0. In certain cases, the PLGA nanoparticles comprise lactic acid:glycolic acid in a ratio selected from the group consisting of: 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 11:89, 12:88, 13:87, 14:86, 15:85, 16:84, 17:83, 18:82, 19:81, 20:8 0, 21:79, 22:78, 23:77, 24:76, 25:75, 26:74, 27:73, 28:72, 29:71, 30:70, 31:69, 32:68, 33:67, 34:66, 35:65, 36:64, 37:63, 38:62, 39:61, 40:60, 41:59, 42:58, 43:57, 44:56, 45:55, 46:54 , 47:53, 48:52, 49:51, 50:50, 51:49, 52:48, 53:47, 54:46, 55:45, 56:44, 57:43, 58:42, 59:41, 60:40, 61:39, 62:38, 63:37, 64:36, 65:35, 66:34, 67:33, 68:32, 69:31, 70:30, 71:29, 72:28 , 73:27, 74:26, 75:25, 76:24, 77:23, 78:22, 79:21, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:4, 98:2, and 100:0. In some cases, the PLGA nanoparticles comprise a 50:50 ratio of lactic acid:glycolic acid.In another case, the PLGA nanoparticles contain lactic acid:glycolic acid in a ratio of 5:95. In certain cases, the ASO comprises or consists of a nucleic acid sequence set forth in SEQ ID NO: 1. In certain cases, the ASO comprises or consists of a nucleic acid sequence useful for treating a neurodegenerative disease.

[0022] In another aspect, the disclosure provides a method for delivering antisense oligonucleotides deeper into the brain of a human subject compared to delivery of a solution of the antisense oligonucleotide in an aqueous buffer. The method includes injecting PLGA nanoparticles encapsulating the antisense oligonucleotide into the subarachnoid space, the antisense oligonucleotide being precomplexed with PEI or another cationic molecule (e.g., cationic peptide, chitosan, hexadecylamine, lauric acid arginate), and the lactic acid:glycolic acid ratio of the PLGA nanoparticles ranges from 2:98 to 100:0. In certain cases, the PLGA nanoparticles comprise lactic acid:glycolic acid in a ratio selected from the group consisting of: 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 11:89, 12:88, 13:87, 14:86, 15:85, 16:84, 17:83, 18:82, 19:81, 20:8 0, 21:79, 22:78, 23:77, 24:76, 25:75, 26:74, 27:73, 28:72, 29:71, 30:70, 31:69, 32:68, 33:67, 34:66, 35:65, 36:64, 37:63, 38:62, 39:61, 40:60, 41:59, 42:58, 43:57, 44:56, 45:55, 46:54 , 47:53, 48:52, 49:51, 50:50, 51:49, 52:48, 53:47, 54:46, 55:45, 56:44, 57:43, 58:42, 59:41, 60:40, 61:39, 62:38, 63:37, 64:36, 65:35, 66:34, 67:33, 68:32, 69:31, 70:30, 71:29, 72:28 , 73:27, 74:26, 75:25, 76:24, 77:23, 78:22, 79:21, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:4, 98:2, and 100:0. In some cases, the PLGA nanoparticles contain a 50:50 ratio of lactic acid:glycolic acid. In other cases, the PLGA nanoparticles contain a 5:95 ratio of lactic acid:glycolic acid.In certain cases, the ASO comprises or consists of a nucleic acid sequence set forth in SEQ ID NO: 1. In certain cases, the ASO comprises or consists of a nucleic acid sequence useful for treating a neurodegenerative disease.

[0023] In certain cases, more of antisense oligonucleotide is delivered to the striatum, thalamus, substantia nigra, and / or cerebellum of the brain, compared with the delivery of the solution of antisense oligonucleotide in aqueous buffer.Efficient delivery and distribution of antisense oligonucleotide can result in reduced administration frequency and improve patient experience and compliance.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the practice or testing of the present invention, methods and materials similar or equivalent to those described herein can be used, but exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0025] Other features and advantages of the invention will become apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0026] [Figure 1] 1 shows the drug release profile of PLGA nanoparticles prepared with scaled-up ASO loading at a flow rate of 8 mL / min over a period of 2 hours. [Diagram 2] 1 shows knockdown levels from ICV injection of nanoparticles in mice. [Diagram 3] 1 shows knockdown in the spinal cord following intrathecal (IT) injection of the nanoparticle formulation. [Figure 4] 1 shows knockdown in the cortex and striatum following IT injection of the nanoparticle formulation. [Diagram 5] 1 depicts the release profile of ASO from PLGA nanoparticles and free ASO over 48 hours. [Figure 6-1] 1 shows knockdown in the spinal cord, cerebellum, cortex, and striatum following T injection of the nanoparticle formulation. [Figure 6-2] 1 shows knockdown in the spinal cord, cerebellum, cortex, and striatum following T injection of the nanoparticle formulation. [Figure 7-1] 1 shows knockdown in the spinal cord, cerebellum, cortex and striatum following IT injection of the nanoparticle formulation. [Figure 7-2] 1 shows knockdown in the spinal cord, cerebellum, cortex and striatum following IT injection of the nanoparticle formulation. [Figure 8] FIG. 1 shows luciferase intensity from the brain following ICV injection of nanoparticle formulations in a reporter mouse model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Delivery of drugs to the central nervous system (CNS) is a central issue in the treatment of neurological diseases because the blood-brain barrier (BBB) ​​provides an effective barricade that prevents most therapeutic molecules from entering the brain. Without access to the brain, the therapeutic effects of these molecules may be compromised or eliminated. Intrathecal (IT) infusion provides a way to directly access the CNS and bypass the BBB. However, the residence time of the therapeutic is often limited because the cerebrospinal fluid (CSF) into which the therapeutic is infused during IT administration is turned over several times per day. Without sufficient exposure to the CNS, the therapeutic utility of the treatment may be limited. To increase the efficacy of IT-administered therapeutics, applicants have sought to improve the distribution of oligonucleotides to target tissues within the CNS, thus extending exposure times and preventing rapid clearance of the drug by improving the distribution of oligonucleotides within the CNS. Applicants reasoned that in this way, a greater amount of the therapeutic could reach the target area in the brain for a longer period of time. Applicants have attempted to improve distribution by encapsulating therapeutic agents in nanoparticles that interact with tissues in a different manner than free negatively charged oligonucleotides, have a longer residence time in the CNS, and release the targeted therapeutic agent from the nanoparticle while the nanoparticle is present in the CNS.

[0028] Polymeric nanoparticles (e.g., PLGA nanoparticles) are usually designed to have slow and controlled release kinetics, and those skilled in the art of designing these drug delivery systems should match the release profile to the therapeutic needs for each particular indication. Most polymeric nanoparticles release their contents over an extended period of time (hours to months) to maximize residence time and control drug delivery. However, the present disclosure is based on the advantage of using nanoparticles that release ASOs rather quickly (hours to days) due to the biological limitations of CNS delivery and the fast turnover of CSF. Applicant finds that by encapsulating oligonucleotides in nanoparticles with a relatively short half-life and ensuring the release of the drug before clearance, it is possible to get more therapeutic agent (e.g., ASO) to deeper regions of the brain along the spinal column. To achieve this, Applicant's approach is to design nanoparticle release rates that allow the particles to travel from the lumbar region of the spine to the brain and then release their contents before the particles are cleared from the CSF. These rates are much faster than those typically used for sustained release nanoparticle formulations. Although encapsulating the drug in nanoparticles increases the retention time in the CSF, the nanoparticles do not remain there forever until completely degraded as most other polymeric nanoparticle (e.g., PLGA nanoparticle) therapies are designed for. Applicant finds that these polymeric nanoparticles are still small enough that they are cleared from the intrathecal space around the CNS, but not as fast as free ASO. Therefore, Applicant refrains from developing polymeric nanoparticles (e.g., PLGA nanoparticles) that release for a very long time. Rather, Applicant relies on polymeric nanoparticles (e.g., PLGA nanoparticles) that release in the order of hours to days to weeks (not weeks to months) to ensure that the therapeutic agent (e.g., ASO) is released and available while the particle still has access to the central nervous system and brain.

[0029] The present disclosure is also based in part on the discovery that administration of nanoparticles to the central nervous system has no toxic effects, and that direct administration of the applicant's polymeric nanoparticles containing antisense oligonucleotides (ASOs) to the central nervous system shows no adverse effects.

[0030] This disclosure provides results from an animal study investigating, among other things, the efficacy and safety of poly(dl-lactide-co-glycolide) (PLGA) nanoparticles for delivery of antisense oligonucleotides (ASO) to the brain. Briefly, PLGA nanoparticles were loaded with Malat-1 ASO and characterized. These particles were then injected intracerebroventricularly (ICV) into mice. These injections were well tolerated and showed knockdown in the lumbar spinal cord cortex, indicating that the ASO was released from the nanoparticles and that the nanoparticles were safe at the doses used in these studies. The nanoparticles were then injected into rats by intrathecal injection. The results of this study show that ASO encapsulated and administered in nanoparticles results in a 1.5-2 fold increase in knockdown in the cortex compared to free unformulated ASO alone. There was an improvement from the particle formulation compared to the free ASO, but more work was needed to examine performance differences between PLGA formulations with different lactate:glycolate ratios. Following refinements to the nanoparticle manufacturing process, improvements were made to supply nanoparticles with different lactate:glycolate ratios for additional rat IT studies that examined the tolerability of the formulation and distribution of oligonucleotides along the spine and brain. Results demonstrated that there was increased knockdown in the cortex from nanoparticles (PLGA 50:50) compared to free unformulated ASO. Further ICV administration in a reporter mouse model with live in vivo imaging showed that the PLGA formulation provided significant improvement compared to the corresponding free unformulated ASO.

[0031] Polymeric nanocarrier composition There are two broad approaches to deliver nucleic acids (e.g., oligonucleotides) to target sites in the body. The first is to chemically modify the nucleic acid, usually with a targeting ligand, while maintaining the molecular nature of the conjugate. The second is to incorporate the nucleic acid into some form of nanocarrier and then determine the tissue distribution and cellular interaction of the oligonucleotide. The major difference between these two approaches is the size of the delivery site on the molecular scale versus the nanoscale. This disclosure focuses on nanoscale delivery. There are various nanoscale systems, including lipid nanocarriers and polymeric nanocarriers. This disclosure relates to polymeric nanocarriers. Polymeric nanocarriers include, for example, PLGA nanoparticles, polymeric micelles (also known as "core-shell" nanoparticles), self-assembled hybrid nanocarriers consisting of a PLGA core and a lipid PEG shell, and nanohydrogels (e.g., PRINT nanohydrogels).

[0032] Nanoparticles are considered one of the most versatile drug delivery systems because they can protect therapeutic agents while efficiently delivering them to target tissues or organs. Several different types of polymeric nanocarriers can be used for the delivery of therapeutic agents (e.g., oligonucleotides such as ASOs). In certain cases, the polymeric nanoparticle is one of poly(l-lactide), poly(glycolide), poly(d,l-lactide) (PLA), poly(dioxanone), poly(d,l-lactide-co-l-lactide), poly(d,l-lactide-co-glycolide), poly(glycolide-co-trimethylene carbonate), poly(caprolactone) ("polycaprolactone"), poly(d,l-lactide-co-glycolide) (PLGA), poly(dioxanone) poly(glycolide-co-trimethylene carbonate), or mixtures thereof. Exemplary lactic acid polymers are described, for example, in EP 1468035, U.S. Pat. No. 6,706,854, WO 2007 / 009919 A2, EP 1907023 A, EP 2263707 A, EP 2147036, EP 0427185, and U.S. Pat. No. 5,610,266.

[0033] The biodegradable polymer PLGA has enormous potential as a drug delivery carrier. In addition, by controlling parameters such as polymer molecular weight, lactide to glycolide ratio, and particle size, it is possible to tailor the overall physical properties of the PLGA-drug matrix to achieve the desired drug loading and release rate. In an aqueous environment, PLGA degrades by hydrolysis of its ester bonds. Thus, the hydrophobicity and crystallinity of the polymer affect its degradation rate, and the more hydrophobic and crystalline the polymer is, the slower it will degrade. Of the two monomers in PLGA, LA is the more hydrophobic, so the more LA present in a PLGA polymer, the more hydrophobic it will be. Also, the more LA present in a PLGA polymer, the more crystalline it will be, and the combination of these two features results in a slower degradation rate for PLGA polymers with a higher LA content and a faster degradation rate for PLGA polymers with a higher GA content. This feature of PLGA is useful and can help determine the most effective type of polymer to choose for a desired release rate.

[0034] In some cases, the disclosure features PLGA nanoparticles comprising a ratio of lactic acid:glycolic acid ranging from 2:98 to 100:0. In certain cases, the disclosure features PLGA nanoparticles comprising a ratio of lactic acid:glycolic acid ranging from 2:98 to 98:2. In some cases, the disclosure features PLGA nanoparticles comprising a ratio of lactic acid:glycolic acid ranging from 5:95 to 95:5. In other cases, the disclosure features PLGA nanoparticles comprising a ratio of lactic acid:glycolic acid ranging from 10:90 to 90:10. In other cases, the disclosure features PLGA nanoparticles comprising a ratio of lactic acid:glycolic acid ranging from 15:85 to 85:15. In still other cases, the disclosure features PLGA nanoparticles comprising a ratio of lactic acid:glycolic acid ranging from 20:80 to 80:20. In some cases, the disclosure features PLGA nanoparticles comprising a ratio of lactic acid:glycolic acid ranging from 25:75 to 75:25. In other cases, the disclosure features PLGA nanoparticles comprising a ratio of lactic acid:glycolic acid ranging from 30:70 to 70:30. In certain cases, the disclosure features PLGA nanoparticles comprising a ratio of lactic acid:glycolic acid ranging from 35:65 to 65:35. In some cases, the disclosure features PLGA nanoparticles comprising a ratio of lactic acid:glycolic acid ranging from 40:55 to 55:45. In some cases, the disclosure features PLGA nanoparticles comprising a ratio of lactic acid:glycolic acid ranging from 5:95 to 85:15.In certain cases, the disclosure features PLGA nanoparticles comprising lactic acid:glycolic acid in a ratio selected from the group consisting of: 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 11:89, 12:88, 13:87, 14:86, 15:85, 16:84, 17:83, 18:82, 19: 81, 20:80, 21:79, 22:78, 23:77, 24:76, 25:75, 26:74, 27:73, 28:72, 29:71, 30:70, 31:69, 32:68, 33:67, 34:66, 35:65, 36:64, 37:63, 38:62, 39:61, 40:60, 41:59, 42:58, 43:57, 44:56, 45:55, 46:54, 47:53, 48:52, 49:51, 50:50, 51:49, 52:48, 53:47, 54:46, 55:45, 56:44, 57:43, 58:42, 59:41, 60:40, 61:39, 62:38, 63:37, 64:36, 65:35, 66:34, 67:33, 68:32, 69:31, 70:30, 71:29, 72: 28, 73:27, 74:26, 75:25, 76:24, 77:23, 78:22, 79:21, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:4, 98:2, and 100:0. In some cases, the disclosure features PLGA nanoparticles that include a 50:50 ratio of lactic acid:glycolic acid. In other cases, the disclosure features PLGA nanoparticles that include a 5:95 ratio of lactic acid:glycolic acid. In yet another instance, the disclosure features PLGA nanoparticles including a ratio of lactic acid:glycolic acid of 85:15.

[0035] In some cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a total charge density of -0.3 to -12.0 mV. In other cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a total charge density of -0.4 to -10.0 mV. In some cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a total charge density of -0.4 to -1.0 mV. In some cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a total charge density of -0.4 to -0.9 mV. In some cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a total charge density of -0.4 to -0.8 mV. In some cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a total charge density of -0.4 to -0.7 mV. In certain cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a total charge density of 0.4 to 0.6 mV. In certain cases, polymeric nanocarriers (e.g., PLGA nanoparticles) have a total charge density of 0.01-0.05 mV.

[0036] In some cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a polydispersity index of 0.2 to 0.9. In certain cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a polydispersity index of 0.2 to 0.8. In other cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a polydispersity index of 0.2 to 0.7. In some cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a polydispersity index of 0.2 to 0.6. In some cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a polydispersity index of 0.2 to 0.5. In some cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a polydispersity index of 0.2 to 0.4. In certain cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a polydispersity index of 0.2 to 0.3.

[0037] In some cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) have a diameter of 100 nm to 1000 nm. In certain cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) have a diameter of 100 nm to 900 nm. In some cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) have a diameter of 100 nm to 800 nm. In certain cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) have a diameter of 100 nm to 700 nm. In other cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) have a diameter of 100 nm to 600 nm. In still other cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) have a diameter of 100 nm to 500 nm. In some cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) have a diameter of 100 nm to 400 nm. In certain cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) have a diameter of 100 nm to 300 nm. In some cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) have a diameter of 100 nm to 250 nm. In other cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) have a diameter of 100 nm to 200 nm.

[0038] In certain cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a diameter of 100-650 nM, a total charge density of -0.4 to -0.6 mV, and a polydispersity index of 0.2 to 0.3. In certain cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a diameter of 100-400 nM, a total charge density of -0.4 to -0.6 mV, and a polydispersity index of 0.2 to 0.3. In certain cases, the polymeric nanocarrier (e.g., PLGA nanoparticles) has a diameter of 200-300 nM, a total charge density of -0.4 to -0.6 mV, and a polydispersity index of 0.2 to 0.3. In certain cases, these polymeric nanocarriers (e.g., PLGA nanoparticles) comprise lactic acid:glycolic acid in a ratio selected from the group consisting of: 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 11:89, 12:88, 13:87, 14:86, 15:85, 16:84, 17:83, 18:84, 19:85, 20:86, 21:87, 22:89, 23:88, 24:89, 25:85, 26:84, 27:83, 28:84, 29:85, 30:89, 31:89, 32:89, 33:89, 34:86, 35:85, 36:84, 37:83, 38:84, 39:85, 40:84, 41:84, 42:84, 43:84, 45:85, 46:84, 47:84, 48:84, 49:85, 50:84, 51:84, 52:84, 53:84, 54:84, 55:84, 56:84, 57:84, 58:84, 59:84, 60:84, 61:84, 62:84, 63:84, 64:84, 65:84, 66:84, 67:84, 68:84, 69:84, 70:84, :82, 19:81, 20:80, 21:79, 22:78, 23:77, 24:76, 25:75, 26:74, 27:73, 28:72, 29:71, 30:70, 31:69, 32:68, 33:67, 34:66, 35:65, 36:64, 37:63, 38:62, 39:61, 40:60, 41:59, 42:58, 43:57, 44:56, 45:59, 46:59, 47:59, 48:59, 49:59, 50:59, 51:59, 52:59, 53:59, 54:59, 55:59, 56:59, 57:59, 58:59, 59:59, 60:59, 61:59, 62:59, 63:59, 64:59, 65:59, 66:59, 67:59, 68:59, 69:59, 70:59, 71:59, 72:59, 73:59, 74:59, 75:59, 76:59, 77:59, 78:59, 79:59, 80:59, 81:59, 82:59, 83:59, 84:59, 85:59, 86:59, 87:59, 88:59, 89:59, 90:59, 9 5:55, 46:54, 47:53, 48:52, 49:51, 50:50, 51:49, 52:48, 53:47, 54:46, 55:45, 56:44, 57:43, 58:42, 59:41, 60:40, 61:39, 62:38, 63:37, 64:36, 65:35, 66:34, 67:33, 68:32, 69:31, 70:30, 71:29, 72:28, 73:27, 74:26, 75:25, 76:24, 77:23, 78:22, 79:21, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:4, 98:2, and 100:0.

[0039] In certain cases, negatively charged (anionic) oligonucleotides are complexed with cationic molecules. Exemplary cationic molecules include synthetic cationic polymers such as polyethyleneimine (PEI), natural cationic polymers such as chitosan, cationic peptides, cationic dendrimers, hexadecylamine, or lauric arginate. In some embodiments, the PEI can be linear PEI or cross-linked PEI. PEI is a linear or branched polymer with multiple titratable amino groups, so it can easily form nanocomplexes with oligonucleotides. In some embodiments, the complex molecule can be PepFect6, a cationic peptide derived from honeybee melittin, a cationic peptide of transactivator of transcription (TAT), a peptide derived from human lactoferrin, or a short amphiphilic sequence. In some embodiments, the cationic dendrimer is a polyamidoamine (PAMAM).

[0040] In some cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) further comprise polyethylene glycol (PEG). Neutral polymers such as PEG can minimize protein binding and uptake by the reticuloendothelial system (RES). Thus, polymeric nanoparticles comprising PEG can increase circulation time. In certain cases, PEG is linked to the polymeric nanocarrier using a cleavable linker or a short lipid anchor. In some cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) further comprise folic acid. Folic acid can be bound to the nanocarrier surface. In some cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) further comprise a small molecule ligand (e.g., anisamide) or an aptamer that targets a site of interest. In some cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) further comprise a transferrin receptor ligand. In some cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) further comprise an anti-transferrin receptor antibody or a fragment thereof. In some cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) further comprise a rabies virus peptide that targets the nanoparticles to neurons. In some cases, the polymeric nanoparticles (eg, PLGA nanoparticles) further comprise a targeting ligand that targets endocytic receptor sites.

[0041] In certain cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) contain a therapeutic agent in addition to the nucleic acid (e.g., an oligonucleotide such as an ASO) encapsulated within the nanoparticle. In certain cases, the therapeutic agent is selected from the group consisting of a small molecule, cDNA, mRNA, siRNA, miRNA, aptamer, ribozyme, and different antisense oligonucleotides.

[0042] Polymeric nanoparticles (e.g., PLGA nanoparticles) can be prepared to encapsulate the nucleic acid (e.g., oligonucleotide such as ASO) to be delivered. In certain cases, the ASO comprises or consists of the sequence shown in SEQ ID NO:1. Such polymeric nanoparticles can include a cationic molecule complexed with the nucleic acid (e.g., oligonucleotide such as ASO). In certain cases, the cationic molecule is PEI. In some cases, such polymeric nanocarriers can transport both a nucleic acid (e.g., ASO) and a second therapeutic agent (e.g., a small molecule drug or another ASO). Thus, polymeric nanoparticles can be used for in vivo co-delivery of therapeutic agents to a target site (e.g., any part of the central nervous system, such as the spinal cord, cortex, striatum, thalamus, substantia nigra, or cerebellum).

[0043] Any of the above-mentioned polymeric nanoparticles (e.g., PLGA nanoparticles) can be formulated for intrathecal delivery to human subjects.In some cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) are administered intrathecally by bolus injection.In some cases, the polymeric nanoparticles (e.g., PLGA nanoparticles) are formulated for delivery to one or more of the striatum, thalamus, substantia nigra, or cerebellum of the brain of human subjects.

[0044] Antisense oligonucleotides Antisense oligonucleotides (ASOs) are synthetic single-stranded nucleic acids that bind to ribonucleic acid (RNA) and thereby alter or reduce expression of a target RNA. They can not only reduce protein expression by degradation of the target transcript, but also restore protein expression or modify proteins through interference with pre-mRNA splicing. The present disclosure encompasses both types of ASOs. In certain cases, the ASOs of the present disclosure are "gapmers". Such ASOs act primarily by selectively cleaving mRNAs with complementary sites via an RNase H-dependent mechanism. They have a central region that supports RNase H activity adjacent to chemically modified ends that increase affinity and / or reduce susceptibility to nucleases. In some cases, the ASOs of the present disclosure are splice-switching oligonucleotides (SSOs) (e.g., nusinersen). SSOs are generally fully modified to remove RNase H activity and allow interaction with nuclear pre-mRNA during splicing. They can be designed to bind to 5' or 3' splice junctions, or to intra-exon splicing enhancer or silencer sites. By binding to such sites, they can modify splicing, for example, by promoting alternative exon usage, exon exclusion, or exon inclusion.

[0045] Ideally, the synthetic oligonucleotides (e.g., ASOs) of the present disclosure should bind to specific sequences on target RNA transcripts and be stable. Synthetic oligonucleotides (e.g., ASOs) are foreign to the cells into which they are introduced, and therefore they are targeted by endogenous nucleases. In order for synthetic oligonucleotides to achieve the level of persistence in cells that may be required to accomplish their tasks, they generally need to be protected from those endogenous nucleases. Synthetic oligonucleotides can be modified by any modification known in the art, including, but not limited to, modifications of the phosphodiester backbone, modifications at the ribose 2'OH group, and modifications of the ribose ring and nucleoside base. For example, modifications of the phosphate backbone can include phosphorothioate (PS) modifications, in which non-bridging phosphate oxygen is replaced with sulfur. In addition, other modifications include phosphorodithioates and phosphonoacetates. See, e.g., U.S. Patent Nos. 6,143,881, 5,587,361, and 5,599,797, which are incorporated by reference. Other modifications include 2'-O-methyl (2'OMe), 2'fluoro (2'F), 2'methoxyethyl (2'-O-MOE), 2'fluora-rabino (FANA), 2'-H, 2'-thiouracil, locked nucleic acid (LNA), constrained ethyl (cEt), bridged nucleic acid (BNA), ethylene-bridged nucleic acid (ENA), hexitol nucleic acid (HNA), altritol nucleic acid (ANA), cyclohexene nucleic acid (CeNA), unlocked nucleic acid (UNA), 4'thio (4'-S), and 3' inverted abasic end caps. In some embodiments, the nucleic acid may be modified by replacing the natural phosphodiester bond with a boranophosphate (PB) bond, a phosphonoacetate (Pac) bond, or a thiophosphonoacetate backbone bond. In some embodiments, the nucleic acid may contain more than one modification. In some embodiments, the nucleic acid may contain more than two modifications.In some cases, the modification of the synthetic oligonucleotide (e.g., ASO) is at least one of a 2'-O-methyl (2'OMe) modification, a 2'fluoro (2'F) modification, an MOE modification, a 2'fluora-rabino (FANA) modification, a 2'-H modification, a 2'-thiouracil modification, a locked nucleic acid (LNA) modification, a bridged nucleic acid (BNA) modification, an ethylene-bridged nucleic acid (ENA) modification, a hexitol nucleic acid (HNA) modification, an altritol nucleic acid (ANA) modification, a cyclohexene nucleic acid (CeNA) modification, an unlocked nucleic acid (UNA) modification, a 4'thio (4'-S) modification, a thiol linkage modification, and a 3' inverted abasic end cap modification.

[0046] In some cases, synthetic oligonucleotides (e.g., ASOs) have one or more phosphorothioate (PS) backbone modifications. Such modifications improve stability and protection from nucleases in blood and tissues. They also promote protein binding, thus aiding in interactions with albumin and other blood proteins, thus slowing renal clearance. This modification supports RNase H activity and can therefore be used in both gapmers and SSOs. In certain cases, ASOs contain phosphorodiamidate morpholino oligomer (PMO) and / or peptide nucleic acid (PNA) modifications. Such modifications generate a neutral backbone and provide high resistance to nucleases. Because such modifications do not support RNase H activity, they are primarily used in SSOs rather than gapmers. Another modification is a change at the 2' sugar position. Such modifications include 2'-O-Me and 2'-O-(2-methoxyethyl) (MOE) modifications. These modifications promote an RNA-like conformation and significantly increase binding affinity to RNA while also providing enhanced nuclease resistance. Oligonucleotides that are fully modified at the 2' position are generally best suited for SSO, as they do not support RNase H activity. However, RNAse H-dependent antisense effects can be achieved by using "gapmers" that contain a central unmodified region of about 7 residues adjacent to the 2' modified region. Another modification that can be effective for oligonucleotides is the use of bridged rings. Locked nucleic acid (LNA) chemistry and constrained ethyl (cEt) as well as tricyclic DNA (tc-DNA) modifications involve bridging of sugar rings. Such modifications promote RNA-like structures, exhibit nuclease resistance, and result in a dramatic increase in binding affinity. These modifications can be used in both gapmers and SSOs. In certain embodiments, the ASOs of the present disclosure include one or more of the modifications described above. In some embodiments, the ASOs of the present disclosure have a PS backbone. In some embodiments, the ASOs of the present disclosure have a mixed PS and phosphodiester backbone. In certain embodiments, the ASOs of the present disclosure have one or more 2'-O-(2-methoxyethyl) (MOE) modifications.In certain embodiments, the ASO of the present disclosure has MOE modification at all residues.In certain embodiments, the ASO of the present disclosure comprises one or more cEt.In certain cases, one or more uracils of the ASO of the present disclosure are replaced by 5-methyl-uracil.In certain cases, all uracils of the ASO of the present disclosure are replaced by 5-methyl-uracil.In certain cases, one or more cytosines of the ASO of the present disclosure are replaced by 5-methyl-cytosine.In certain cases, all cytosines of the ASO of the present disclosure are replaced by 5-methyl-cytosine.

[0047] Non-limiting examples of ASOs encompassed by this disclosure are provided in Table I. [Table 7]

[0048] For other non-limiting exemplary ASOs encompassed by this disclosure, see Evers et al., Advanced Drug Delivery Reviews, 87:90-103 (2015) (see, e.g., Table 2 and the references cited therein), Bennett et al., Annu. Rev. Pharmacol. Toxicol., 61:831-52 (2021) (see, e.g., Table 1 and the references cited therein), Silva et al., Brain, 143;407-429 (2020) (see, e.g., Table 2 and the references cited therein), US10,385,341, US9,683,235, and US10,407,680, the contents of all of which are incorporated by reference in their entireties.

[0049] The ASOs described herein are encapsulated in a polymeric nanocarrier (e.g., PLGA). In certain cases, the ASOs described herein are complexed with a cationic molecule (e.g., PEI).

[0050] Pharmaceutical Compositions The polymeric nanoparticles disclosed herein can be combined with a pharma- ceutically acceptable carrier to form a pharmaceutical composition. As will be understood by those skilled in the art, the carrier can be selected based on the route of administration described below, the location of the target problem, the drug to be delivered, the course of drug delivery, and the like.

[0051] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any brand of fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. In one embodiment, the conjugate is suspended in a carrier fluid that includes 1% (w / v) sodium carboxymethylcellulose and 0.1% (v / v) TWEEN® 80. Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by terminal sterilization of solid compositions which can be dissolved or dispersed in sterile water or a selected injectable diluent prior to administration.

[0052] It should be understood that the exact dosage of the polymeric nanoparticles containing a nucleic acid agent (e.g., ASO) is selected by the individual physician in consideration of the patient being treated, and generally, dosage and administration are adjusted to provide the patient being treated with an effective amount of the nucleic acid agent nanoparticles. As used herein, an "effective amount" of a nanoparticle containing a nucleic acid agent (e.g., ASO) refers to the amount necessary to induce a desired biological response. As will be understood by those skilled in the art, the effective amount of a polymeric nanoparticle (e.g., PLGA nanoparticle) containing a nucleic acid agent (e.g., ASO) can vary depending on factors such as the desired biological endpoint, the drug to be delivered, the target tissue, and the route of administration. For example, an effective amount of a polymeric nanoparticle containing a nucleic acid agent (e.g., ASO) can be an amount that reduces tumor size by a desired amount over a desired period of time. Additional factors that may be considered include the severity of the disease state, the age, weight, and sex of the patient being treated, diet, time and frequency of administration, drug combinations, reaction sensitivities, and tolerance / response to the therapy.

[0053] The polymeric nanoparticles of the present disclosure may be formulated into unit dosage forms for ease of administration and uniformity of dosage. The expression "unit dosage form" as used herein refers to a physically discrete unit of nanoparticles appropriate for the patient to be treated. However, it will be understood that the total daily usage of the composition will be determined by the attending physician within the scope of sound medical judgment. For any nanoparticle, the therapeutically effective amount can be estimated initially either in cell culture assays or animal models, usually mice, rabbits, dogs, or pigs. Animal models are also used to achieve a desired concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The therapeutic efficacy and toxicity of the nanoparticles can be determined according to standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose that is therapeutically effective in 50% of the population) and the LD 50 (the dose that is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, or the ratio, LD 50 / ED 50The therapeutic index can be expressed as: Pharmaceutical compositions exhibiting large therapeutic indices can be useful in some embodiments. The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for human use.

[0054] In some embodiments, compositions suitable for freezing are contemplated that include the polymeric nanoparticles disclosed herein, and a solution suitable for freezing, such as a monosaccharide, disaccharide, or polysaccharide, such as sucrose and / or trehalose, and / or a salt and / or cyclodextrin solution, is added to the nanoparticle suspension. The sugar (e.g., sucrose or trehalose) may act as a cryoprotectant, for example, to prevent particles from agglomerating upon freezing. For example, nanoparticle formulations are provided herein that include a plurality of the disclosed nanoparticles, sucrose, an ionic halide, and water, where the nanoparticles / sucrose / water / ionic halide are about 3-40% / 10-40% / 20-95% / 0.1-10% (w / w / w / w) or about 5-10% / 10-15% / 80-90% / 1-10% (w / w / w / w). For example, such a solution can include nanoparticles disclosed herein, about 5% to about 20% by weight sucrose, and an ionic halide such as sodium chloride at a concentration of about 10 to 100 mM. In another example, provided herein is a nanoparticle formulation including a plurality of the disclosed nanoparticles, trehalose, cyclodextrin, and water, where the nanoparticles / trehalose / water / cyclodextrin is about 3-40% / 1-25% / 20-95% / 1-25% (w / w / w / w) or about 5-10% / 1-25% / 80-90% / 10-15% (w / w / w / w).

[0055] Method of service The present disclosure features a method for delivering nucleic acids (e.g., oligonucleotides, such as antisense oligonucleotides) to the central nervous system (CNS) of a human subject. Access to the CNS is a difficult challenge due to the blood-brain barrier (BBB). The BBB is composed of tightly connected endothelial cells supported by a network of pericyte and astrocytic processes and is impermeable to small molecules such as sucrose. The BBB is also largely impermeable to oligonucleotides.

[0056] One way to address this delivery problem is by direct administration of oligonucleotides. This can be done, for example, by intrathecal injection. When administered intrathecally, oligonucleotides are widely distributed within the CNS and taken up by both neurons and glial cells.

[0057] In certain cases, polymeric nanocarriers (e.g., PLGA nanoparticles) containing nucleic acids (e.g., antisense oligonucleotides) are administered to human subjects by intrathecal injection. In some cases, the intrathecal injection is a bolus injection. In certain embodiments, the antisense oligonucleotides are encapsulated within PLGA nanoparticles. In some cases, the antisense oligonucleotides are complexed with cationic molecules (e.g., PEI, chitosan, hexadecylamine, lauric arginate, or cationic peptides). In some cases, the lactic acid:glycolic acid ratio of the PLGA nanoparticles ranges from 2:98 to 98:2. In certain cases, the PLGA nanoparticles comprise lactic acid:glycolic acid in a ratio selected from the group consisting of: 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 11:89, 12:88, 13:87, 14:86, 15:85, 16:84, 17:83, 18:82, 19:81, 20:8 0, 21:79, 22:78, 23:77, 24:76, 25:75, 26:74, 27:73, 28:72, 29:71, 30:70, 31:69, 32:68, 33:67, 34:66, 35:65, 36:64, 37:63, 38:62, 39:61, 40:60, 41:59, 42:58, 43:57, 44:56, 45:55, 46:54 , 47:53, 48:52, 49:51, 50:50, 51:49, 52:48, 53:47, 54:46, 55:45, 56:44, 57:43, 58:42, 59:41, 60:40, 61:39, 62:38, 63:37, 64:36, 65:35, 66:34, 67:33, 68:32, 69:31, 70:30, 71:29, 72:28 , 73:27, 74:26, 75:25, 76:24, 77:23, 78:22, 79:21, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:4, 98:2, and 100:0.

[0058] The present disclosure features the delivery of polymeric nanoparticle carriers that release their encapsulated cargo (e.g., ASO) with "fast" release kinetics. "Fast" refers to release of the cargo within about 0.1 hours to about 1 week of intrathecal injection of the polymeric nanoparticle carrier. In some embodiments, the cargo is released within 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day of intrathecal injection of the polymeric nanoparticle carrier. In other embodiments, the cargo is released within 0.1 hours, 0.2 hours, 0.3 hours, 0.4 hours, 0.5 hours, 0.6 hours, 0.7 hours, 0.8 hours, 0.9 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, or 48 hours of intrathecal injection of the polymeric nanoparticle carrier.

[0059] The compositions and delivery methods disclosed herein allow for increased amounts of nucleic acids (e.g., antisense oligonucleotides) delivered to the brain of a human subject compared to free, unformulated ASO delivery.

[0060] In addition, the compositions and delivery methods disclosed herein allow for an increased time for a delivered nucleic acid (e.g., ASO) to be present and active in the CNS of a human subject compared to unformulated ASO delivery.

[0061] Additionally, the compositions and delivery methods disclosed herein allow for delivery of nucleic acids (e.g., ASOs) deeper into the brain of a human subject compared to unformulated ASO delivery. In some cases, more nucleic acid (e.g., ASO) is delivered to the striatum, thalamus, substantia nigra, and / or cerebellum of the human subject's brain compared to unformulated ASO delivery.

[0062] Treatment methods The disclosure features a method of treating a CNS disorder in a human subject in need of treatment thereof. The method includes administering to the subject a therapeutically effective amount of a polymeric nanocarrier composition described herein. In some cases, the CNS disorder is a synucleinopathy or tauopathy. In certain cases, the CNS disorder is spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, Angelman syndrome, frontotemporal dementia (FTD), Creutzfeldt-Jakob disease, spinocerebellar ataxia type 3 (SCA3), or Menk's disease. In some cases, the polymeric nanocarrier comprises an antisense oligonucleotide comprising or consisting of a sequence set forth in SEQ ID NO:1. In some cases, the polymeric nanocarrier further comprises an additional therapeutic agent (e.g., a small molecule compound). In some cases, the polymeric nanocarrier comprises an ASO at a dose of 0.05 mg / kg to 25 mg / kg. In some cases, the polymeric nanocarrier comprises an ASO at a dose of 0.05 mg / kg to 20 mg / kg. In some cases, the polymeric nanocarrier comprises an ASO at a dose of 0.05 mg / kg to 15 mg / kg. In some cases, the polymeric nanocarrier comprises an ASO at a dose of 0.05 mg / kg to 10 mg / kg. In some cases, the polymeric nanocarrier comprises an ASO at a dose of 0.05 mg / kg to 5 mg / kg. In some cases, the polymeric nanocarrier comprises an ASO at a dose of 0.05 mg / kg to 4 mg / kg. In some cases, the polymeric nanocarrier comprises an ASO at a dose of 0.05 mg / kg to 3 mg / kg. In some cases, the polymeric nanocarrier comprises an ASO at a dose of 0.05 mg / kg to 2 mg / kg. In some cases, the polymeric nanocarrier comprises an ASO at a dose of 0.05 mg / kg to 1 mg / kg. The therapeutically effective amount can be readily determined by a medical provider based on, among other things, the age, sex, and stage of disease of the human subject being treated. In some cases, the polymeric nanocarrier is administered by intrathecal (IT) injection. In certain cases, the IT injection is a bolus injection.

[0063] In some cases, the disclosure features a method of treating spinal muscular atrophy (SMA) in a human subject. In another case, a method is provided for increasing the inclusion of exon 7 in SMN2 messenger ribonucleic acid (mRNA) transcripts in a human subject having a loss of both functional copies of the SMN1 gene. In yet another case, a method is featured for increasing the inclusion of exon 7 in SMN2 messenger ribonucleic acid (mRNA) transcripts in a human subject having a mutation in the SMN1 gene that causes a functional SMN protein deficiency. In one embodiment of these methods, the human subject is administered a CNS delivery composition (e.g., PLGA nanoparticles) comprising an ASO that can be used to treat SMA (e.g., nusinersen) by injection into the intrathecal space of the human subject. In one embodiment, the antisense oligonucleotide encapsulated in the polymeric nanocarrier administered to the human subject is a nucleobase sequence Me U Me CA Me C Me U Me U Me U Me CA Me UAA Me U.G. Me C Me comprising or consisting of UGG (SEQ ID NO:1), each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage, and each nucleoside of the oligonucleotide is a 2'-methoxyethyl nucleoside; Me U is 5-methyl-uracil, Me C is 5-methylcytosine.

[0064] In certain cases, the intrathecal injection is by bolus injection.

[0065] The following examples are provided to more fully illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, they are merely for illustrative purposes and are not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without the exercise of inventive ability and without departing from the scope of the invention. EXAMPLES

[0066] Example 1: Study 1 - Materials and Methods Study 1 is described in Examples 1-6. material PLGA lactide:glycolide (50:50), ester terminated, average 100,000 Da PLGA lactide:glycolide (85:15), ester terminated, 190,000~240,000Da PLGA L-lactide:glycolide (5:95), 190,000~210,000Da Polyethyleneimine (PEI), linear, 2.5 kDa, Sigma 764604 Ethyl Acetate Malat-1 antisense oligonucleotide: [ka] (wherein "o" is a phosphodiester (if "o" is not labeled with phosphorothioate, Me U is 5-methyl-uracil, Me C is 5-methyl-cytosine, the underlined nucleosides are MOE), 7 kDa Phosphate Buffered Saline, 1x, pH 7.4, Life Technologies 10010023 Polyvinyl alcohol Modified polyethersulfone hollow fiber filter module, 750kDa cutoff ·Water for injection 2% Brij® S100 Surfactant Malic acid buffer, pH 3

[0067] PLGA Nanoparticle Preparation Protocol Malat-1-loaded poly(dl-lactide-co-glycolide) (PLGA) particles were prepared using a double emulsion solvent evaporation technique. Three different types of PLGA polymers (PLGA lactide:glycolide (50:50), PLGA lactide:glycolide (85:15), and PLGA lactide:glycolide (5:95)) were used to fabricate nanoparticles. Because the three polymers have different ratios of lactic acid (LA) to glycolic acid (GA), the release rate of antisense oligonucleotides (ASO) should vary, with the slowest releasing nanoparticles resulting from having the highest LA content.

[0068] Briefly, polyvinyl alcohol (PVA) solution was made by dissolving PVA in phosphate buffered saline (PBS). PLGA was dissolved in ethyl acetate. Polyethylenimine (PEI) was dissolved in water. ASO was dissolved in PBS at pH 7.4. ASO and PEI solutions were mixed together in a 1:1 molar ratio. PEI is a positively charged polymer that is complexed to the negatively charged ASO to prevent charge repulsion and allow encapsulation in the negatively charged PLGA. The ASO-PEI complex was then mixed with the PLGA solution and sonicated to create a water-in-oil emulsion. This emulsion was then added to a PVA solution, which acts as a stabilizer for the nanoparticles, and sonicated to create a water-in-oil-in-water emulsion. The ethyl acetate was then removed by evaporation. The emulsion was purified and buffer exchanged by tangential flow filtration (TFF) using PBS. The resulting emulsions were characterized by several analytical techniques to measure particle size, polydispersity, and ASO loading, and then stored at -20°C until administration to animals.

[0069] Example 2: Characterization of ASO-loaded PLGA nanoparticles The results presented here summarize the characterization of nanoparticles made with each type of PLGA when Malat-1 ASO was encapsulated within the particles. The size distributions shown in Table 1 indicate that particles made with 85:15 PLGA had the largest hydrodynamic diameter and those made with 50:50 PLGA were the smallest. Particles made with 85:15 and 5:95 PLGA were nearly neutral, while those made with 50:50 were slightly negative. The majority of the particles had a polydispersity (PDI) of less than 0.3, indicating a narrow size distribution. Triplicate measurements were performed for each formulation and the average readings are reported. [Table 1]

[0070] Example 3: Malat-1 ASO concentration in nanoparticles After extraction of the ASO from the particles, the concentration of Malat-1 ASO in the nanoparticles was determined by UV absorbance at 260 nm. The UV readings and corresponding ASO concentrations are shown in Table 2. [Table 2]

[0071] Example 4: In vitro release of ASO from PLGA nanoparticles The in vitro release kinetics of ASO-loaded nanoparticles was investigated using particles made from three different PLGA compositions, 5:95, 50:50, and 85:15. The release kinetics was performed on a SOTAX USB IV dissolution apparatus in an open system. The mechanical and chemical properties, swelling behavior, resistance to hydrolysis, and subsequent biodegradation rate of the polymer are directly affected by the crystallinity of PLGA, which further depends on the molar ratio of the individual monomeric components in the copolymer chain. Crystalline PGA, when copolymerized with PLA, reduces the crystallinity of PLGA, and as a result, increases the rate of hydrolysis and degradation. The results of the release of ASO from the two groups of nanoparticles can be seen in Figure 1. Free ASO (not encapsulated in nanoparticles) at a concentration of 6 mg / mL was used as a control. Using a flow rate of 8 mL / min, nanoparticles made with 5:95 PLGA were found to have the fastest release, followed by 50:50 and 85:15.

[0072] Example 5: Intraventricular delivery of PLGA nanoparticles in mice An efficacy study comparing the effects of unformulated Malat-1 ASO and nanoparticle-encapsulated Malat-1 in C57B6 mice was performed by injecting mice with intracerebroventricular (ICV) solution. Ten mice were injected in each of five different groups (50 mice in total): a buffer PBS control group, an unformulated Malat-1 ASO group (50 μg), and three PLGA nanoparticle groups described by their LA:GA ratios of 85:15, 50:50, and 5:95, containing approximately 50 μg of Malat1 ASO. Animals were followed for one week after ICV injection. The formulation was well tolerated with no observable safety signals. Knockdown levels are shown for different regions of the CNS in Figure 2.

[0073] In mice injected with the nanoparticle formulation, significant knockdown was observed in the lumbar spinal cord region, indicating that the Malat-1 ASO was released from the nanoparticles and was able to act on the intended target. Animals administered nanoparticles with a 50:50 LA:GA ratio showed higher knockdown compared to 85:15 and 5:95 PLGA nanoparticles, especially in the lumbar spinal cord cortex, indicating that the ASO was released from the 50:50 nanoparticles, which had better distribution and provided more effective delivery compared to the other nanoparticle groups.

[0074] Example 6: Intrathecal delivery of PLGA nanoparticles in rats To test the nanoparticle delivery of Malat-1 ASO in the intrathecal (IT) space, the same formulation was prepared and injected intrathecally into rats. A similar experimental design to the mouse ICV study in Example 5 was used for this rat study. A total of 50 rats were used in this experiment, with 10 rats in each of the five dose cohorts: a buffer PBS control group, an unformulated Malat-1 ASO group (150 μg), and three PLGA nanoparticle groups, 85:15, 50:50, and 5:95. After two weeks, the rats were sacrificed and the percent knockdown of Malat-1 expression in various regions of the CNS and brain was measured. No safety issues were observed in the rats after the solution was injected. More than 90% signal knockdown in the spinal cord region in all groups (Figure 3) indicates that the injection was successful.

[0075] Malat-1 expression was also measured in different regions of the brain, especially the cerebral cortex (Figure 4). This data indicates that ASOs were delivered from PLGA particles and reached deeper regions of the brain, such as the striatum. This experiment also demonstrated that administration by IT injection was safe.

[0076] In conclusion, ASO-encapsulated nanoparticles with three different release rates were created and injected into mice by ICV injection. Knockdown in these mice showed that ASO was released from the nanoparticles and was still active. Furthermore, no safety signals were recorded. These same formulations were injected intrathecally into rats. Knockdown in these rats further showed that ASO was released from the nanoparticles and could reach deeper regions of the brain.

[0077] Example 7: Study 2 - Materials and Methods Study 2 is described in Examples 7-11. material PLGA lactide:glycolide (50:50), ester terminated, average 100,000 Da PLGA lactide:glycolide (85:15), ester terminated, 190,000~240,000Da PLGA L-lactide:glycolide (5:95), 190,000~210,000Da Polyethyleneimine (PEI), linear, 2.5 kDa, Sigma 764604 Ethyl Acetate Malat-1 antisense oligonucleotide: [ka] (wherein "o" is a phosphodiester (if "o" is not labeled with phosphorothioate, Me U is 5-methyl-uracil, Me C is 5-methyl-cytosine, the underlined nucleosides are MOE), 7 kDa Phosphate Buffered Saline, 1x, pH 7.4, Life Technologies 10010023 Polyvinyl alcohol Modified polyethersulfone hollow fiber filter module, 750kDa cutoff ·Water for injection

[0078] PLGA Nanoparticle Preparation Protocol Malat-1 ASO-loaded poly(dl-lactide-co-glycolide) (PLGA) particles were prepared using a double emulsion solvent evaporation technique. Three different types of PLGA polymers (PLGA lactide:glycolide (50:50), PLGA lactide:glycolide (85:15), and PLGA lactide:glycolide (5:95)) were used to fabricate nanoparticles.

[0079] The same protocol in Example 1 was followed for the enhanced precomplexation step in ASO-PEI conjugation. Briefly, PLGA polymer was dissolved in ethyl acetate and mixed with PEI-ASO precomplex to form a water-in-oil emulsion. Prior to making the primary emulsion, PEI was dissolved in deionized water, heated to 80°C, and mixed at 300 rpm. The temperature of the dissolved PEI solution was reduced to 60°C to form the ASO-PEI precomplex. The water-in-oil emulsion was further emulsified with 2.5% w / v PVA solution to form a water-in-oil emulsion. The final emulsion was stirred at ambient conditions for 18 hours to remove the solvent. The final product was purified and the buffer was exchanged by tangential flow filtration (TFF) using PBS. These resulting nanoparticles were characterized by several analytical techniques to measure size, polydispersity, and ASO loading, and then stored frozen at -20°C until administration to animals.

[0080] Example 8: Characterization of ASO-loaded PLGA nanoparticles All particles were less than 300 nm with PDI values ​​less than 0.25, indicating a monodisperse size distribution (see Table 3). Particles with a LA:GA ratio of 5:95 had the smallest hydrodynamic diameter (176 nm) and particles with a LA:GA ratio of 85:15 had the largest particle size (288.6 nm). Near neutral charge was observed for all three formulations. [Table 3]

[0081] Example 9: ASO loading in nanoparticles The amount of Malat-1 ASO concentration in the nanoparticles was determined by extracting the ASO from the nanoparticles and measuring the UV absorbance at 260 nm on a SoloVPE. The measured ASO concentrations are shown in Table 4. [Table 4]

[0082] Example 10: In vitro release of ASO from PLGA nanoparticles The in vitro release kinetics of ASO-loaded nanoparticles were measured in a closed loop configuration using a fully automated flow-through cell lysis apparatus (USP4, Sotax). A cell with an internal diameter of 22.4 mm was used. A UV spectrophotometer at a wavelength of 260 nm was used to measure the ASO concentration of the dissolution fluid in real time. The temperature was maintained at 37°C during the process. The flow rate of PBS dissolution medium through the cells was 16 mL / min. The dissolution apparatus pumped PBS and recirculated it through the cells, and as the ASO was released from the nanoparticles, changes in concentration were detected. From this, a cumulative release curve was generated and can be seen in Figure 5. Unencapsulated ASO at a concentration of 2.9 mg / mL was used as a control. Nanoparticles made with a LA:GA ratio of 5:95 were found to have the fastest release. This is expected since this polymer has the lowest LA concentration and is therefore the least crystalline and hydrophobic. Nanoparticles made with a ratio of 85:15 released the slowest due to the higher content of glycolic acid and therefore more hydrophilic.

[0083] Example 11: Intrathecal delivery of PLGA nanoparticles in rats The gene regulatory effect of Malat-1 ASO-loaded PLGA nanoparticles was tested in Sprague-Dawley rats by injecting the solution intrathecally and measuring knockdown of the Malat-1 gene in different sections of brain tissue. Ten rats were injected in each of five different groups, including a buffer PBS control group, an unformulated free Malat-1 group (75 μg), and the three nanoparticle groups mentioned above, 85:15, 50:50, 5:95, all containing approximately 75 μg of Malat-1. The animals were monitored for two weeks after IT injection. The formulation was well tolerated with no observable safety issues. The knockdown levels are shown for various regions of the CNS in Figure 6. The signal knockdown of over 90% in the spinal cord region in all groups (Figure 6) indicates that the injections were well performed, as a near complete knockdown in this region would be expected with a successful injection. Malat-1 knockdown in the cortex and striatum indicates that ASO was released from the particles as it was distributed through the CNS (Figure 6). PLGA particles with a composition of 85:15 showed a lower knockdown percentage compared to 50:50 and 5:95. There was increased knockdown from the 5:95 and 50:50 particles compared to the control dose of unformulated ASO.

[0084] In conclusion, PLGA nanoparticles encapsulating Malat-1 ASO with different release rates were administered intrathecally to rats. Particles with fast and medium release rates showed improved knockdown compared to unformulated ASO. Another rat IT study was tested to confirm the effect of particle degradation rate on ASO knockdown in the CNS.

[0085] Example 12: Study 3 - Materials and Methods Study 3 is described in Examples 12-15. material PLGA lactide:glycolide (50:50), ester terminated, average 40,000 Da PLGA lactide:glycolide (75:25), ester terminated, 40,000 Da PLA L-lactide, 40,000Da Polyethyleneimine (PEI), linear, 2.5 kDa, Sigma 764604 Ethyl Acetate Malat-1 antisense oligonucleotide: [ka] (wherein "o" is a phosphodiester (if "o" is not labeled with phosphorothioate, Me U is 5-methyl-uracil, Me C is 5-methyl-cytosine, the underlined nucleosides are MOE), 7 kDa Phosphate Buffered Saline, 1x, pH 7.4, Life Technologies 10010023 Brij(R) S100% Modified polyethersulfone hollow fiber filter module, 500kDa cutoff ·Water for injection 10% sucrose

[0086] PLGA Nanoparticle Preparation Protocol In this study, Malat-1 ASO-loaded PLGA nanoparticles were prepared using a double emulsion solvent evaporation technique. Three different types of PLGA polymers were used: PLGA lactide:glycolide (50:50), PLGA lactide:glycolide (75:15), and PLA lactide:glycolide (100:0). As previously described, the PLGA polymer was dissolved in ethyl acetate and mixed with the PEI-ASO (2.8:1) precomplex to form a water-in-oil emulsion. The water-in-oil emulsion was further emulsified with 0.2% w / v Brij® S 100% solution to form a water-in-oil emulsion. The final emulsion was stirred at ambient conditions for 18 hours to remove the solvent. The final product was purified and buffer exchanged by tangential flow filtration (TFF) using PBS, followed by addition to 10% sucrose formulation to prevent any nonspecific aggregation during freeze / thaw cycles. The resulting nanoparticles were characterized by several analytical techniques to measure size, polydispersity, and ASO loading, and then stored frozen at -20°C until administration to animals.

[0087] Example 13: Characterization of ASO-encapsulated PLGA nanoparticles The particle size was consistent for each batch and evenly distributed with a PDI less than 0.2 (Table 5). The PLGA 75:25 composition achieved a negative charge compared to the other formulations. [Table 5]

[0088] Example 14: ASO concentration in nanoparticles The amount of Malat-1 ASO concentration in the nanoparticles was determined by extracting the ASO from the nanoparticles and measuring the UV absorbance at 260 nm on the SoloVPE. The measured ASO concentrations are shown in Table 6. [Table 6]

[0089] Example 15: Intrathecal delivery of PLGA nanoparticles in rats The knockdown effect of Malat-1 loaded PLGA nanoparticles was tested in Sprague-Dawley rats by injecting the solution intrathecally and measuring knockdown of the Malat-1 gene in different sections of brain tissue. Ten rats were injected in each of five different groups, including a buffer PBS control group, a free Malat-1 group (75 μg), and the three nanoparticle groups mentioned above 100:0, 75:25, 50:50, all containing approximately 75 μg of Malat-1. The animals were allowed to survive for two weeks after IT injection. The formulation was well tolerated with no observable safety issues. The knockdown levels are shown for various regions of the CNS in Figure 7. More than 90% signal knockdown in the spinal cord region of all groups (Figure 7) indicates that the injections were well performed, as a near complete knockdown in this region would be expected with a successful injection. Malat-1 knockdown in the cortex and striatum indicates that ASO was released from the particles as it was distributed through the CNS (Figure 7). All nanoparticle formulations showed similar knockdown kinetics in the cortex and striatum compared to the corresponding unformulated ASO. The results showed that the PLGA formulations provided at least similar treatment when compared to unformulated ASO. PLGA 50:50 nanoparticles show even slightly higher efficacy compared to unformulated ASO and other nanoparticle formulations. This study also confirmed the safe delivery of PLGA nanoparticles by IT administration. Current intrathecal administration methods in rodents only provide a single time point to assess knockdown levels in the CNS. Therefore, the next study investigated the impact of PLGA nanoparticles by determining ASO uptake in CNS tissues using real-time imaging methods.

[0090] Example 16: Study 4 - Materials and Methods Study 4 is described in Examples 16-19.

[0091] material PLGA lactide:glycolide (50:50), ester terminated, average 200,000 Da Polyethyleneimine (PEI), linear, 2.5 kDa, Sigma 764604 Ethyl acetate β-globin antisense oligonucleotide: [ka] (In the formula, Underlined The nucleoside has a 2'-O-(2-methoxyethyl) (MOE) modification), 7 kDa Phosphate Buffered Saline, 1x, pH 7.4, Life Technologies 10010023 Brij(R) S100% Modified polyethersulfone hollow fiber filter module, 500kDa cutoff 10% sucrose

[0092] PLGA Nanoparticle Preparation Protocol Using a double emulsion solvent evaporation technique, β-globin ASO-loaded PLGA nanoparticles were prepared with PLGA lactide:glycolide (50:50). Precomplexed β-globin ASO-PEI solutions were mixed in 2% Brij® S100 surfactant, followed by sonication at 100% for 60 seconds and 80% for 60 seconds. To achieve targeted ASO loading in PLGA particles, a secondary emulsion was created by using malic acid buffer at pH 3 to enhance the stability of the precomplexed medium in the primary emulsion. The pH of the final solution was then adjusted to 7.2 by dilution in PBS. The suspension was characterized by several analytical techniques to measure particle size, polydispersity, and ASO loading, and then stored at -20°C before use in animals.

[0093] Example 17: Characterization of PLGA nanoparticles encapsulating β-globin ASO The particle size of the β-globin ASO-loaded PLGA nanoparticles was measured using the same method previously used for the Malat-1 formulation. The results showed that the β-globin-loaded nanoparticles were approximately 264 nm and had a uniformly distributed PDI value of 0.19.

[0094] Example 18: β-globin ASO concentration in nanoparticles The concentration of β-globin encapsulated in the nanoparticles was determined by extracting the ASO from the nanoparticles and measuring the UV absorbance at 260 nm on a SoloVPE. The measured β-globin ASO concentration was 3.3 mg / mL.

[0095] Example 19: In vivo live imaging in a reporter mouse model to assess ASO uptake The increased potency of ASO-loaded nanoparticles was investigated using a luciferase reporter mouse model. The AAV reporter construct was designed to read out splice correction by bioluminescence imaging. The luciferase reporter gene in the construct is split by a beta-globin intron, which is spliced ​​in the presence of the beta-globin ASO, resulting in expression of the luciferase gene. The AAV construct was administered through IV injection on postnatal day 0 to achieve widespread expression of AAV in the brain. In 6- to 8-week-old mice, the ASO formulation was injected ICV at a dose equivalent to 33 μg of ASO, and overhead bioluminescence imaging was performed at multiple time points using an IVIS imager. Each image was taken 10 min after intraperitoneal injection of D-luciferin substrate. Figure 8 shows the fold change in bioluminescence from baseline values ​​prior to ASO administration. Bioluminescence signals in animals administered encapsulated beta-globin are significantly higher than those administered by unformulated ASO at any given time point during the 2-week imaging study. The results confirm that PLGA nanoparticles were able to provide faster and higher ASO uptake in the brain for an extended period of time.

[0096] Other embodiments While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A central nervous system (CNS) delivery composition comprising a polymeric nanocarrier and an antisense oligonucleotide, wherein the antisense oligonucleotide is encapsulated within the polymeric nanocarrier and the antisense oligonucleotide is directly precomplexed with a cationic molecule.

2. 2. The CNS delivery composition of claim 1, wherein the polymeric nanocarrier is selected from the group consisting of poly(l-lactide), poly(glycolide), poly(d,l-lactide) (PLA), poly(dioxanone), poly(d,l-lactide-co-l-lactide), poly(d,l-lactide-co-glycolide), poly(glycolide-co-trimethylene carbonate), poly(caprolactone) ("polycaprolactone"), poly(d,l-lactide-co-glycolide) (PLGA), poly(dioxanone)poly(glycolide-co-trimethylene carbonate), and mixtures thereof.

3. The CNS delivery composition of claim 1 , wherein the polymeric nanocarrier is PLGA.

4. 4. The CNS delivery composition of claim 3, wherein the composition is a PLGA nanoparticle comprising a ratio of lactic acid:glycolic acid in the range of 2:98 to 100:

0.

5. The CNS delivery composition of any one of claims 1 to 4, wherein the cationic molecule is a cationic peptide.

6. The CNS delivery composition of any one of claims 1 to 4, wherein the cationic molecule is chitosan, hexadecylamine, or lauric arginate.

7. The CNS delivery composition of any one of claims 1 to 5, wherein the cationic molecule is polyethyleneimine (PEI).

8. The CNS delivery composition of claim 7 , wherein the PEI is linear PEI or cross-linked PEI.

9. The CNS delivery composition of any one of claims 1 to 8, further comprising a therapeutic agent.

10. 10. The CNS delivery composition of claim 9, wherein the therapeutic agent is selected from the group consisting of a small molecule, cDNA, mRNA, siRNA, miRNA, an aptamer, and a ribozyme.

11. The CNS delivery composition of any one of claims 1 to 10, formulated for intrathecal delivery to a human subject.

12. The CNS delivery composition of any one of claims 1 to 11, wherein the antisense oligonucleotide is a gapmer or a splice-switching antisense oligonucleotide.

13. The CNS delivery composition of any one of claims 1 to 11, wherein the antisense oligonucleotide consists of the nucleic acid sequence set forth in SEQ ID NO:

1.

14. The antisense oligonucleotide is comprised of 18 linked nucleosides, the oligonucleotide having the nucleobase sequence Ｍｅ U Ｍｅ CA Ｍｅ C Ｍｅ U Ｍｅ U Ｍｅ U Ｍｅ CA Ｍｅ U.A.A. Ｍｅ U.G. Ｍｅ C Ｍｅ a nucleic acid base sequence consisting of UGG (SEQ ID NO: 1), each internucleoside bond of said oligonucleotide is a phosphorothioate bond, and each nucleoside of said oligonucleotide is a 2'-methoxyethyl nucleoside; Ｍｅ U is 5-methyl-uracil; Ｍｅ The CNS delivery composition of any one of claims 1 to 11, wherein C is 5-methylcytosine.

15. 15. The CNS delivery composition of any one of claims 1 to 14 for treating a CNS disorder in a human subject in need thereof.

16. 16. The CNS delivery composition of claim 15, wherein the CNS delivery composition is administered by intrathecal injection.

17. 15. The CNS delivery composition of claim 14 for treating spinal muscular atrophy (SMA) in a human subject in need thereof, wherein the CNS delivery composition increases the inclusion of exon 7 in SMN2 messenger ribonucleic acid (mRNA) transcripts in human subjects having a loss of both functional copies of the SMN1 gene, or increases the inclusion of exon 7 in SMN2 messenger ribonucleic acid (mRNA) transcripts in human subjects having a mutation in the SMN1 gene that causes a functional SMN protein deficiency, wherein the CNS delivery composition is administered by injection into the intrathecal space of the human subject.

18. The CNS delivery composition of any one of claims 15 to 17, wherein the injection is a bolus injection.

19. 1. A composition for delivering an antisense oligonucleotide to the CNS of a human subject, comprising the antisense oligonucleotide encapsulated within PLGA nanoparticles, the PLGA nanoparticles having a lactic acid:glycolic acid ratio ranging from 2:98 to 100:0, the antisense oligonucleotide being precomplexed with PEI or another cationic molecule, and the composition being administered by intrathecal injection.

20. 20. The composition of claim 19, wherein the human subject has a CNS disorder.

21. 21. The composition of claim 20, wherein the CNS disorder is SMA, ALS, Parkinson's disease, Alzheimer's disease, Huntington's disease, Angelman syndrome, frontotemporal dementia (FTD), Creutzfeldt-Jakob disease, spinocerebellar ataxia type 3 (SCA3), Menkes disease, a synucleinopathy, or a tauopathy.

22. The composition of any one of claims 19 to 21, wherein the antisense oligonucleotide is delivered to the CNS of the human subject 0.1 hours to 1 week after administration.

23. The composition of any one of claims 19 to 21, wherein the antisense oligonucleotide is delivered to the CNS of the human subject 1 to 6 days after administration.

24. The composition of any one of claims 19 to 21, wherein the antisense oligonucleotide is delivered to the cortex of the human subject within 0.1 to 48 hours after administration.

25. The composition of any one of claims 19 to 21, wherein the antisense oligonucleotide is delivered to the striatum of the human subject within 6 hours after administration.

26. The composition according to any one of claims 19 to 25, wherein the other cationic molecule is a cationic peptide, chitosan, hexadecylamine, or lauric arginate.

27. The composition of any one of claims 19 to 25, wherein the PEI is linear PEI or crosslinked PEI.

28. 1. A composition for increasing the amount of an antisense oligonucleotide delivered to the brain of a human subject in need thereof compared to delivery of a solution of the antisense oligonucleotide in an aqueous buffer, the composition comprising PLGA nanoparticles encapsulating the antisense oligonucleotide, the antisense oligonucleotide being precomplexed with PEI or another cationic molecule, the PLGA nanoparticles having a lactic acid:glycolic acid ratio in the range of 2:98 to 100:0, and the composition being injected intrathecally.

29. 1. A composition for delivering an antisense oligonucleotide deeper into the brain of a human subject compared to a solution of the antisense oligonucleotide in an aqueous buffer, comprising PLGA nanoparticles encapsulating the antisense oligonucleotide, the antisense oligonucleotide being precomplexed with PEI or another cationic molecule, the PLGA nanoparticles having a lactic acid:glycolic acid ratio ranging from 2:98 to 100:0, and the composition being injected intrathecally.

30. 30. The composition of claim 29, wherein more of the antisense oligonucleotide is delivered to the striatum, thalamus, substantia nigra, and / or cerebellum of the brain compared to a solution of the antisense oligonucleotide in the aqueous buffer.

31. 1. A composition for reducing the number of administrations of an antisense oligonucleotide to the spinal cord and / or brain of a human subject compared to a solution of the antisense oligonucleotide in an aqueous buffer, comprising PLGA nanoparticles encapsulating the antisense oligonucleotide, the antisense oligonucleotide being precomplexed with PEI or another cationic molecule, the PLGA nanoparticles having a lactic acid:glycolic acid ratio in the range of 2:98 to 100:0, and the composition being injected intrathecally.

32. The composition of any one of claims 28 to 31, wherein the PEI is linear PEI or crosslinked PEI.

33. The composition of any one of claims 28 to 31, wherein the additional cationic molecule is a cationic peptide, chitosan, hexadecylamine, or lauric arginate.

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