Antisense oligonucleotide (ASO)-based methods for substrate reduction therapy for lysosomal storage diseases

Abstract

The present invention relates to a method of treating a lysosomal storage disease in a subject by substrate reduction therapy (SRT), the method including administering to the subject a therapeutically effective amount of at least one antisense oligonucleotide (ASO) capable of affecting expression of an enzyme in a glycosaminoglycan (GAG) biosynthesis pathway.

Description

[0001] ANTISENSE OLIGONUCLEOTIDE (ASO)-BASED METHODS FOR SUBSTRATE REDUCTION THERAPY FOR LYSOSOMAL STORAGE DISEASES FIELD OF THE INVENTION

[0002] The present invention is generally directed to methods for RNA-based substrate reduction therapy (SRT). More specifically, the invention relates to an antisense oligonucleotide (ASO)-based method for SRT for treating lysosomal storage diseases.

[0003] BACKGROUND OF THE INVENTION

[0004] Mucopolysaccharidosis type IIIA (MPS3A), also known as Sanfilippo syndrome type A, is a rare, recessive, inherited lysosomal storage disorder characterized by a deficiency in the enzyme heparan N-sulfatase, or N-sulfoglucosamine sulfohydrolase (SGSH). This enzyme is crucial for the degradation of heparan sulfate, a glycosaminoglycan (GAG). The deficiency of SGSH leads to the accumulation of heparan sulfate in lysosomes, resulting in progressive cellular and tissue damage.

[0005] Patients with MPS3 A typically present with normal development for the first few years of life, followed by a rapid decline in cognitive and motor functions. Symptoms include severe behavioral problems, sleep disturbances, and progressive intellectual disability. Physical symptoms may be less pronounced compared to other mucopolysaccharidoses, but hepatosplenomegaly, coarse facial features, and skeletal abnormalities are common. The disease course is relentlessly progressive, leading to severe neurological deterioration and premature death, often in the second decade of life.

[0006] Substrate reduction therapy (SRT) is a therapeutic approach designed to reduce the synthesis of substrates that accumulate due to enzyme deficiencies, thereby alleviating the pathological effects of these accumulations. In the context of MPS3 A, SRT aims to reduce the production of glycosaminoglycans, particularly heparan sulfate.

[0007] Traditionally, SRT utilizes small molecules that inhibit enzymes involved in the synthesis pathways of the accumulated substrates. For example, inhibitors targeting the enzymes responsible for GAG synthesis can reduce the intracellular and extracellular buildup of these substances. This can potentially mitigate cellular dysfunction and delay the progression of symptoms.

[0008] Several challenges exist in developing effective SRT for MPS3 A. These include ensuring sufficient inhibition of substrate synthesis without causing off-target effects, achieving adequate distribution of the therapeutic agents to the central nervous system (CNS), and managing longterm treatment adherence and effectiveness. Nonetheless, SRT represents a promising avenue for treating lysosomal storage disorders by addressing the root cause of substrate accumulation. Several SRT based therapies are already in clinical use, including the glucose analog Miglustat and the ceramide analog eliglustat for Gaucher disease treatment.

[0009] Previous experiments with substrate reduction therapy (SRT) in MPS3A patients have shown mixed results, often highlighting the challenges associated with this therapeutic approach. One notable attempt involved the use of genistein, an isoflavone that inhibits the synthesis of GAGs by interfering with the tyrosine kinase activity of epidermal growth factor receptors. Preclinical studies in MPS3 A models demonstrated that genistein could reduce GAG accumulation and improve some aspects of disease pathology. These promising results led to early-phase clinical trials in patients with MPS3 A.

[0010] However, the clinical trials with genistein in MPS3 A patients did not achieve the expected therapeutic success. The primary reasons for this failure included insufficient penetration of the blood-brain barrier (BBB), leading to inadequate concentrations of the drug in the CNS, which is the primary site of pathology in MPS3 A. Additionally, the dosing required to achieve a therapeutic effect was associated with adverse effects, limiting the drug's tolerability. The variability in response among patients also suggested that genistein alone might not be potent enough to significantly alter the disease course. Moreover, long-term adherence to the therapy posed challenges, as the benefits did not clearly outweigh the difficulties associated with chronic treatment.

[0011] Another SRT approach involved small molecule inhibitors targeting key enzymes in the GAG biosynthesis pathway, such as glucosylceramide synthase inhibitors. Although these inhibitors showed potential in reducing GAG levels in vitro and in animal models, their translation to human clinical trials encountered similar obstacles. Effective targeting and delivery to the CNS remained a significant hurdle, and systemic side effects due to the broad inhibition of GAG synthesis pathways further complicated their use.

[0012] Antisense oligonucleotides (ASOs) are short, synthetic strands of nucleic acids designed to bind to specific RNA sequences, thereby modulating gene expression. One specialized form of ASOs is gapmers, which are used to degrade target RNA molecules through an RNase H-mediated mechanism. Gapmers consist of a central DNA region flanked by modified RNA nucleotides that enhance binding affinity and nuclease resistance.

[0013] Gapmers function by hybridizing to complementary RNA sequences, forming RNA-DNA hybrids that are recognized and cleaved by RNase H. This results in the degradation of the target RNA, leading to a decrease in the production of the corresponding protein. This mechanism is particularly useful for reducing the levels of pathogenic proteins in diseases caused by overexpression or gain-of-function mutations.

[0014] Gapmers offer several advantages, including high specificity for target sequences, the ability to modulate gene expression without altering the genome, and the potential for systemic delivery. ASOs, including gapmers, were also shown to be efficiently distributed in the CNS following intrathecal injections.

[0015] Accordingly, the use of ASO and specifically gapmers for substrate reduction therapy may solve issues related with previously used approaches.

[0016] SUMMARY OF INVENTION

[0017] The following embodiments are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

[0018] According to some embodiments, there is provided a method of treating a lysosomal storage disease in a subject by substrate reduction therapy (SRT), the method including administering to the subject a composition including therapeutically effective amount of at least one antisense oligonucleotide (ASO) capable of affecting expression of an enzyme in a glycosaminoglycan (GAG) biosynthesis pathway.

[0019] In some embodiments, the lysosomal storage disease is selected from mucopolysaccharidosis type IIIA (MPS3A), mucopolysaccharidosis type IIIB (MPS3B); mucopolysaccharidosis type I (MPSI), mucopolysaccharidosis type II (MPSII), and multiple sulfatase deficiency (MSD).

[0020] In some embodiments, the GAG is heparan sulfate.

[0021] In some embodiments, the enzyme is encoded by a gene selected from exostosin glycosyltransferase 1 (EXT1), exostosin glycosyltransferase 2 (EXT2), exostosin-like glycosyltransferase 2 (EXTL2), exostosin-like glycosyltransferase 3 (EXTL3), xylosyltransferase 1 (XYLT1), and combinations thereof.

[0022] In some embodiments, the ASO includes at least one phosphorothioate (PS) bond and / or at least one 2'-O-methoxy ethyl (MOE) group. In some embodiments, the ASO is a gapmer. In some embodiments, the ASO has a length of about 14-30 nucleotides.

[0023] In some embodiments, the ASO targets a sequence at least at least 90%, 95%, or 99% identical to a sequence identical to a sequence selected from sequences in Table 1. In some embodiments, the ASO targets a sequence at least at least 90%, 95%, or 99% identical to a sequence selected from SEQ ID Nos: 1-55.

[0024] In some embodiments, the ASO includes a sequence at least at least 90%, 95%, or 99% identical to a sequence selected from ASO sequences presented in Table 2. In some embodiments, the ASO sequence includes a sequence at least at least 90%, 95%, or 99% identical to a sequence selected from ASO sequences set forth in SEQ ID Nos: 61-115.

[0025] In some embodiments, the ASO is a modified ASO selected from modified ASOs presented in Table 3. In some embodiments, the ASO is a modified ASO selected from modified ASOs defined by sequences set forth in SEQ ID Nos: 121-175.

[0026] In some embodiments, the ASO is selected from EXT1 1313 (SEQ ID NO: 133), EXT1 1315 (SEQ ID NO: 134), EXT1J248 (SEQ ID NO: 124), EXT2J577 (SEQ ID NO: 152), EXTL2 816 (SEQ ID NO: 157), EXTL3_3013 (SEQ ID NO: 165), XYLT1_1147 (SEQ ID NO: 167), EXT 1_2915 (SEQ ID NO: 148), EXT1 1309 (SEQ ID NO: 131), andEXTl_1311 (SEQ ID NO: 132)

[0027] In some embodiments, the method causes a reduction of at least about 30% in expression levels of the enzyme.

[0028] In some embodiments, the method causes a reduction of at least about 30% in levels of the GAG.

[0029] In some embodiments, the method causes a reduction of at least about 30% in levels of a neuroinflammation marker selected from IL1 0 (interleukin- 1 beta), AIF1 (allograft Inflammatory Factor 1), GFAP (glial fibrillary acidic protein), and / or Serpin3n.

[0030] In some embodiments, the composition is a pharmaceutical composition, further including a pharmaceutically acceptable carrier.

[0031] In some embodiments, the administration is intrathecal administration.

[0032] In some embodiments, there is provided an ASO for use in a method of treating a lysosomal storage disease in a subject by SRT, wherein the ASO is capable of affecting expression of an enzyme in a GAG biosynthesis pathway.

[0033] In some embodiments, there is provided a gapmer antisense oligonucleotide (ASO) capable of affecting expression of an enzyme in a glycosaminoglycan (GAG) biosynthesis pathway, wherein the gapmer has a length of about 14-30 nucleotides, and includes at least one phosphorothioate (PS) bond and about 8-12 nucleotides modified by the addition of an 2'-O-m ethoxy ethyl (MOE) group.

[0034] In some embodiments, the gapmer is selected from EXT1 1248, EXT2 1577, EXTL2 816, EXTL3 3013, XYLT1 1147, EXT1 2915, EXT1 1309, EXT1 1311, EXT1 1313, and EXT1 1315. In some embodiments, the gapmer is selected from sequences set forth in SEQ ID Nos: 124, 152, 157, 165, 167, 148, 131, 132, 133, and 134

[0035] In addition to the exemplary embodiments described above, further embodiments will become apparent by reference to the figures and by study of the following detailed descriptions.

[0036] BRIEF DESCRIPTION OF DRAWINGS

[0037] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0038] Fig- 1 shows heparan sulfate (HS )biosynthesis scheme, highlighting genes encoding key enzymes which are targeted by ASOs in this therapeutic approach (XYLT1, EXT1, EXT2, EXTL2, EXTL3) (taken from Kreuger et al. 2012, Heparan sulfate biosynthesis: regulation and variability, J Histochem Cytochem 60(12):898-907). HS - Heparan Sulfate. NDST - N-deacetylase / N-sulfotransferase. OST- O-sulfotransferases. NS- N-sulfated.

[0039] Fig- 2 shows degradation of heparan sulphate scheme, including genes encoding enzymes and syndromes related to their malfunction, (taken from Kong et al, 2022, Orphanet J Rare Dis.

[0040] 17(1): 112). MPS: mucopolysaccharidosis; IDS: Iduronate-2-sulfatase ; IDUA: a-L-iduronohydrolase; GNS: glucosamine (N-acetyl)-6-sulfatase; SGSH: N-sulfoglucosamine sulfohydrolase; HGSNAT: Heparan-a-glucosaminide N-acetyltransf erase; NAGLU: N-Acetylglucosaminidase; GUSB: p -Glucuronidase.

[0041] Figs. 3A-3H show screening of design ASOs against target genes in HEK293 cells by measuring relative mRNA transcription levels after transfection. Fig 3A. ASOs targeting EXTL3.

[0042] Fig. 3B. ASOs targeting XYLT1. Fig. 3C. ASOs targeting EXTL2. Fig. 3D. ASOs targeting EXT1. Fig. 3E. ASOs targeting EXT2. Final concentration of ASO was 50-200nM (indicated on the X axis). Expression levels were normalized to GAPDH levels, and compared to untreated cells (set to 1.0). SCR=mock ASO, ns= not signification. N=3 per wells, ANOVA test, *p-value <0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001. Figs. 3F-3H show screening of optimized EXT 1 -targeting ASOs, by measuring relative mRNA transcription levels (qRT-PCR) GM01881A (patient-derived fibroblasts) after transfection. Figs. 3F-3G show EXT1 levels in following treatment with various amounts of the indicated ASOs. Fig. 3H. shows EXT1 levels in a dose-response curve following treatment with selected ASOs (EXT1 1248, EXT1 1315, EXT1 1313, EXT1 1311, EXT1 1309, and EXT1 2915), in 6.25-100 nM concentrations.

[0043] Figs. 4A-4G show immunofluorescence staining of GM01881A fibroblasts. Cells were stained with mouse monoclonal anti-heparan sulfate antibody (10E4 epitope, Tx-Red) and DAPI for nuclei (blue). Fig. 4A. Untreated patient fibroblasts (control). Fig.4B. Patient fibroblasts treated with 100 nM of mock ASO (control). Fig. 4C-4G. Patient fibroblasts treated with 100 nM of the respective ASO: Fig. 4C. EXT1 (EXT1_1248); Fig. 4D. EXT2 (EXT2_1577); Fig. 4E.

[0044] EXTL2 (EXTL2 816); Fig. 4F. EXTL3 (EXTL3 3013); Fig. 4G. XYLT1 (EYLT1 1147). Scale bar = 150 pm.

[0045] Fig- 5 shows glycosaminoglycan (GAG) quantification in comparison to DNA normalization to control, following ASO treatments. GM01881A (abbreviated as GM) are fibroblasts from a MPS3A patient, where ATTC is a normal human donor fibroblasts cell line (WT). Fibroblasts were treated with ASOs targeting EXT1 (EXT1 1248) and EXTL3 (EXTL3 3013), and GAGs were quantified using 9-dimethylmethylene blue (DMMB), showing an accumulation of GAGs in the patient-derived cell line in comparison to WT (normal healthy donor fibroblasts acquired from ATCC), and reduction following ASO treatment (Amount in nGrams normalized per lOOng DNA / miL cells, N=3).

[0046] Figs. 6A-6G show that lysosomal stress is increased in patient-derived fibroblasts compared to WT cells, and reduced following a 72 hours treatment with ASOs targeting key enzymes in the HS biosynthesis pathway. Lysosomal stress is identified with Lysotracker (red), labeling acidic organelles. Patient-derived fibroblasts were treated with lOOnM ASO, and stained 72 hours posttreatment. Fig. 6A. untreated GM fibroblasts; Fig. 6B. ATCC-CTL (control); Figs. 6C-6G: GM fibroblasts treated with the indicated ASO: Fig. 6C. EXT1_1248; Fig. 6D. EXT2_1577; Fig. 6E.

[0047] EXTL2 816; Fig. 6F. EXTL3_3013; Fig. 6G. XYLT1_1147.

[0048] Figs. 7A-7E show that lysosomal stress is increased in neurons differentiated from patient-derived iPSC, compared to untreated cells, and reduced following 5 -days treatment with ASOs targeting key enzymes in the HS biosynthesis pathway. Lysosomal stress is identified with LysoTracker™ (red), labeling acidic organelles. Patient-derived neurons were treated with 2uM ASO, and stained 5 days post-treatment. Fig. 7A. untreated induced neurons (iNeurons). Fig. 7B.

[0049] Mock ASO. Fig. 7C. EXTl_1248:2pM. Fig. 7D. EXTL2_816:2pM. Fig. 7E.

[0050] XYLYl l 147:2pM.

[0051] Figs. 8A-8B show transcript levels in brains harvested from 3 -month old homozygote MPSIIIA mouse models, treated with and EXT 1 -targeting ASO (EXT1 1313) or vehicle (aCSF) for 2 months (4 injections), comparing with WT littermates. Numbers under the X axis are animal numbers (F=female). HM: homozygote; HM: heterozygote; aCSF: artificial CSF (vehicle). Fig.

[0052] 8A shows Extl transcript levels following the injection, as measured by qRT-PCR. Fig. 8B shows reduction in the neuroinflammation markers IL 10, AIF1, GFAP, and Serpin3n following the treatment, as measured by qRT-PCR. DETAILED DESCRIPTION OF THE INVENTION

[0053] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0054] The present invention is directed to the use of a novel RNA-based substrate reduction therapy (SRT) approach for treating storage diseases by utilizing antisense oligonucleotides (ASOs) specifically designed to target and reduce the expression of key enzymes involved in producing the accumulating metabolites. In the case of mucopolysaccharidosis type IIIA (MPS3 A) which is specifically demonstrated herein, the target enzymes are those participating in heparan sulfate biosynthesis, particularly exostosin glycosyltransferase 1 (EXT1) and exostosin-like glycosyltransferase 2 (EXTL2). The ASOs, modified with phosphor othioate and 2'-O-m ethoxy ethyl groups for enhanced stability and CNS penetration, aim to decrease heparan sulfate production, thereby mitigating disease progression.

[0055] Preclinical evaluations presented herein demonstrate the potential of this approach to effectively reduce substrate accumulation in both peripheral tissues and the CNS, offering a promising therapeutic strategy for MPS3A and potentially other related lysosomal storage disorders.

[0056] EXT1 and EXTL2 were identified by the inventors as the most promising targets for treating MPS3A by reducing heparan sulfate production by the new RNA-based SRT approach, through an extensive review of existing literature and a series of experimental studies directed to several key enzymes in its biosynthesis pathway, encoded by the genes XYLT1, EXT1, EXT2, EXTL2, and EXTL3.

[0057] In the described experiments, the ASOs designed to target EXT1 and EXTL2 are gapmers, i.e., are a specific type of ASO that facilitates the degradation of target mRNA through RNase H-mediated cleavage. These gapmers were modified with phosphorothioate (PS) backbones and 2'-O-methoxyethyl (MOE) modifications, for increased stability, nuclease resistance, and affinity to the target. The PS backbone modification replaced a non-bridging oxygen with sulfur in the phosphate linkage, enhancing the nuclease resistance and binding affinity of the ASOs. The 2'-MOE modification further increases the stability and affinity by adding a methoxyethyl group to the ribose sugar, improving the pharmacokinetic properties and reducing off-target effects. Such PS-MOE chemistries are already in clinical use today, used in drugs such as nusinersen for spinal muscular atrophy, which highlights their efficacy and safety in therapeutic applications. By binding to mRNA transcripts of the target genes, the ASOs reduce the production of enzymes encoded by these genes, thereby decreasing the synthesis of heparan sulfate. This reduction alleviates the pathological accumulation of heparan sulfate in cells and tissues, addressing the root cause of MPS3A and potentially mitigating the severe neurological and systemic manifestations of the disease. The ASOs can be injected intrathecally to ensure their arrival to the CNS.

[0058] ASOs offer a targeted approach for directly decreasing the overall production of heparan sulfate and glycosaminoglycans (GAGs) in general, the accumulation of which causes the symptoms in lysosomal storage diseases. The benefits of this approach include a potentially more effective reduction of the substrate in both peripheral tissues and the central nervous system, potentially mitigating the neurological symptoms that are not adequately addressed by current treatments. Additionally, ASOs with phosphorothioate (PS) and 2'-O-methoxyethyl (MOE) modifications are known for their enhanced stability and improved pharmacokinetic properties, which translates to more efficient and sustained therapeutic effects with potentially fewer side effects. This innovative therapy thus holds promise for improving the quality of life for patients by offering a more precise and effective means of reducing heparan sulfate (or other GAGs) accumulation.

[0059] Some of the advantages of using ASOs include a high specificity with rather rare off-target effects; effective penetration into the central nervous system (CNS); a sustained action, since the ASOs remain stable and active in the CNS for extended periods; and reduced systemic exposure when administered directly to the CNS. Furthermore, ASOs have been clinically approved and are well tolerated for treating the CNS, as opposed, e.g., to siRNA.

[0060] The same principle demonstrated here for MPS3 A may be also used for treating other storage diseases such as other subtypes of MPSIII, including MPS3B; MPSI, caused by the accumulation of GAGs, including heparan sulfate, leading to various systemic and neurological symptoms; MPSII, another lysosomal storage disorder characterized by the buildup of GAGs; and multiple sulfatase deficiency (MSD), a rare genetic disorder where the deficiency of multiple sulfatases leads to the accumulation of sulfated GAGs, including heparan sulfate. Targeting EXT1 and EXTL2 might help manage glycosaminoglycan levels in the above diseases.

[0061] The examples provided herein demonstrate the ability of the ASOs used in the invention to reduce mRNA levels of the targeted genes in cell lines, and to reduce levels of GAGs in fibroblasts from patients. Finally, lysosomal stress is reduced in cells from patients following ASO treatment. Methods of treating storage disease

[0062] Accordingly, in some embodiments, there is provided a method of treating a lysosomal storage disease in a subject by substrate reduction therapy (SRT), the method including administering to the subject a composition including a therapeutically effective amount of at least one antisense oligonucleotide (ASO) capable of affecting expression of an enzyme in a glycosaminoglycan (GAG) biosynthesis pathway.

[0063] The term “capable of affecting expression”, as used herein, means at least partially inhibiting or reducing expression of the enzyme. The expression is affected by the ASO of the invention binding to RNA transcripts encoding the enzyme and causing degradation of the RNA or inhibiting translation. In some embodiments, affecting expression means reducing expression levels.

[0064] The term “lysosomal storage disease”, as used herein relates to a group of inherited metabolic disorders characterized by defects in lysosomal function. These defects typically arise from mutations in genes encoding lysosomal enzymes, transporters, or associated proteins, leading to incomplete degradation and subsequent accumulation of substrates — such as lipids, glycoproteins, or mucopolysaccharides — within lysosomes. The progressive buildup of undegraded material disrupts normal cellular processes, resulting in multisystem pathology that often affects the nervous system, liver, spleen, and skeletal structures. Lysosomal storage diseases are generally inherited in an autosomal recessive manner, though some (e.g., Fabry disease, Hunter syndrome) follow X-linked inheritance.

[0065] In some embodiments, the lysosomal storage disease is selected from mucopolysaccharidosis type IIIA (MPS3A), mucopolysaccharidosis type IIIB (MPS3B); mucopolysaccharidosis type I (MPSI), mucopolysaccharidosis type II (MPSII), and multiple sulfatase deficiency (MSD).

[0066] In some embodiments, the lysosomal storage disease is MPS3 A.

[0067] The term “substrate reduction therapy (SRT)”, as used herein, relates to a pharmacological approach used in the treatment of certain metabolic and lysosomal storage diseases. The therapy functions by reducing the synthesis of the specific substrate that accumulates due to the enzyme deficiency characteristic of the disorder.

[0068] In some embodiments, the GAG is a GAG which accumulates in a lysosomal storage disease. In some embodiments, the GAG is selected from Keratan Sulfate (accumulating in mucopolysaccharidosis IV, or Morquio's syndrome), chondroitin sulfate, and dermatan sulfate (which accumulate in mucopolysaccharidosis type 6).

[0069] In some embodiments, the GAG is heparan sulfate. In some embodiments, the GAG biosynthesis pathway enzyme is encoded by a gene selected from exostosin glycosyltransferase 1 (EXT1), exostosin glycosyltransferase 2 (EXT2), exostosin-like glycosyltransferase 2 (EXTL2), exostosin-like glycosyltransferase 3 (EXTL3), xylosyltransferase 1 (XYLT1), and combinations thereof. In some embodiments, the GAG biosynthesis pathway enzyme is EXT1 and / or EXTL2.

[0070] EXT1 and EXT2 encode exostosin- 1 and exostosin-2, two endoplasmic reticulum-resident type II transmembrane glycosyltransferases. EXTL2 and EXTL3 encode the exostosin-like 2 and exostosin-like 3 glycosyltransferases. XYLT1 encodes xylosyltransferase, which catalyzes the transfer of UDP -xylose to serine residues within xylosyltransferase recognition sequences of target proteins.

[0071] The term “antisense oligonucleotide (ASO)”, as used herein, encompasses single stranded oligonucleotides having a length of about 14-30 nucleotides and a sequence complementary to an mRNA which it targets and reduce expression of. ASOs are short, synthetic, single-strand oligonucleotides, that can bind to target RNA and modulate its expression. They can be divided to two major categories - steric-blockers and RNAse-H recruiting ASOs (gapmers). An ASO may include DNA or RNA nucleotides, as well as modified nucleotides. Gapmers can efficiently recruit and localize RNase H for mRNA degradation, by utilizing the “gapmer” design.

[0072] A “gapmer”, as used herein, relates to an ASO having several (e.g. 5) 2' -methoxy ethyl (2'-MOE)-modified ribonucleotides (RNA) at each terminus and a central region of 2'-deoxynucleotide (DNA) nucleotides (8-10 bases). While the flanking 2' -MOE ends prevent nuclease cleavage of the ASO, the chimeric gapmer ASO design directs RNase Hl to the central gap made of DNA where it performs specific mRNA degradation, as RNase Hl shows high specificity for DNA-RNA duplexes. It is noted that siRNA, being double stranded, is not encompassed by the definition of an ASO.

[0073] Accordingly, in some embodiments, the ASO includes at least one deoxyribonucleotide (DNA). In some embodiments, the ASO includes at least one ribonucleotide (RNA). In some embodiments, at least one of the nucleotides of the ASO is modified.

[0074] In some embodiments, at least one of the ASO nucleotides is modified to further include a 2'-O-methoxyethyl (MOE) group.

[0075] In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the ASO nucleotides are modified nucleotides. In some embodiments, the ASO includes at least 1, 2, 3, 4, or 5 modified nucleotides both at the 5 ’-end and at the 3 ’-end. In some embodiments, the ASO includes 5 modified nucleotides both at the 5 ’-end and at the 3 ’-end.

[0076] In some embodiments, the modified nucleotides are modified by including a group selected from a methyl group (as in 5 ’-methylcytosine), a 2'-O-m ethoxy ethyl (2'-M0E), a methylene group between the 2'-0 and 4'-C of the nucleotide (locked nucleic acid, LNA), constrained ethyl (cET), 2'-O-methylation (20me), and combinations thereof.

[0077] In some embodiments, the modified nucleotides are modified by including a 2'-O-methoxyethyl (MOE) group. In some embodiments, all of the RNA nucleotides in the ASO are modified by adding a 2'-O-methoxyethyl (MOE) group.

[0078] In some embodiments, the ASO includes at least one 5 ’-methylcytosine. In some embodiments, all of the cytosines in the ASO are 5’-methylcytosines.

[0079] In some embodiments, all of the nucleotides modified by adding a 2’ -MOE group are RNA nucleotides.

[0080] In some embodiments, the ASO includes a central region (i.e., not at the 5’ or the 3’ ends) including only DNA nucleotides. In some embodiments, the central region has a length of about 5-10, or 8-10 nucleotides. In some embodiments, the central region has a length of about 10 nucleotides.

[0081] In some embodiments, the ASO includes at least one PS bond . In some embodiments, the ASO includes PS bonds instead of all of the phosphodiester bonds in the ASO. In some embodiments, the ASO includes no phosphodiester bonds. In some embodiments, all bonds between nucleotides of the ASO are PS bonds.

[0082] In some embodiments, the ASO has a length of about 14-30 nucleotides. In some embodiments, the ASO has a length of about 20-30 nucleotides. In some embodiments, the ASO has a length of about 15-25 nucleotides. In some embodiments, the ASO has a length of about 15-20 nucleotides. In some embodiments, the ASO has a length of about 20-25 nucleotides. In some embodiments, the ASO has a length of about 20 nucleotides.

[0083] In some embodiments, the ASO is a gapmer.

[0084] In some embodiments, the ASO targets a sequence at least 90%, 95%, or 99% identical to a sequence selected from sequences in Table 1. In some embodiments, the ASO targets a sequence at least 90%, 95%, or 99% identical to a sequence selected from SEQ ID Nos: 1-55. In some embodiments, the ASO targets a sequence at least at least 90%, 95%, or 99% identical to a sequence selected from SEQ ID Nos: 4, 32, 37, 45, 47, 28, 11, 12, 13, and 14.

[0085] In some embodiments, the ASO includes a sequence at least 90%, 95%, or 99% identical to a sequence selected from sequences defined in Table 2. In some embodiments, the ASO sequence includes a sequence at least 90%, 95%, or 99% identical to a sequence selected from SEQ ID Nos: 61-115 In some embodiments, the ASO sequence includes a sequence at least 90%, 95%, or 99% identical to a sequence selected from SEQ ID Nos: 64, 92, 97, 105, 107, 88, 71, 72, 73, and 74. In some embodiments, the ASO is a modified ASO selected from modified ASOs presented in Table 3. In some embodiments, the ASO is a modified ASO selected from modified ASOs defined by SEQ ID Nos: 121-175. In some embodiments, the ASO is a modified ASO selected from modified ASOs defined by SEQ ID Nos: 124, 152, 157, 165, 167, 148, 131, 132, 133, and 134

[0086] In some embodiments, the ASO is selected from EXT1 1248, EXT2 1577, EXTL2 816, EXTL3 3013, XYLT1 1147, EXIT 2915, EXT1 1309, EXT1 1311, EXT1 1313, and EXT1 1315.

[0087] In some embodiments, the ASO targets a gene encoding EXT1. In some embodiments, the ASO is selected from EXT1 1248, EXIT 2915, EXT1 1309, EXT1 1311, EXT1 1313, and EXT1 1315. In some embodiments, the ASO is EXT1 1248 (SEQ ID NO: 124). In some embodiments, the ASO is EXT1 2915 (SEQ ID NO: 148). In some embodiments, the ASO is EXT1 1309 (SEQ ID NO: 131). In some embodiments, the ASO is EXT1 1311 (SEQ ID NO: 132). In some embodiments, the ASO is EXT1 1313 (SEQ ID NO: 133). In some embodiments, the ASO is EXT1_1315 (SEQ ID NO: 134).

[0088] In some embodiments, the ASO targets a gene encoding EXT2. In some embodiments, the ASO is EXT2_1577 (SEQ ID NO: 152).

[0089] In some embodiments, the ASO targets a gene encoding EXTL2. In some embodiments, the ASO is EXTL2 816 (SEQ ID NO: 157).

[0090] In some embodiments, the ASO targets a gene encoding EXTL3. In some embodiments, the ASO is EXTL3 3013 (SEQ ID NO: 165).

[0091] In some embodiments, the ASO targets a gene encoding XYLT1. In some embodiments, the ASO is XYLTl l 147 (SEQ ID NO: 167).

[0092] The phrase “the ASO targets a sequence”, as used herein, means that the ASO is capable of binding to an RNA molecule including the targeted sequence, thereby causing inhibition of translation, either by steric inhibition or by causing degradation of the target RNA. While the ASO is generally complementary to the target sequence (sufficient for binding), it does not have to be 100% complementary. In some embodiments, the ASO is at least about 90%, 95%, or 99% complementary to the target sequence. In other words, in some embodiments, the ASO is at least about 90%, 95%, or 99% identical to a sequence complementary to the target sequence. The term “complementary”, as used herein, means having a matching base to the base of the target sequence at a corresponding position, according to standard base pairing: A-T / U, C-G.

[0093] In some embodiments, the method causes reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in expression levels of the enzyme. In some embodiments, the method causes reduction of at least about 30% in expression levels of the enzyme. In some embodiments, the method causes reduction of at least about 50% in expression levels of the enzyme.

[0094] In some embodiments, the method causes a reduction in levels of the GAG. In some embodiments, the method causes a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in levels of the GAG. In some embodiments, the method causes reduction of at least about 30% in in levels of the GAG. In some embodiments, the method causes reduction of at least about 50% in in levels of the GAG.

[0095] In some embodiments, the method causes a reduction in neuroinflammation markers levels. In some embodiments, the method causes a reduction in levels of a neuroinflammation marker selected from IL1 0 (interleukin- 1 beta), AIF1 (allograft Inflammatory Factor 1), GFAP (glial fibrillary acidic protein), and Serpin3n. In some embodiments, the method causes a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in neuroinflammation markers levels. In some embodiments, the method causes a reduction of at least about 30% in neuroinflammation markers levels. In some embodiments, the method causes a reduction of at least about 50% in neuroinflammation markers levels.

[0096] In some embodiments, the composition is a pharmaceutical composition, further including a pharmaceutically acceptable carrier.

[0097] The term “treating” or “treatment”, as used herein, refers to means of obtaining a desired physiological effect. The effect may be therapeutic in terms of partially or completely curing a disease and / or symptoms attributed to the disease. The term includes inhibiting the disease, i.e. arresting its development; or ameliorating the disease, i.e. causing regression of the disease, e.g., by eliminating or ameliorating its symptoms.

[0098] The term "therapeutically effective amount" as used herein means an amount of a therapeutic that will elicit the biological or medical response of a tissue, system, animal or human that is being sought. The amount must be effective to achieve the desired therapeutic effect, depending inter alia, on the type and severity of the condition to be treated and the treatment regime. The therapeutically effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person skilled in the art will know how to properly conduct such trials to determine the effective amount. As generally known, an effective amount depends on a variety of factors including the affinity of a ligand to its binding partner, its distribution profile within the body, a variety of pharmacological parameters such as half-life in the body, on undesired side effects, if any, and on factors such as age and gender, etc.

[0099] Pharmaceutical compositions in accordance with the present invention may be formulated in any conventional manner using one or more physiologically or pharmaceutically acceptable carriers or excipients. The carrier(s) must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition, not being deleterious to the recipient thereof, and not significantly interfering with the activity of the compound of the invention, or of any other active ingredient in the pharmaceutical composition.

[0100] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the active agent is administered. The carriers in the pharmaceutical composition may include a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.

[0101] Methods of administration include, but are not limited to, parenteral, e.g., intravenous, intraperitoneal, intramuscular, subcutaneous; mucosal (e.g., oral, sublingual, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, intracerebroventricular, topical, and intradermal routes. Administration can be systemic or local. In certain embodiments, the pharmaceutical composition is adapted for oral administration. In some embodiments, the administration is intrathecal (IT) administration. In some embodiments, the administration is intracerebral (IC) administration. In some embodiments, the administration is intracerebroventricular (ICV) administration. In some embodiments, the administration is selected from IV, IC, ICV, and IT administration.

[0102] While the administration may be any type of administration, the advantages of the present invention are more pronounced when the administration is directly to the CNS. Accordingly, in some embodiments, the administration is intrathecal.

[0103] ASOs and ASOs for use

[0104] In some embodiments, there is provided an ASO for use in a method of treating a lysosomal storage disease in a subject by SRT, wherein the ASO is capable of affecting expression of an enzyme in a GAG biosynthesis pathway.

[0105] In some embodiments, there is provided a use of an ASO in the preparation of a medicament for treating a lysosomal storage disease in a subject by substrate reduction therapy (SRT), wherein the ASO is directed against an enzyme in a GAG biosynthesis pathway.

[0106] In some embodiments, there is provided a gapmer ASO capable of affecting expression of an enzyme in a GAG biosynthesis pathway, wherein the gapmer ASO has a length of about 14-30 nucleotides, and includes at least one PS bond and about 8-12 nucleotides modified by the addition of an MOE group. Definitions and embodiments mentioned above and which may be relevant to the ASOs or for the ASOs for use also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. For terms used herein, unless stated otherwise, their definition and embodiments are intended to be the same as above (mutatis mutandis).

[0107] In some embodiments, the ASO targets a gene encoding an enzyme in a heparan sulphate biosynthesis pathway. In some embodiments, the ASO targets genes encoding EXT1 and / or EXTL2.

[0108] In some embodiments, the ASO includes only PS bonds instead of phosphodiester bonds. In some embodiments, the ASO includes about 10 nucleotides modified by the addition of an MOE group. In some embodiments, the nucleotides modified by the addition of an MOE group are RNA nucleotides flanking a sequence of DNA nucleotides. In some embodiments, the sequence of DNA nucleotides flanked by the modified RNA nucleotides is about 6-10, or 8-10 nucleotides long.

[0109] In some embodiments, the ASO targets a sequence at least at least 90%, 95%, or 99% identical to a sequence identical to a sequence selected from sequences in Table 1. In some embodiments, the ASO targets a sequence at least at least 90%, 95%, or 99% identical to a sequence identical to a sequence selected from SEQ ID Nos: 1-55. In some embodiments, the ASO targets a sequence at least at least 90%, 95%, or 99% identical to a sequence identical to a sequence selected from SEQ ID Nos: 4, 32, 37, 45, 47, 28, 11, 12, 13, and 14.

[0110] In some embodiments, the ASO includes a sequence at least 90%, 95%, or 99% identical to a sequence selected from sequences defined in Table 2. In some embodiments, the ASO sequence includes a sequence at least 90%, 95%, or 99% identical to a sequence selected from SEQ ID Nos: 61-115 In some embodiments, the ASO sequence includes a sequence at least 90%, 95%, or 99% identical to a sequence selected from SEQ ID Nos: 64, 92, 97, 105, 107, 88, 71, 72, 73, and 74.

[0111] In some embodiments, the ASO is a modified ASO selected from modified ASOs presented in Table 3. In some embodiments, the ASO is a modified ASO selected from modified ASOs defined by SEQ ID Nos: 121-175. In some embodiments, the ASO is a modified ASO selected from modified ASOs defined by SEQ ID Nos: 124, 152, 157, 165, 167, 148, 131, 132, 133, and 134

[0112] In some embodiments, the ASO is selected from EXT1 1248, EXT2 1577, EXTL2 816, EXTL3 3013, XYLT1 1147, EXT1 2915, EXT1 1309, EXT1 1311, EXT1 1313, and EXT1 1315.

[0113] In some embodiments, the ASO targets a gene encoding EXT1. In some embodiments, the ASO is selected from EXT1 1248, EXT1 2915, EXT1 1309, EXT1 1311, EXT1 1313, and EXT1 1315. In some embodiments, the ASO is EXT1 1248 (SEQ ID NO: 124). In some embodiments, the ASO is EXT1 2915 (SEQ ID NO: 148). In some embodiments, the ASO is EXT1 1309 (SEQ ID NO: 131). In some embodiments, the ASO is EXT1 1311 (SEQ ID NO: 132). In some embodiments, the ASO is EXT1 1313 (SEQ ID NO: 133). In some embodiments, the ASO is EXT1_1315 (SEQ ID NO: 134).

[0114] In some embodiments, the ASO targets a gene encoding EXT2. In some embodiments, the ASO is EXT2_1577 (SEQ ID NO: 152).

[0115] In some embodiments, the ASO targets a gene encoding EXTL2. In some embodiments, the ASO is EXTL2 816 (SEQ ID NO: 157).

[0116] In some embodiments, the ASO targets a gene encoding EXTL3. In some embodiments, the ASO is EXTL3 3013 (SEQ ID NO: 165).

[0117] In some embodiments, the ASO targets a gene encoding XYLT1. In some embodiments, the ASO is XYLTl l 147 (SEQ ID NO: 167).

[0118] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0119] The term "a" and "an" refers to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0120] The term "about", when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about”, unless indicated otherwise.

[0121] The term “sequence”, referring to a nucleotide sequence such as a DNA or RNA sequence, as used herein, relates to a sequence of nucleotides and may include different types of nucleotides, such as DNA nucleotides, RNA nucleotides, and synthetic, or modified, nucleotides.

[0122] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

[0123] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0124] EXAMPLES

[0125] Materials and Methods

[0126] Table 1: List of target sequences for ASOs

[0127]

[0128]

[0129] Table 2: List of ASO sequences

[0130]

[0131]

[0132] The ASOs of Table 2 were further modified as follows: the phosphodiester bond was replaced with a phosphorothioate (PS) bond, the first and last five nucleotides in each ASO are RNA nucleotides modified by a 2'-O-methoxy ethyl (MOE) group, and the cytosines were modified to 5’-methylcytosines. The modified ASOs are presented in Table 3.

[0133] Table 3: List of modified ASOs

[0134]

[0135]

[0136]

[0137] * - PS bond; eN = 2'MOE modified base; X - 5’mC (methyl cytosine); Z - 2'MOE-5’mC; the unmodified nucleotides are DNA (in bold).

[0138] Immunocytochemistry

[0139] Patient-derived fibroblasts were fixated with or without treatment with 4% paraformaldehyde (PF A; Electron Microscopy Sciences) for 15 min, and subsequently washed 3 times with PBS. Cells were permeabilized with 0.25% Triton X-100 (sigma) for 15 min, washed with PBS and subsequently blocking was performed with 1% BSA and 2% serum in PBS-T for 30 min. Fixed and permeabilized neurons were incubated with HS antibody (10E4 epitope, Tx-Red) 1:100 in 1% BSA for 1 hour at room temperature. After an additional washing step, cells were incubated with secondary antibody 1:1000 (abl50077, abeam) in 1% BSA for 1 hour. DAPI (D9542, sigma) was used to visualize the nucleus, and images were acquired using EVOS microscope.

[0140] Lysotracker

[0141] Patient-derived fibroblasts were stained using LysoTracker™ Deep Red (Invitrogen) with accordance with the manufacturer's protocol.

[0142] GAG quantification by DM MB

[0143] qRT-PCR

[0144] RNA isolation from tissue culture was carried using ReliaPrep(TM) RNA Cell Miniprep System (Promega), according to the manufacturer’ s instructions. 1000 nanograms of isolated RNA of each sample was reverse transcribed to cDNA by using High Capacity cDNA RT Synthesis Kit (Applied biosystems). Quantitative real-time PCR was performed on BioRad CFX96 system in technical triplicate per sample by adding 5ul cDNA (5 ng / uL) and primer pairs to SYBR Green Master Mix (Applied Biosystems). GAPDH was used as a housekeeping gene and relative quantification (RQ) values (RQmin / RQmax) were determined using CFX Maestro system.

[0145] ASO design

[0146] ASOs used in this study were synthesized by Ella Biotech (PS-MOE ASOs ) and include PS backbone modifications and flanking 2-MOE residues in a gapmer structure. ASOs were diluted in DDW (stock: 100 pM) and used as indicated for each experiment.

[0147] Example 1: ASO targeting reduces heparan sulfate (HS) accumulation and lysosomal stress To decrease the levels of HS accumulated in patient-derived cells, libraries of gapmer ASOs were first designed, targeting enzymes in the HS biosynthesis pathway: exostosin glycosyltransferase 1 (EXT1), exostosin glycosyltransferase 2 (EXT2), exostosin-like glycosyltransferase 2 (EXTL2), exostosin-like glycosyltransferase 3 (EXTL3), xylosyltransferase 1 (XYLT1). HEK293 cells were transfected with increasing concentrations of ASOs, and harvested 72 hours post-treatment. RNA was extracted and analyzed by qRT-PCR. Next. Candidate ASOs, showing >50% reduction in their target's expression, were tested in MPS3A patients-derived fibroblasts (GM01881A) by transfecting cells with 50 / 100 nM ASO and harvesting 72 hours pot transfection (Fig. 3).

[0148] Further optimization of specific EXTl-directed ASOs was done in GM01881A fibroblasts (Figs. 3F-3G) including dose-response experiments (Fig. 3H). Fibroblasts were transfected with increasing concentrations (25-100 nM) of candidate EXT 1 -targeting ASOs. Cells were harvested 72 hours post-treatment and EXT1 levels were analyzed by qRT-PCR.

[0149] For a visualization of the effects of the ASOs on the fibroblasts, GM01881 A fibroblasts were treated with 100 nM of the following ASOs: EXT1J248, EXT2J577, EXTL2_816, EXTL3 3013, and XYLT1 1147. HS levels were measured in treated cells and in control untreated cells or mock treated cells (treated by an unrelated ASO) by two different methods - HS staining (Fig. 4) and dimethylmethylene Blue Assay (DMMB) for the Quantification of Sulfated GAGs (Fig 5). By both methodologies, reduction of HS biosynthesis enzymes (mainly EXT1 and EXTL2) decreased HS accumulation, in contrast to the mock treatment.

[0150] To test for the effect of HS reduction on lysosomal dysfunction, lysosomal stress was measured as a functional outcome for treatment. Quantification of lysosomal activity was done by applying LysoTracker™, a fluorescent dye that selectively stains acidic organelles, LysoTracker™ to patient-derived fibroblasts, either untreated, or following 72 hours of treatment with EXT1_1248, EXT2_1577, EXTL2_816, EXTL3_3013, or XYLT1 1147 (Fig.6). As can be seen, ASO treatments significantly reduced lysosomal stress following reduction of expression of EXT 1, EXT2, EXTL2, EXTL3, or XYLT1.

[0151] The same procedure was also done using patient-derived induced neurons (iNeurons) differentiated from patient-derived iPSC, instead of fibroblasts. Quantification of lysosomal activity was done by applying LysoTracker™ on untreated cells, or following 5 days treatment with different ASOs, targeting EXT1, EXTL2 or XYTL1 (Figs. 7C-7E, respectively).

[0152] Example 2: Intracerebroventricular injection (ICV) of ASO targeting mouse Extl reduces neuroinflammation in an MPSIIIA mouse model

[0153] An SGSH mouse model is a mouse model with a deficiency in the Sgsh gene, which produces the N-sulfoglucosamine sulfohydrolase (sulfamidase) enzyme. SHSG mice were injected by ICV administration with the EXT1 1313 ASO - 4 injections in the span of 2 months. Following 2 months of treatment, brains were harvested for analysis, showing dramatic reduction in Extl levels (10-25% residual expression). (Fig 8A). Subsequently, neuroinflammation markers were analysed using qRT-PCR, showing significant reduction towards WT levels in hallmark neuroinflammation genes - IL1 0 (interleukin- 1 beta), AIF1 (allograft Inflammatory Factor 1), GFAP (glial fibrillary acidic protein), and Serpin3n (Fig. 8B).

Claims

CLAIMSWhat is claimed is:

1. A method of treating a lysosomal storage disease in a subject by substrate reduction therapy (SRT), the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of at least one antisense oligonucleotide (ASO) capable of affecting expression of an enzyme in a glycosaminoglycan (GAG) biosynthesis pathway.

2. The method of claim 1, wherein the lysosomal storage disease is selected from mucopolysaccharidosis type IIIA (MPS3A), mucopolysaccharidosis type IIIB (MPS3B); mucopolysaccharidosis type I (MPSI), mucopolysaccharidosis type II (MPSII), and multiple sulfatase deficiency (MSD).

3. The method of claim 1, wherein the GAG is heparan sulfate.

4. The method of claim 1 wherein the enzyme is encoded by a gene selected from exostosin glycosyltransferase 1 (EXT1), exostosin glycosyltransferase 2 (EXT2), exostosin-like glycosyltransferase 2 (EXTL2), exostosin-like glycosyltransferase 3 (EXTL3), xylosyltransf erase 1 (XYLT1), and combinations thereof.

5. The method of claim 1, wherein the ASO comprises at least one phosphorothioate (PS) bond and / or at least one 2'-O-methoxyethyl (MOE) group.

6. The method of claim 1, wherein ASO is a gapmer.

7. The method of claim 1, wherein the ASO has a length of about 14-30 nucleotides.

8. The method of claim 1 , wherein the ASO targets a sequence at least 90% identical to a sequence selected from SEQ ID Nos: 1-55.

9. The method of claim 1, wherein the ASO sequence comprises a sequence at least 90% identical to a sequence selected from ASO sequences set forth in SEQ ID Nos: 61-115.

10. The method of claim 1, wherein the ASO is a modified ASO selected from modified ASOs defined by SEQ ID Nos: 121-175.

11. The method of claim 1, wherein the ASO is selected from EXT1 1313 (SEQ ID NO: 133), EXT1 1315 (SEQ ID NO: 134), EXT1J248 (SEQ ID NO: 124), EXT2_1577 (SEQ ID NO:152), EXTL2 816 (SEQ ID NO: 157), EXTL3_3013 (SEQ ID NO: 165), XYLT1 1147 (SEQ ID NO: 167), EXT1_2915 (SEQ ID NO: 148), EXT1_13O9 (SEQ ID NO: 131), and EXT1 1311 (SEQ ID NO: 132).

12. The method of claim 1, wherein the method causes a reduction of at least about 30% in expression levels of the enzyme.

13. The method of claim 1, wherein the method causes a reduction of at least about 30% in levels of the GAG.

14. The method of claim 1, wherein the method causes a reduction of at least about 30% in levels of a neuroinflammation marker selected from IL I P (interleukin- 1 beta), AIF1 (allograft Inflammatory Factor 1), GFAP (glial fibrillary acidic protein), and / or Serpin3n.

15. The method of claim 1, wherein the administration is intracerebroventricular (ICV) or intrathecal administration.

16. A gapmer antisense oligonucleotide (ASO) capable of affecting expression of an enzyme in a glycosaminoglycan (GAG) biosynthesis pathway, wherein the gapmer has a length of about 14-30 nucleotides, and comprises at least one phosphorothioate (PS) bond and about 8-12 nucleotides modified by the addition of an 2'-O-methoxyethyl (MOE) group.

17. The gapmer of claim 16, wherein the gapmer is selected from sequences set forth in SEQ ID Nos: 133, 134, 124, 152, 157, 165, 167, 148, 131, and 13218. An antisense oligonucleotide (ASO) for use in a method of treating a lysosomal storage disease in a subject by substrate reduction therapy (SRT), wherein the ASO is capable of affecting expression of an enzyme in a glycosaminoglycan (GAG) biosynthesis pathway.

19. The ASO of claim 18, wherein the lysosomal storage disease is selected from mucopolysaccharidosis type IIIA (MPS3A), mucopolysaccharidosis type IIIB (MPS3B); mucopolysaccharidosis type I (MPSI), mucopolysaccharidosis type II (MPSII), and multiple sulfatase deficiency (MSD).

20. The ASO of claim 18 or 19, wherein the GAG is heparan sulfate.

21. The ASO of any one of claims 18-20, wherein the enzyme is encoded by a gene selected from exostosin glycosyltransferase 1 (EXT1), exostosin glycosyltransferase 2 (EXT2), exostosin-like glycosyltransferase 2 (EXTL2), exostosin-like glycosyltransferase 3 (EXTL3), xylosyltransf erase 1 (XYLT1), and combinations thereof.

22. The ASO of any one of claims 18-21, comprising at least one phosphorothioate (PS) bond and / or at least one 2'-O-methoxyethyl (MOE) group.

23. The ASO of any one of claims 18-22, wherein ASO is a gapmer.

24. The ASO of any one of claims 18-23, having a length of about 14-30 nucleotides.

25. The ASO any one of claims 18-24, wherein the ASO targets a sequence at least 90% identical to a sequence selected from SEQ ID Nos: 1-55.

26. The ASO of claim 25, wherein the ASO sequence comprises a sequence at least 90% identical to a sequence selected from ASO sequences set forth in SEQ ID Nos: 61-115.

27. The ASO of claim 26, wherein the ASO is a modified ASO selected from modified ASOs defined by SEQ ID Nos: 121-175.

28. The ASO of claim 27, wherein the ASO is selected from EXT1_1313 (SEQ ID NO: 133), EXT1 1315 (SEQ ID NO: 134), EXT1J248 (SEQ ID NO: 124), EXT2_1577 (SEQ ID NO: 152), EXTL2 816 (SEQ ID NO: 157), EXTL3_3013 (SEQ ID NO: 165), XYLT1_1147 (SEQ ID NO: 167), EXT1_2915 (SEQ ID NO: 148), EXT1 1309 (SEQ ID NO: 131), and EXT1 1311 (SEQ ID NO: 132).

29. The ASO of any one of claims 18-28, wherein the method causes a reduction of at least about 30% in expression levels of the enzyme.

30. The ASO of any one of claims 18-29, wherein the method causes a reduction of at least about 30% in levels of the GAG.

31. The ASO of any one of claims 18-30, wherein the method causes a reduction of at least about 30% in levels of a neuroinflammation marker selected from IL1 0 (interleukin- 1 beta), AIF1 (allograft Inflammatory Factor 1), GFAP (glial fibrillary acidic protein), and / or Serpin3n.

32. The ASO of any one of claims 18-31, wherein the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

33. The ASO of any one of claims 18-32, wherein the administration is intracerebroventricular(ICV) or intrathecal administration.

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