Treatment of mucopolysaccharidosis ii with recombinant human iduronate-2-sulfatase (IDS)

Abstract

Compositions and methods are described for the delivery of recombinant human iduronate-2-sulfatase (IDS) produced by human neuronal or glial cells to the cerebrospinal fluid of the central nervous system (CNS) of a human subject diagnosed with mucopolysaccharidosis II (MPS II).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 148,093, filed February 10, 2021, U.S. Provisional Application No. 63 / 180,361, filed April 27, 2021, U.S. Provisional Application No. 63 / 210,610, filed June 15, 2021, U.S. Provisional Application No. 63 / 242,250, filed September 9, 2021, and U.S. Provisional Application No. 63 / 256,805, filed October 18, 2021, the content of each of which is incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] This application incorporates by reference a Sequence Listing submitted with this application as text file entitled “Sequence_Listing_12656-146-228.TXT” created on January 27, 2022 and having a size of 172,391 bytes. 1. INTRODUCTION

[0003] Compositions and methods are described for the delivery of recombinant human iduronate-2-sulfatase (IDS) produced by human neuronal or glial cells to the cerebrospinal fluid (CSF) of the central nervous system (CNS) of a human subject diagnosed with mucopolysaccharidosis II (MPS II). 2. BACKGROUND OF THE INVENTION

[0004] Hunter syndrome / MPS II is a rare X-linked recessive genetic disease occurring in 0.5 to 1.3 per 100,000 male live births. This progressive and devastating disease is caused by genetic mutation in the IDS gene leading to deficiency of the lysosomal storage enzyme iduronate-2-sulfatase, an enzyme required for the lysosomal catabolism of heparan sulfate and dermatan sulfate. With no or very little I2S, the protein is unable to perform its usual lysosomal exohydrolase function which leads to the accumulation of ubiquitous polysaccharides, called GAGs (glycosaminoglycans), in tissues and organs of MPS II patients resulting in the characteristic storage lesions and diverse disease sequelae. Morbidity and mortality are high in this patient population; death has been reported to occur at a mean age of 11.7 years in patients with the severe phenotype (characterized by neurocognitive deterioration) and 21.7 years in patients with a mild or attenuated phenotype. (Young et al., 1982, A clinical and genetic study of Hunter’s syndrome. 2 Differences between the mild and severe forms. J. Medical Genetics 19:408-411). The majority (two-thirds) of patients are reported to have the severe form of this disease. (Wraith JE, et al., 2007, Enzyme replacement therapy in patients who have mucopolysaccharidosis I and are younger than 5 years: Results of a multinational study of recombinant human alpha-L-Iduronidase (Laronidase). Pediatrics 120(l):E37-E46). While the disease primarily affects boys, affected females have been reported as a result of non-random x-inactivation and / or mutation in both alleles of the gene. (Martin et al., 2008, Recognition and diagnosis of mucopolysaccharidosis II (Hunter Syndrome). Pediatrics 121:e377).

[0005] Patients with MPS II appear normal at birth, but signs and symptoms of disease typically present between the ages of 18 months and 4 years in the severe form and between the ages of 4 and 8 years in the attenuated form. Signs and symptoms common to all affected patients include short stature, coarse facial features, macrocephaly, macroglossia, hearing loss, hepato-and splenomegaly, dystosis multiplexjoint contractures, spinal stenosis and carpal tunnel syndrome. Frequent upper respiratory and ear infections occur in most patients and progressive airway obstruction is commonly found, leading to sleep apnea and often death. Cardiac disease is a major cause of death in this population and is characterized by valvular dysfunction leading to right and left ventricular hypertrophy and heart failure. Death is generally attributed to obstructive airway disease or cardiac failure.

[0006] In severe forms of the disease, early developmental milestones may be met, but developmental delay is readily apparent by 18-24 months. Some patients fail hearing screening tests in the first year and other milestones are delayed, including ability to sit unsupported, ability to walk, and speech. Developmental progression begins to plateau between 3 and 5 years of age, with regression reported to begin around 6.5 years. Of the -50% of children with MPS II who become toilet trained, most, if not all, will lose this ability as the disease progresses. (Wraith et al., 2007, supra, Martin et al., 2008, supra).

[0007] Patients with significant neurologic involvement exhibit severe behavioral disturbances including hyperactivity, obstinacy, and aggression beginning in the second year of life and continuing until age 8-9, when neurodegeneration attenuates this behavior. (Muenzer, et al., 2009, Mucopolysaccharidosis I: Management and Treatment Guidelines, Pediatric 123(1): 19-29).

[0008] Seizures are reported in over half of severely affected patients who reach the age of 10, and by the time of death most patients with CNS involvement are severely mentally handicapped and require constant care. (Wraith et al., 2007, supra, Martin et al., 2008, supra). Although patients with attenuated disease exhibit normal intellectual functioning, MRI imaging reveals gross brain abnormalities in all patients with MPS II including white matter lesions, enlarged ventricles, and brain atrophy. (Muenzer, et al., 2009, supra).

[0009] Enzyme replacement therapy (ERT) with recombinant idursulfase produced by HT1080 (fibrosarcoma) cells (Elaprase®, Shire Human Genetic Therapies) is the only approved product for the treatment of Hunter syndrome and is administered as a weekly infusion. (ELAPRASE (idursulfase) injection [package insert], Lexington, MA: Shire Human Genetic Therapies, Inc; 2013, available at http: / / pi.shirecontent.com / PI / PDFs / Elaprase_USA_ENG.pdf). While weekly treatment with intravenous (IV) ERT (recombinant idursulfase) has demonstrated improvement in the systemic manifestations of MPS II, patients and their caregivers have the added burden of living with these weekly ERT infusions through life thereby impacting patient quality of life.

[0010] ERT as currently administered does not cross the blood brain barrier and is therefore unable to address the unmet need in patients with severe disease, i.e., MPS II with CNS / neurocognitive and behavioral involvement. In a recent clinical trial designed to address this problem, idursulfase (Elaprase) formulated for intrathecal administration was administered once monthly to pediatric patients using an intrathecal drug delivery device implanted into the spine (insertion of the catheter at the level of L4 / L5 with implantation of the access port via an incision on the lower ribs). The patients also received concurrent i.v. idursulfase once weekly. See Muenzer et al., 2016, Genetics in Med 18: 73-81, esp. p. 74; abstract available at https: / / www.ncbi.nlm.nih.gov / pubmed / 25834948?dopt=Abstract). Device malfunction led to partial revision, total surgical revision, or removal in 6 of the 12 (50%) of the treated patients. Notably, 12 of 14 SAEs (serious adverse events) were device-related (complication of device insertion, device dislocation / connection issue, device breakage / malfunction / failure, implant site infection, procedural pain, and wound dehiscence). (Muenzer et al., 2016, p. 75, col. 2 and Fig. 1). Device breakage and catheter migration from the spinal canal was exacerbated by the high activity level of this pediatric population. (Muenzer et al., 2016 at p.78 Discussion). 3. SUMMARY OF THE INVENTION

[0011] The invention involves the delivery of recombinant human iduronate-2-sulfatase (rhIDS) produced by human cells including but not limited to neuronal or glial cells to the cerebrospinal fluid (CSF) of the central nervous system (CNS), and cells of the liver for systemic distribution in a human subject diagnosed with mucopolysaccharidosis II (MPS II), including, but not limited to patients diagnosed with Hunter syndrome.

[0012] In a preferred embodiment, the treatment is accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding human IDS (hIDS), or a derivative of hIDS, to the CSF of a patient (human subject) diagnosed with MPS II, so that a permanent depot of (a) transduced neuronal and / or glial cells is generated that continuously supplies the transgene product to the CNS, and (b) transduced liver cells that supply the transgene product systemically. The rhIDS secreted from the neuronal / glial cell depot into the CSF, and from the liver depot systemically will be endocytosed by other CNS and liver cells, respectively, resulting in “cross-correction” of the enzymatic defect in the recipient cells. Moreover, it has been found, unexpectedly, that the administration of the viral vector to the CSF results in systemic delivery of the vector, and that the depot of transduced neural and glial cells in the CNS can deliver the recombinant enzyme to both the CNS and systemically, which may reduce or eliminate the need for systemic treatment, e.g., weekly i.v. injections of the enzyme.

[0013] In an alternative embodiment, the hIDS can be produced by human neuronal or glial cells in cell culture (e.g., bioreactors) and administered as an enzyme replacement therapy (“ERT”), e.g., by injecting the enzyme - into the CSF, directly into the CNS, and / or systemically. However, the gene therapy approach offers several advantages over ERT since systemic delivery of the enzyme will not result in treating the CNS because the enzyme cannot cross the blood brain barrier; and, unlike the gene therapy approach of the invention, direct delivery of the enzyme to the CSF and / or CNS would require repeat injections which are not only burdensome, but pose a risk of infection.

[0014] The hIDS encoded by the transgene can include, but is not limited to human IDS (hIDS) having the amino acid sequence of SEQ ID NO. 1 (as shown in FIG. 1), and derivatives of hIDS having amino acid substitutions, deletions, or additions, e.g., including but not limited to amino acid substitutions selected from corresponding non-conserved residues in orthologs of IDS shown in FIG. 2, with the proviso that such mutations do not include replacement of the cysteine residue at position 84 (C84) which is required for enzyme activity (Millat et al., 1997, Biochem J 326: 243-247); or a mutation that has been identified in severe, severe-intermediate, intermediate, or attenuated MPS II phenotypes e.g., as shown in FIG. 3, or as reported by Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-761 (reporting “attenuated” mutants R48P, A85T, W337R, and the truncated mutant Q531X; and “severe” mutants P86L, S333L, S349I, R468Q, R468L); Millat et al., 1998, BBA 1406: 214-218 (reporting “attenuated” mutants P480L and P480Q; and “severe” mutant P86L); and Bonucelli et al., 2001, BBA 1537:233-238, each of which is incorporated by reference herein in its entirety.

[0015] For example, amino acid substitutions at a particular position of hIDS can be selected from among corresponding non-conserved amino acid residues found at that position in the IDS orthologs aligned in FIG. 2, with the proviso that such substitutions do not include any of the deleterious mutations shown in FIG. 3 or as reported by Sukegawa-Hayasaka et al., 2006, supra', Millat et al., 1998, supra, or Bonucelli et al., 2001, supra, each of which is incorporated by reference herein in its entirety. The resulting transgene product can be tested using conventional assays in vitro, in cell culture or test animals to ensure that the mutation does not disrupt IDS function. Preferred amino acid substitutions, deletions or additions selected should be those that maintain or increase enzyme activity, stability or half-life of IDS, as tested by conventional assays in vitro, in cell culture or animal models for MPS II. For example, the enzyme activity of the transgene product can be assessed using a conventional enzyme assay with, for example, 4-Methylumbelliferyl a-L-idopyranosiduronic acid 2-sulfate or 4-methylumbelliferyl sulfate as the substrate (see, e.g., Lee et al., 2015, Clin. Biochem. 48(18):1350-1353, Dean et al., 2006, Clin. Chern. 52(4):643-649 for exemplary IDS enzyme assays that can be used, each of which is incorporated by reference herein in its entirety). The ability of the transgene product to correct MPS II phenotype can be assessed in cell culture; e.g., by transducing MPS II cells in culture with a viral vector or other DNA expression construct encoding hIDS or a derivative; by adding the transgene product or a derivative to MPS II cells in culture; or by co-culturing MPS II cells with human neuronal / glial host cells engineered to express and secrete rhIDS or a derivative, and determining correction of the defect in the MPS II cultured cells, e.g., by detecting IDS enzyme activity and / or reduction in GAG storage in the MPS II cells in culture (see, e.g., Stroncek et al., 1999, Transfusion 39(4):343-350, which is incorporated by reference herein in its entirety). In a preferred embodiment, the reduction in GAG storage is reduction in heparan sulfate (HS) storage. In another embodiment, the reduction in GAG storage is reduction in dermatan sulfate (DS) storage. In another embodiment, the reduction in GAG storage is reduction in both HS storage and DS storage.

[0016] Animal models for MPS II have been described that can be used to assess the therapeutics described herein. For example, a knockout mouse model (IDS-knockout) of MPS II was engineered by replacing exons 4 and 5 of the IDS gene with the neomycin resistance gene. (Garcia et al., 2007, J Inherit Metab Dis 30: 924-34). This IDS-knockout mouse exhibits many of the characteristics of MPS II, including skeletal abnormalities, hepatosplenomegaly, elevated urinary and tissue GAG, and brain storage lesions (Muenzer et al., 2001, Acta Paediatr Suppl 91:98-99) and was used to assess the effect of enzyme replacement therapy in MPS II in support of clinical trials for ERT. This mouse model, therefore, is a relevant model for studying the effects of gene therapy delivering rIDS produced by neuronal or glial cells as a treatment for MPS II (see, e.g., Polito and Cosma, 2009, Am. J. Hum. Genet. 85(2):296-301, which is incorporated by reference herein in its entirety).

[0017] Preferably, the hIDS transgene produced by the human neuronal / glial cells should be controlled by expression control elements that function in neurons and / or glial cells, e.g., the CB7 promoter (a chicken P-actin promoter and CMV enhancer), and can include other expression control elements that enhance expression of the transgene driven by the vector (e.g., chicken P-actin intron and rabbit P-globin poly A signal). The cDNA construct for the hIDS transgene should include a coding sequence for a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced CNS cells. Such signal peptides used by CNS cells may include but are not limited to: Oligodendrocyte-myelin glycoprotein (hOMG) signal peptide: MEYQILKMSLCLFILLFLTPGILC (SEQ ID NO:2) Cellular repressor of ElA-stimulated genes 2 (hCREG2) signal peptide: MSVRRGRRPARPGTRLSWLLCCSALLSPAAG (SEQ ID NO:3) V-set and transmembrane domain containing 2B (hVSTM2B) signal peptide: MEQRNRLGALGYLPPLLLHALLLFVADA (SEQ ID NO:4) Protocadherin alpha-1 (hPCADHAl) signal peptide: MVFSRRGGLGARDLLLWLLLLAAWEVGSG (SEQ ID NO:5) FAM19A1 (TAFA1) signal peptide: MAMVSAMSWVLYLWISACA (SEQ ID NO:6) Interleukin-2 signal peptide: MYRMQLLSCIALILALVTNS (SEQ ID NO: 14) Signal peptides may also be referred to herein as leader sequences or leader peptides.

[0018] The recombinant vector used for delivering the transgene should have a tropism for cells in the CNS, including but limited to neurons and / or glial cells. Such vectors can include non-replicating recombinant adeno-associated virus vectors (“rAAV”), particularly those bearing an AAV9 or AAVrhlO capsid are preferred. AAV variant capsids can be used, including but not limited to those described by Wilson in US Patent No. 7,906,111 which is incorporated by reference herein in its entirety, with AAV / hu.31 and AAV / hu.32 being particularly preferred; as well as AAV variant capsids described by Chatterjee in US Patent No. 8,628,966, US Patent No. 8,927,514 and Smith et al., 2014, Mol Ther 22: 1625-1634, each of which is incorporated by reference herein in its entirety. However, other viral vectors may be used, including but not limited to lentiviral vectors, vaccinia viral vectors, or non-viral expression vectors referred to as “naked DNA” constructs.

[0019] In one embodiment, Construct 1 can be used for delivering the transgene. Construct 1 is a recombinant adeno-associated virus serotype 9 capsid containing human iduronate-2-sulfatase expression cassette wherein expression is driven by a hybrid of the cytomegalovirus (CMV) enhancer and the chicken beta actin promoter (CB7), wherein the IDS expression cassette is flanked by inverted terminal repeats (ITRs) and the transgene includes the chicken beta actin intron and a rabbit beta-globin polyadenylation (polyA) signal. In a preferred embodiment, the ITRs are AAV2 ITRs. In one embodiment, Construct 1 comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO:45.

[0020] Pharmaceutical compositions suitable for administration to the CSF comprise a suspension of the rhIDS vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients. In certain embodiments, the pharmaceutical compositions are suitable for intrathecal administration. In certain embodiments, the pharmaceutical compositions are suitable for intraci sternal administration (injection into the cistema magna). In certain embodiments, the pharmaceutical compositions are suitable for injection into the subarachnoid space via a Cl-2 puncture. In certain embodiments, the pharmaceutical compositions are suitable for intracerebroventricular administration. In certain embodiments, the pharmaceutical compositions are suitable for administration via lumbar puncture. In some embodiments, the pharmaceutical composition comprising the rAAV of the present disclosure comprises sodium chloride at a concentration of about 8.77 g / L, magnesium chloride 6-hydrate, at a concentration of about 0.244 g / L, potassium chloride at a concentration of about 0.224 g / L, calcium chloride dihydrate at a concentration of about 0.206 g / L, dextrose anhydrous at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.001% (volume / volume), sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L.

[0021] Therapeutically effective doses of the recombinant vector should be administered to the CSF via intrathecal administration (i.e., injection into the subarachnoid space so that the recombinant vectors distribute through the CSF and transduce cells in the CNS). In some embodiments, the recombinant vector is administered in a solution comprising sodium chloride at a concentration of about 8.77 g / L, magnesium chloride 6-hydrate, at a concentration of about 0.244 g / L, potassium chloride at a concentration of about 0.224 g / L, calcium chloride dihydrate at a concentration of about 0.206 g / L, dextrose anhydrous at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.001% (volume / volume), sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L. This can be accomplished in a number of ways - e.g., by intracranial (cisternal or ventricular) injection , or injection into the lumbar cistern. For example, intraci sternal (IC) injection (into the cistema magna) can be performed by CT-guided suboccipital puncture; or injection into the subarachnoid space can be performed via a Cl-2 puncture when feasible for the patient; or lumbar puncture (typically diagnostic procedures performed in order to collect a sample of CSF) can be used to access the CSF. Alternatively, intracerebroventricular (ICV) administration (a more invasive technique used for the introduction of antiinfective or anticancer drugs that do not penetrate the blood-brain barrier) can be used to instill the recombinant vectors directly into the ventricles of the brain. Alternatively, intranasal administration may be used to deliver the recombinant vector to the CNS.

[0022] CSF concentrations can be monitored by directly measuring the concentration of rhIDS in the CSF fluid obtained from occipital or lumbar punctures, or estimated by extrapolation from concentrations of the rhIDS detected in the patient’s serum.

[0023] In certain embodiments, the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain magnetic resonance imaging (MRI) of the human subject’s brain. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x 1010 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.9 * 1010 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x io10 GC / g brain mass as determined by MRI. In certain embodiments, the dose of the recombinant nucleotide expression vector is 1.3 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a PolyA PCR assay). In certain embodiments, the dose of the recombinant nucleotide expression vector is 1.9 x 1010 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments, the dose of the recombinant nucleotide expression vector is 6.5 x 1010 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A PCR assay). In certain embodiments, the dose of the recombinant nucleotide expression vector is 9.6 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments, the dose of the recombinant nucleotide expression vector is 2.0 x 1011 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A PCR assay). In certain embodiments, the dose of the recombinant nucleotide expression vector is 2.9 x 1011 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x io10 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x io11 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x io11 GC / g brain mass brain mass (e.g., as determined by MRI). In some embodiments, a transgene-specific assay or a PolyA-specific assay is used to calculate the dose of the recombinant nucleotide expression vector to be administered to a subject. In certain embodiments, the human subject’s brain mass is converted from the human subject’s brain volume by multiplying the human subject’s brain volume in cm3 by a factor of 1.046 g / cm3, wherein the human subject’s brain volume is obtained from the human subject’s brain MRI.

[0024] By way of background, human IDS is translated as a 550 amino acid polypeptide that contains eight potential N-glycosylation sites (N31, N115, N144, N246, N280, N325, N513 and N537) depicted in FIG.l and includes a 25 amino acid signal sequence which is cleaved during processing. An initial 76 kDa intracellular precursor is converted into a phosphorylated 90 kDa precursor after modification of its oligosaccharide chains in the Golgi apparatus. This precursor is processed by glycosylation modifications and proteolytic cleavage through various intracellular intermediates to a major 55 kDa form. To summarize, after removal of the 25 aa signal sequence, proteolytic processing involves N-terminal proteolytic cleavage downstream of N31 removing a propeptide of eight amino acids (residues 26-33), and C-terminal proteolytic cleavage upstream of N513 which releases an 18 kDa polypeptide and produces a 62 kDa intermediate that is converted to a 55 kDa mature form. Further proteolytic cleavage yields a 45 kDa mature form located in the lysosomal compartment. (See FIG. 4 for diagram reproduced from Millat et al., 1997, Exp Cell Res 230: 362-367 (“Millat 1997”); Millat et al. 1997, Biochem J. 326: 243-247 (“Millat 1997a”); and Froissart et al., 1995, Biochem J. 309:425-430, each of which is incorporated by reference herein in its entirety).

[0025] A formylglycine modification of C84 (shown in bold in FIG. 1) required for enzyme activity probably occurs as an early post-translational or co-translational event, most probably in the endoplasmic reticulum. (See, Millat 1997a, citing Schmidt et al., 1995, Cell 82: 271-278). Post-translational processing continues in the Golgi to include addition of complex sialic acidcontaining glycans and acquisition of mannose-6-phosphate residues which tag the enzyme for delivery to the lysosomal compartment. (See, Clarke, 2008, Expert Opin Pharmacother 9: 311317 for a concise review which is incorporated by reference herein in its entirety). While no single glycosylation site is essential for IDS stability, glycosylation at position N280 is important for cellular internalization and lysosomal targeting via the mannose-6-phosphate (M6P) receptor. (Chung et al., 2014, Glycoconj J 31:309-315 at p. 310, first column). In the normal physiologic state, IDS is produced at very low levels and very little, if any, enzyme is secreted from the cell. (Clarke, 2008, supra).

[0026] The invention is based, in part, on the following principles: (i) Neuronal and glial cells in the CNS are secretory cells that possess the cellular machinery for post-translational processing of secreted proteins - including glycosylation, mannose-6-phosphorylation, and tyrosine-O-sulfation - robust processes in the CNS. See, e.g., Sleat et al., 2005, Proteomics 5: 1520-1532, and Sleat 1996, J Biol Chern 271: 19191-98 which describes the human brain mannose-6-phosphate glycoproteome and notes that the brain contains more proteins with a much greater number of individual isoforms and mannose-6-phosphorylated proteins than found in other tissues; and Kanan et al., 2009, Exp. Eye Res. 89: 559567 and Kanan & Al-Ubaidi, 2015, Exp. Eye Res. 133: 126-131 reporting the production of tyrosine-sulfated glycoproteins secreted by neuronal cells, each of which is incorporated by reference in its entirety for post-translational modifications made by human CNS cells. (ii) The human brain produces multiple isoforms of natural / native IDS. In particular, N-terminal sequencing of human brain mannose-6-phosphorylated glycoproteins revealed that the N-terminal sequence of the mature 42 kDa chain of hIDS varies in the brain, starting at positions 34 or 36 as follows: T34DALNVLLI; and A36LNVLLIIV. (Sleat, 2005, Proteomics 5: 1520-1532, Table S2). Two of the eight N-linked glycosylation sites, namely N280 and N116, were found to be mannose-6-phophorylated in IDS obtained from human brain. (Sleat et al., 2006, Mol & Cell Proeomics 5.4: 686-701, reported at Table V). (iii) During processing of hIDS, two polypeptides, 76 kDa and 90 kDa, are secreted by neural and glial cells, but only the 90 kDa polypeptide is mannose-6-phosphorylated, which is necessary for secreted forms of the enzyme to achieve cross correction. (See, Millat, 1997, Fig. 1 results for transduced lymphoblastoid cells, and Froissart 1995, Fig. 4 showing similar results for transduced fibroblasts -in culture medium, only the 90 kDa form is phosphorylated). Interestingly, it has been demonstrated that recombinant IDS produced by neuronal and glial cells may be endocytosed by recipient CNS cells more avidly than recombinant IDS produced by other cells such as kidney. Daniele 2002 (Biochimica et Biophysica Acta 1588(3):203-9) demonstrated M6P-receptor mediated endocytosis of recombinant IDS from conditioned media of transduced neuronal and glial cell cultures by a recipient population of non-transduced neuronal and glial cells which properly processed the precursor to the 45 kDa mature active form. Uptake of the recombinant IDS produced by the neuronal and glial cell lines (74% endocytosis) far exceeded uptake of the enzyme produced by a kidney cell line (5.6% endocytosis). In each case, uptake was inhibited by M6P, indicating that recombinant IDS uptake was M6P-receptor mediated. (See Daniele 2002, Tables 2 and 4 and accompanying description in Results at pp. 205-206 summarized in Table 1 below). Table 1. Summary of Results Reported in Daniele 2002 Cell Line Source of rIDS Media Enzyme Units Recipient Cells: Units Recovered % Endocytosis (mean value) Neuronal Glial Kidney (transfected’ 35 U 1.7U 2.2 U 5.6% Neuronal 12 U 8.8 U 8.8 U 74% Glial ^d-transduced) 14 U 10.5 U 10.5 U 74% (iv) The gene therapy approach described herein should result in the continuous secretion of an hIDS glycoprotein precursor of about 90 kDa as measured by polyacrylamide gel electrophoresis (depending on the assay used) that is enzymatically active. First, the enzyme responsible for the formylglycine modification of C84 which is required for IDS activity — the FGly-Generating Enzyme (FGE, aka SUMF1) — is expressed in the cerebral cortex of the human brain (gene expression data for SUMF1 may be found, for example, at GeneCards, accessible at http: / / www.genecards.org). Second, the secreted glycosylated / phosphorylated rIDS produced by transduced neurons and glial cells in situ should be taken up and correctly processed by untransduced neural and glial cells in the CNS. Without being bound to any theory, it appears that the secreted rhIDS precursor produced in situ by gene therapy may be more avidly endocytosed by recipient cells in the CNS than would traditional recombinant enzymes used for ERT if administered to the CNS. For example, Elaprase® (made in HT1080, a fibrosarcoma cell line) is a purified protein reported to have a molecular weight of about 76 kDa - not the 90 kDa species secreted by neuronal and glial cells that appears to be more heavily phosphorylated. While the eight N-linked glycosylation sites are reported to be fully occupied in Elaprase® and contain two bis-mannose-6-phosphate terminated glycans as well as complex highly sialylated glycans, the post-translational modification of C84 to FGly, which is an absolute requirement for enzyme activity, is only about 50%. (Clarke, 2008, Expert Opin Pharmacother 9:311-317; Elaprase® Full Prescribing Information and EMA filing). Another recombinant product, Hunterase® is made in CHO cells. While reported to have higher FGly and activity than Elaprase®, mannose-6-phosphorylation and uptake did not differ. (Chung, 2014, Glycoconj J 31:309-315). (v) The extracellular IDS efficacy in vivo depends on uptake (cell and lysosome internalization) through M6P and its active site formylglycine (FGly), which is converted from C84 through post-translational modification by formylglycine-generating enzyme. As shown above in Table 1, brain cells (neuronal and glial cells) show higher enzyme activities when incubated with IDS precursor media secreted by transduced neuronal and glial cells than with IDS precursor media secreted by genetically engineered kidney cells. The resultant five-fold increase in activity can likely be attributed to the efficient uptake of IDS (See Daniele 2002, Tables 2 and 4). Commercial forms of IDS, which are generated by CHO cells or HT-1080 cells, have a FGly content of about about 50% to 70%, which determines the enzyme activity. However, neuronal and glial cells may improve upon this activity, due to improvement of IDS uptake. (vi) The cellular and subcellular trafficking / uptake of lysosomal proteins, including IDS, is through M6P. IDS from brain cells may contain higher M6P content, as reported in Daniele 2002, and in Sleat, Proteomics, 2005 (indicating that the human brain contains more (in both a quantitative and qualitative sense) Man6-P glycoproteins than other tissues.). It is possible to measure the M6P content of an IDS precursor, as done in Daniele 2002. In the presence of inhibitory M6P (e.g., 5 mM), the uptake of IDS precursor generated by non-neuronal or non-glial cells, such as the genetically engineered kidney cells of Daniele 2002, is predicted to decrease to levels close to that of the control cells, as was shown in Daniele 2002. While in the presence of inhibitory M6P, the uptake of IDS precursor generated by brain cells, such as neuronal and glial cells, is predicted to remain at a high level, as was shown in Daniele 2002, where the uptake was four times higher than control cells and comparable to the level of IDS activity (or uptake) of IDS precursor generated by genetically engineered kidney cells without the presence of inhibitory M6P. This assay allows for a way to predict the M6P content in IDS precursor generated by brain cells, and, in particular, to compare the M6P content in IDS precursors generated by different types of cells. The gene therapy approach described herein should result in the continuous secretion of an hIDS precursor that may be taken up into neuronal and glial cells at a high level in the presence of inhibitory M6P in such an assay. (vii) The M6P content and uptake of IDS precursor may also be demonstrated by 90 kDa and 76 kDa gel bands (e.g., SDS-PAGE gel bands). The 90 kDa is reported to be highly glycosylated / phosphorylated and contains M6P, while 76 kDa is not. A very broad gel band with a range from 76 kDa to 95 kDa and with an average MW of SO-85 kDa, similar to the IDS precursor gel band generated from genetically engineered kidney cells (Daniele 2002, Figure 1), may be contrasted with a gel band of IDS precursor generated from brain cells. In Daniele 2002, the gel band cannot be obtained due to unsuccessful immunoprecipitation of the IDS precursor. The gene therapy approach described herein should result in the continuous secretion of an hIDS precursor that differs from the IDS precursor gel band generated from genetically engineered kidney cells. (viii) The M6P content of commercial IDS precursor is 2 to 2.5 mol / mol, majority of which is present in a form of di-phosphorylated glycans. Although in average, every IDS precursor is phosphorylated, a normal distribution of glycans will have some IDS precursor with 2, 1 and 0 of di-phosphorylated M6P glycans assuming multiple phosphorylation sites. Uptake rate should be significant higher with multiple phosphorylation. (ix) The glycosylation of hIDS by human cells of the CNS will result in the addition of glycans that can improve stability, half-life and reduce unwanted aggregation of the transgene product. Significantly, the glycans that are added to hIDS of the invention include 2,6-sialic acid, incorporating Neu5 Ac (“NANA”) but not its hydroxylated derivative, NeuGc (N-Glycolylneuraminic acid, i.e., “NGNA” or “Neu5Gc”). Such glycans are not present in recombinant IDS products, such as Hunterase®, made in CHO cells because CHO cells do not have the 2,6-sialyltransferase required to make this post-translational modification; nor do CHO cells produce bisecting GlcNAc, although they do add Neu5Gc (NGNA) as sialic acid not typical (and potentially immunogenic) to humans instead of Neu5Ac (NANA). See, e.g., Dumont et al., 2016, Critical Rev in Biotech 36(6): 1110-1122 (Early Online pp. 1-13 at p. 5); and Hague et al., 1998 Electrophor 19:2612-2630 (“[t]he CHO cell line is considered ‘phenotypically restricted,’ in terms of glycosylation, due to the lack of an a2,6-sialyl-transferase”). Moreover, CHO cells can also produce an immunogenic glycan, the a-Gal antigen, which reacts with anti-a-Gal antibodies present in most individuals, and at high concentrations can trigger anaphylaxis. See, e.g., Bosques, 2010, Nat Biotech 28: 1153-1156. The human glycosylation pattern of the rhIDS of the invention should reduce immunogenicity of the transgene product and improve efficacy. (x) Immunogenicity of a transgene product could be induced by various factors, including the immune condition of the patient, the structure and characteristics of the infused protein drug, the administration route, and the duration of treatment. Process-related impurities, such as host cell protein (HCP), host cell DNA, and chemical residuals, and product-related impurities, such as protein degradants and structural characteristics, such as glycosylation, oxidation and aggregation (sub-visible particles), may also increase immunogenicity by serving as an adjuvant that enhances the immune response. The amounts of process-related and product-related impurities can be affected by the manufacturing process: cell culture, purification, formulation, storage and handling, which can affect commercially manufactured IDS products. In gene therapy, proteins are produced in vivo, such that process-related impurities are not present and protein products are not likely to contain product-related impurities / degradants associated with proteins produced by recombinant technologies, such as protein aggregation and protein oxidation. Aggregation, for example, is associated with protein production and storage due to high protein concentration, surface interaction with manufacturing equipment and containers, and the purification process with certain buffer systems. But these conditions that promote aggregation are not present when a transgene is expressed in vivo. Oxidation, such as methionine, tryptophan and histidine oxidation, is also associated with protein production and storage, caused, for example, by stressed cell culture conditions, metal and air contact, and impurities in buffers and excipients. The proteins expressed in vivo may also oxidize in a stressed condition, but humans, like many organisms, are equipped with an antioxidation defense system, which not only reduces the oxidation stress, but can also repairs and / or reverses the oxidation. Thus, proteins produced in vivo are not likely to be in an oxidized form. Both aggregation and oxidation could affect the potency, PK (clearance) and can increase immunogenicity concerns. The gene therapy approach described herein should result in the continuous secretion of an hIDS precursor with a reduced immunogenicity compared to commercially manufactured products. (xi) In addition to the N-linked glycosylation sites, hIDS contains a tyrosine (“Y”) sulfation site (PSSEKY165ENTKTCRGPD). (See, e.g., Yang et al., 2015, Molecules 20:2138-2164, esp. at p. 2154 which is incorporated by reference in its entirety for the analysis of amino acids surrounding tyrosine residues subjected to protein tyrosine sulfation. The “rules” can be summarized as follows: Y residues with E or D within +5 to -5 position of Y, and where position -1 of Y is a neutral or acidic charged amino acid - but not a basic amino acid, e.g., R, K, or H that abolishes sulfation). While not intending to be bound by any theory, sulfation of this site in hIDS may improve stability of the enzyme and binding affinity for substrate. Tyrosine-sulfation of hIDS - a robust post-translational process in human CNS cells - should result in improved processing and activity of transgene products. The significance of tyrosine-sulfation of lysosomal proteins has not been elucidated; but in other proteins it has been shown to increase avidity of proteinprotein interactions (antibodies and receptors), and to promote proteolytic processing (peptide hormone). (See, Moore, 2003, J Biol. Chern. 278: 24243-46; and Bundegaard et al., 1995, The EMBO J 14: 3073-79). The tyrosylprotein sulfotransferase (TPST1) responsible for tyrosine-sulfation (which may occur as a final step in IDS processing) is apparently expressed at higher levels (based on mRNA) in the brain (gene expression data for TPST1 may be found, for example, at the EMBL-EBI Expression Atlas, accessible at http: / / www.ebi.ac.uk / gxa / home). Such post-translational modification, at best, is under-represented in CHO cell products. Unlike human CNS cells, CHO cells are not secretory cells and have a limited capacity for post-translational tyrosine-sulfation. (See, e.g., Mikkelsen & Ezban, 1991, Biochemistry 30: 1533-1537, esp. discussion at p. 1537).

[0027] For the foregoing reasons, the production of rhIDS by human neuronal and / or glial cells should result in a “biobetter” molecule for the treatment of MPS II accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding rhIDS to the CSF of a patient (human subject) diagnosed with an MPS II disease (including but not limited to Hunter) to create a permanent depot in the CNS that continuously supplies a fully human-glycosylated, mannose-6-phosphorylated, sulfated transgene product secreted by the transduced CNS cells. The hIDS transgene product secreted from the depot into the CSF will be endocytosed by cells in the CNS, resulting in “cross-correction” of the enzymatic defect in the MPS II recipient cells.

[0028] It is not essential that every rhIDS molecule produced either in the gene therapy or protein therapy approach be fully glycosylated, phosphorylated, and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation (including 2,6-sialylation and mannose-6-phophorylation) and sulfation to demonstrate efficacy. The goal of gene therapy treatment of the invention is to slow or arrest the progression of disease. Efficacy may be monitored by measuring cognitive function (e.g., prevention or decrease in neurocognitive decline); reductions in biomarkers of disease (such as GAG) in CSF and or serum; and / or increase in IDS enzyme activity in CSF and / or serum. Signs of inflammation and other safety events may also be monitored.

[0029] As an alternative, or an additional treatment to gene therapy, the rhIDS glycoprotein can be produced in human neural or glial cell lines by recombinant DNA technology and the glycoprotein can be administered to patients diagnosed with MPS II systemically and / or into the CSF for ERT). Human cell lines that can be used for such recombinant glycoprotein production include but are not limited to HT-22, SK-N-MC, HCN-1 A, HCN-2, NT2, SH-SY5y, hNSCl 1, or ReNcell VM (see, e.g., Dumont et al., 2016, Critical Rev in Biotech 36(6): 1110-1122 “Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives” which is incorporated by reference in its entirety for a review of the human cell lines that could be used for the recombinant production of the rHuGlylDS glycoprotein). To ensure complete glycosylation, especially sialylation, and tyrosine-sulfation, the cell line used for production can be enhanced by engineering the host cells to co-express a-2,6-sialyltransferase (or both a-2,3-and a-2,6-sialyltransferases) and / or TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation.

[0030] While the delivery of rhIDS should minimize immune reactions, the clearest potential source of toxicity related to CNS-directed gene therapy is generating immunity against the expressed rhIDS protein in human subjects who are genetically deficient for IDS and, therefore, potentially not tolerant of the protein and / or the vector used to deliver the transgene.

[0031] Thus, in a preferred embodiment, it is advisable to co-treat the patient with immune suppression therapy — especially when treating patients with severe disease who have close to zero levels of IDS. Immune suppression therapies involving a regimen of tacrolimus or rapamycin (sirolimus) in combination with mycophenolic acid, or other immune suppression regimens used in tissue transplantation procedures can be employed. Such immune suppression treatment may be administered during the course of gene therapy, and in certain embodiments, pre-treatment with immune suppression therapy may be preferred. Immune suppression therapy can be continued subsequent to the gene therapy treatment, based on the judgment of the treating physician, and may thereafter be withdrawn when immune tolerance is induced; e.g., after 180 days.

[0032] Combinations of delivery of the rhIDS to the CSF accompanied by delivery of other available treatments are encompassed by the methods of the invention. The additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment. Available treatments for MPS II that could be combined with the gene therapy of the invention include but are not limited to enzyme replacement therapy using Elaprase® administered systemically or to the CSF; and / or HSCT therapy.

[0033] In one aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising delivering to the CSF of the human subject a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or human glial cells,wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain MRI of the human subject’s brain.

[0034] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising, in the following order: (a) delivering to the CSF of the human subject a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or human glial cells; (b) measuring level of heparan sulfate in the CSF of the human subject; and (c) comparing the level of heparan sulfate in the CSF of the human subject with level of heparan sulfatae in a reference population; wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain magnetic resonance imaging (MRI) of the human subject’s brain. In certain embodiments, the reference population consists of: (a) at least 1, 2, 3, 4, 5, 10, 25, 50, 75, 100, 200, 250, 300, 400, 500, or 1000 individual healthy people without MPS II, preferably of similar age, weight, and / or of the same gender as the human subject.

[0035] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising, in the following order: (a) taking a first measurement of the level of heparan sulfate in the CSF of the human subject; (b) delivering to the CSF of the human subject a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or human glial cells; and (c) after a period of time, taking a second measurement of the level of heparan sulfate; wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain magnetic resonance imaging (MRI) of the human subject’s brain. In certain embodiments, the period of time is about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 moths, 4 months, 5 months, 6 months, 7 months, 8 months, 11 months, or 1 year. 2022221284   28 Aug 2026 [0035A] In another aspect, provided herein is a method of treating mucopolysaccharidosis II (MPS II) in a human subject, the method comprising: a) measuring the level of D2S6 in a biological sample from the human subject;and b) delivering an effective amount of an active human iduronate-2-sulfatase (hIDS) to the central nervous system (CNS) of the human subject if the level of D2S6 in the biological sample is higher than a reference level, wherein the reference level is the level of D2S6 from one or more healthy subjects or one or more non-neuronopathic MPS II subjects. [0035B] In another aspect, provided herein is method of treating mucopolysaccharidosis II (MPS II) in a human subject, the method comprising: a) measuring the levels of D2S6 and total heparan sulfate disaccharides (HS) in a biological sample from the human subject, wherein the total HS comprises disaccharides D2S6, D0A0, D0S0, and D0A6; and b) delivering an effective amount of an active human iduronate-2-sulfatase (hIDS) to the central nervous system (CNS) of the human subject if the ratio between the levels of D2S6 and total HS is higher than a reference ratio, or the ratio between the levels of D2S6 and total HS is at least about 20%, wherein the reference ratio is the ratio between the levels of D2S6 and total HS from one or more healthy subjects or one or more non-neuronopathic MPS II subjects. [0035C] In another aspect, provided herein is the use of an active human iduronate-2-sulfatase (hIDS) in the manufacture of a medicament for treating mucopolysaccharidosis II (MPS II) in a human subject, wherein the level of D2S6 has been measured in a biological sample from the human subject and the level of D2S6 in the biological sample is higher than a reference level, wherein the hIDS is formulated to be delivered to the central nervous system (CNS) of the human subject, and wherein the reference level is the level of D2S6 from one or more healthy subjects or one or more non-neuronopathic MPS II subjects. [0035D] In another aspect, provided herein is the use of an active human iduronate-2-sulfatase (hIDS) in the manufacture of a medicament for treating mucopolysaccharidosis II (MPS II) in a human subject, wherein the levels of D2S6 and total heparan sulfate disaccharides (HS) have been measured in a biological sample from the human subject and 2022221284   28 Aug 2026 the ratio between the levels of D2S6 and total HS in the biological sample is higher than a reference ratio or the ratio between the levels of D2S6 and total HS is at least about 20%, wherein the hIDS is formulated to be delivered to the central nervous system (CNS) of the human subject, and wherein the reference ratio is the ratio between the levels of D2S6 and total HS from one or more healthy subjects or one or more non-neuronopathic MPS II subjects. [0035E] In another aspect, provided herein is a method of treating mucopolysaccharidosis II (MPS II) in a human subject in need thereof, the method comprising delivering an effective amount of an active human iduronate-2-sulfatase (hIDS) to the central nervous system (CNS) of the human subject, and measuring the level of D2S6 in a biological sample from the human subject after the active hIDS has been delivered to the CNS of the human subject. [0035F] In another aspect, provided herein is the use of an active human iduronate-2-sulfatase (hIDS) in the manufacture of a medicament for treating mucopolysaccharidosis II (MPS II) in a human subject in need thereof, wherein the hIDS is formulated to be delivered to the central nervous system (CNS) of the human subject and wherein the level of D2S6 is to be measured in a biological sample from the human subject after the active hIDS has been delivered to the CNS of the human subject. [0035G] In another aspect, provided herein is a method of treating mucopolysaccharidosis II (MPS II) in a human subject in need thereof, the method comprising delivering an effective amount of an active human iduronate-2-sulfatase (hIDS) to the central nervous system (CNS) of the human subject, measuring the level of D2S6 in a first cerebrospinal fluid (CSF) sample from the human subject before delivering the active hIDS to the human subject, and measuring the level of D2S6 in a second CSF sample from the human subject after delivering the active hIDS to the human subject. [0035H] In another aspect, provided herein is the use of an active human iduronate-2-sulfatase (hIDS) in the manufacture of a medicament for treating mucopolysaccharidosis II (MPS II) in a human subject in need thereof, wherein the hIDS is formulated to be delivered to the central nervous system (CNS) of the human subject, wherein the level of D2S6 has been measured in a first cerebrospinal fluid (CSF) sample from the human subject before the active hIDS has been delivered to the human subject and the level of D2S6 is to be measured in a second CSF sample from the human subject after the active hIDS has been delivered to the human subject.

[0036] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is delivered to lysosomes of cells in the CNS of the human subject.

[0037] In certain embodiments of the method for treating described herein, the human subject’s brain mass is converted from the human subject’s brain volume by multiplying the human subject’s brain volume in cm3 by a factor of 1.046 g / cm3, wherein the human subject’s brain volume is determined by brain MRI of the subject’s brain.

[0038] In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x 1010 GC / g brain mass as determined by MRI, or about 6.5 x 1010 GC / g brain mass as determined by MRI. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x io11 GC / g brain mass as determined by MRI. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x io10 GC / g brain mass as determined by MRI. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x io10 GC / g brain mass as determined by MRI. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x io11 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x 1011 GC / g brain mass as determined by MRI. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x 1011 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x 1010 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay).

[0039] In various embodiments of the method for treating described herein, the human subject is 5 years old or older and less than 18 years old. In specific embodiments, the human subject is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 years old. In specific embodiments, the human subject is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 years old. In specific embodiments, the human subject is 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18 or 18-19 years old. In specific embodiments, the human subject is about 56, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18 or 18-19 years old. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x io10 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose according to Table 7.

[0040] In various embodiments of the method for treating described herein, the human subject is 4 months old or older and less than 5 years old. In specific embodiments, the human subject is 4, 5, 6, 7, 8, 9, 10, or 11 months old. In specific embodiments, the human subject is about 4, 5, 6, 7, 8, 9, 10, or 11 months old. In specific embodiments, the human subject is 4-5, 56, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months old. In specific embodiments, the human subject is about 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months old. In specific embodiments, the human subject is 1, 2, 3, 4, or 5 years old. In specific embodiments, the human subject is about 1, 2, 3, 4, or 5 years old. In specific embodiments, the human subject is 1-2, 2-3, 3-4, 4-5, or 5-6 years old. In specific embodiments, the human subject is about 1-2, 2-3, 3-4, 4-5, or 5-6 years old. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x io10 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x io10 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x 1010 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x io10 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x io11 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x io11 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x io11 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x io11 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose chosen from Dose 1 or Dose 2 according to Table 5. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose according to Table 6.

[0041] In some embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered via intraci sternal (IC) administration. In other embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered via intracerebroventricular (ICV) administration.

[0042] In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a volume that does not exceed 10% of the total cerebrospinal fluid volume of the human subject.

[0043] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is secreted at a detectable level.

[0044] In certain embodiments of the method for treating described herein, the human neuronal or human glial cells carry at least one mutation in the endogenous gene encoding human IDS precursor.

[0045] In certain embodiments of the method for treating described herein, the human neuronal or human glial cells are transduced with a recombinant adeno-associated virus vector (rAAV).

[0046] In a preferred embodiment, the recombinant nucleotide expression vector is an AAV9 or AAVrhlO vector.

[0047] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is expressed under the control of a CB7 promoter.

[0048] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is expressed from a cDNA encoding human IDS precursor.

[0049] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is about 90 kDa as measured by polyacrylamide gel electrophoresis.

[0050] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor contains a formylglycine.

[0051] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor (a) is a2,6-sialylated; (b) does not contain detectable NeuGc; (c) does not contain detectable a-Gal antigen; (d) contains tyrosine-sulfation; and / or (e) is mannose-6-phosphorylated.

[0052] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO. 1.

[0053] In certain embodiments provided herein, the method further comprising administering an immune suppression therapy to the human subject before or concurrently with the human IDS precursor treatment and optionally continuing immune suppression therapy thereafter.

[0054] In some embodiments, the immune suppression therapy comprises administering one or more corticosteroids, sirolimus, and / or tacrolimus. In a specific embodiment, the one or more corticosteroids are methylprednisolone and / or prednisone.

[0055] In some embodiments, the method further comprises administering one or more antibiotics to the human subject before or concurrently with the immune suppression therapy. In a specific embodiment, the one or more antibiotics are trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atovaquone.

[0056] In some embodiments, the method further comprises administering one or more antifungal therapies to the human subject before or concurrently with the immune suppression therapy.

[0057] In some embodiments, the method further comprises a step of measuring one or more of the following biomarkers after administration of the recombinant nucleotide expression vector: (a) level of glycosaminoglycans (GAGs) in CSF; (b) level of iduronate-2-sulfatase (I2S) in CSF; (c) level of GAGs in plasma; (d) level of I2S in plasma; (e) level of leukocyte I2S enzyme activity; and (f) level of GAGs in urine. In a specific embodiment, the GAGs in CSF comprise heparin sulfate in CSF. In another specific embodiment, the GAGs in CSF are heparin sulfate in CSF. In another specific embodiment, the GAGs in plasma comprise heparin sulfate in plasma. In another specific embodiment, the GAGs in plasma are heparin sulfate in plasma. In another specific embodiment, the GAGs in urine comprise heparin sulfate in urine. In another specific embodiment, the GAGs in urine are heparin sulfate in urine. In a specific embodiment, the step of measuring comprises mearing level of heparin sulfate in CSF. In another specific embodiment, the step of measuring comprises measuring level of leukocyte I2S enzyme activity

[0058] In another aspect, provided herein is a method of treating a human subject diagnosed with MPS II, comprising: (a) administering a therapeutically effective amount of an rAAV encoding hIDS to the human subject, wherein the human subject was treated with ERT or is being treated with ERT; and (b) discontinuing ERT treatment in the human subject if the level of at least one biomarker in a biological sample from the human subject is lower than a reference, wherein the biological sample was obtained from the human subject after the administering, and wherein the at least one biomarker comprises D2S6, HS, and / or total GAG (e.g., as measured in urine). In some embodiments, the at least one biomarker is about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% lower than the reference before ERT is discontinued. In some embodiments ERT is recombinant idursulfase. In some embodiments, the subject diagnosed with MPS II has hepatosplenomegaly.

[0059] In another aspect, provided herein is a method of treating a human subject diagnosed with MPS II, comprising: (a) administering a therapeutically effective amount of an rAAV encoding hIDS to the human subject, wherein the human subject was treated with ERT or is being treated with ERT; and (b) discontinuing ERT treatment in the human subject if the level of at least one biomarker in a biological sample from the human subject is higher than a reference, wherein the biological sample was obtained from the human subject before the administering, and wherein the at least one biomarker is an anti-IDS antibody. In some embodiments, the at least one biomarker is about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 150%, 250%, 500%, 750%, or 1000% higher than the reference before ERT is discontinued. In some embodiments ERT is recombinant idursulfase. In some embodiments, the subject diagnosed with MPS II has hepatosplenomegaly.

[0060] In some embodiments, the reference is the level of the at least one biomarker in a biological sample obtained from the human subject prior to administration of the rAAV to the human subject. In some embodiments, the reference is the level of the at least one biomarker in a biological sample obtained from a subject diagnosed with MPS II but not receiving ERT. In some embodiments, the reference is a predetermined value. In some embodiments, ERT treatment is discontinued 52 weeks after the rAAV is administered to the human subject. In some embodiments, the biological sample is CSF, urine, plasma, or serum.

[0061] In another aspect, provided herein is a method of treating a human subject diagnosed with MPS II comprising: (a) discontinuing ERT treatment in the human subject,wherein the human subject was treated with ERT or is being treated with ERT; and (b) administering a therapeutically effective amount of an rAAV encoding hIDS to the human subject, wherein the administering is after ERT treatment is discontinued in the human subject. In some embodiments, the ERT treatment is discontinued about or at least about 1 year, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 3 weeks, 2 weeks, 1 week, 10 days, 5 days, or one day before the rAAV encoding hIDS is administered to the human subject. In some embodiments, the ERT treatment is discontinued if the level of at least one biomarker is not detected in a biological sample from the human subject. In some embodiments, the at least one biomarker is an anti-AAV antibody. In some embodiments, the anti-AAV antibody is an anti-AAV9 antibody. In some embodiments, the biological sample is serum. In some embodiments, the human subject has hepatosplenomegaly. In some embodiments, the ERT is enzyme replacement therapy with recombinant idursulfase.

[0062] In another aspect, provided herein is a method of determining efficacy or monitoring efficacy of MPS II treatment in a human subject, comprising administering a therapeutically effective amount of an rAAV encoding hIDS to the human subject, wherein a decrease in the level of D2S6 in a biological sample from the human subject as compared to a reference is indicative of efficacy of the MPS II treatment in the human subject, wherein the biological sample was obtained from the human subject at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 days, or 1, 2, 3, 4, 5, 6, 7, 8, 10, 16, 20, 24, 30, 35, 40, 45, 48, 50, 52, 56, 104 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years after the administering step. In some embodiments, the human subject was treated with ERT prior to the administration of the rAAV encoding hIDS to the human subject. In some embodiments, the human subject received ERT treatment after the administration of the rAAV encoding hIDS to the human subject. In some embodiments, the ERT is enzyme replacement therapy with recombinant idursulfase. In some embodiments, the biological sample is CSF. In some embodiments, the patient is a pediatric patient.

[0063] In some embodiments, efficacy of the treatment is demonstrated by a decrease in the level of D2S6 of about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% as compared to the reference (eg, the D2S6 level in the same patient prior to the administering step). In some embodiments, the efficacy of MPS II treatment is an improvement in at least one subtest of the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III) as compared to a reference. In some embodiments, the at least one subtest is age equivalence score, cognitive developmental quotient (DQ), expressive language DQ, receptive language DQ, gross motor DQ, and / or fine motor DQ. In some embodiments, the improvement is an improvement in DQ of about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100. In some embodiments, the age equivalence score is increased by about or at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 monts, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, or more than 24 months. In some embodiments, the reference is the score of the at least one subtest of the BSID-III obtained prior to the administering. In some embodiments, the reference is an average score of the at least one subtest of the BSID-III obtained from human subjects with MPS II of the same age as the human subject. In some embodiments, the reference is the level of D2S6 in a biological sample obtained from the human subject prior to the administering.

[0064] In some embodiments, the rAAV is administered intrathecally to the human subject. In some embodiments, the rAAV is administered to the human subject in a solution comprising: (a) sodium chloride at a concentration of about 8.77 g / L, (b) magnesium chloride, at a concentration of about 0.244 g / L, (c) potassium chloride at a concentration of about 0.224 g / L, (d) calcium chloride at a concentration of about 0.206 g / L, (e) dextrose at a concentration of about 0.793 g / L, (f) poloxamer 188 at a concentration of about 0.001% (volume / volume), (g) sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and (h) sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L.

[0001] In one aspect provided herein is a method of identifying or diagnosing a subject as having neuronopathic MPS II, wherein the method comprises: (a) determining the level of one or more heparan sulfate disaccharide(s) in a biological sample from the subject; (b) identifying or diagnosing the subject as having neuronopathic MPS II if the level of the one or more heparan sulfate disaccharide(s) is elevated as compared to a reference level; and (c) administering a therapeutically effective amount of an rAAV encoding hIDS to the subject identified or diagnosed as having neuronopathic MPS II. In some embodiments, the one or more heparan sulfate disaccharide(s) comprises one or more of D0A0, D0S0, D0A6, D2S6, or a combination thereof. In some embodiments, the one or more heparan sulfate disaccharide(s) is D2S6.

[0002] In one aspect provided herein is a method of identifying or diagnosing a subject as having neuronopathic MPS II, wherein the subject is identified or diagnosed as having neuronopathic MPS II if the level of D2S6 in a biological sample from the subject is elevated as compared to a reference level, and wherein a therapeutically effective amount of an rAAV encoding hIDS is administerd to the subject identified or diagnosed as having neuronopathic MPS II.

[0003] In some embodiments, the biological sample is cerebrospinal fluid. In some embodiments, the subject is presymptomatic or has no visible or detectable MPS II symptom. In some embodiments, the subject has MPS II. In some embodiments, the reference level is the level of the at least one or more heparan sulfate disaccharide(s) in a biological sample from one or more healthy subjects and / or from one or more non-neuronopathic subjects. In some embodiments, the reference level is the level of D2S6 in a biological sample from one or more healthy subjects and / or from one or more non-neuronopathic subjects. In some embodiments, the biological sample from one or more healthy subjects and / or from one or more non-neuronopathic subjects is a CSF sample. In some embodiments, the reference level is a pre-determined level. In some embodiments, the level of D2S6 is about or at least about 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, or higher than 40% of the total heparan sulfate disaccharides (HS) in the biological sample from the subject. In some embodiments, the level of D2S6 is about or at least about 20% of the total heparan sulfate disaccharides (HS) in the biological sample from the subject. In some embodiments, the level of one or more heparan sulfate disaccharide(s) or the level of D2S6 in the biological sample from the subject is about or at least about 50 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 270 ng / mL, 280 ng / mL, 290 ng / mL, 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, or more than 400 ng / mL. In some embodiments, the level of one or more heparan sulfate disaccharide(s) or the level of D2S6 in the biological sample from the subject is about or at least about 100 ng / mL, 110 ng / / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, or more than 200 ng / mL. In some embodiments, the level of the one or more heparan sulfate disaccharide(s) or the level of D2S6 in the biological sample from the subject is elevated by about or at least about 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 270 ng / mL, 280 ng / mL, 290 ng / mL, 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, 410 ng / mL, 420 ng / mL, 430 ng / mL, 440 ng / mL, 450 ng / mL, 460 ng / mL, 470 ng / mL, 480 ng / mL, 490 ng / mL, 500 ng / mL, or more than 500 ng / mL as compared to the reference level.

[0004] In one aspect provided herein is a method of determining efficacy or monitoring efficacy of MPS I treatment in a human subject, comprising administering a therapeutically effective amount of an rAAV encoding human IDUA to the human subject, wherein a decrease in the level of I0S6 in a biological sample from the human subject as compared to a reference is indicative of efficacy of the MPS I treatment in the human subject, wherein the biological sample was obtained from the human subject after the administering.

[0005] In some aspects, the biological sample is plasma. In some aspects, the human subject was treated with ERT prior to the administering and / or received ERT treatment after the administering. In some aspects, the ERT is enzyme replacement therapy with recombinant idursulfase. In some aspects, the decrease in the level of I0S6 is a decrease of about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% as compared to the reference. In some aspects, the reference is the level of I0S6 in a biological sample obtained from the human subject prior to the administering. In some aspects, the reference is a predetermined value. In some aspects, the reference is the level of I0S6 in a biological sample obtained from another human subject diagnosed with MPS I or a population of human subjects diagnosed with MPS I. In some aspects, the efficacy of MPS I treatment is an improvement in at least one subtest of the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III) as compared to a reference. In some aspects, the at least one subtest is age equivalence score, cognitive developmental quotient (DQ), expressive language DQ, receptive language DQ, gross motor DQ, and / or fine motor DQ. In some aspects, the reference is the score of the at least one subtest of the BSID-III obtained from the human subject prior to the administering. In some aspects, the reference is an average score of the at least one subtest of the BSID-III obtained from human subjects with MPS I of the same age as the human subject.

[0006] In some aspects, the rAAV is administered to the human subject in a solution comprising: (a) sodium chloride at a concentration of about 8.77 g / L, (b) magnesium chloride, at a concentration of about 0.244 g / L, (c) potassium chloride at a concentration of about 0.224 g / L, (d) calcium chloride at a concentration of about 0.206 g / L, (e) dextrose at a concentration of about 0.793 g / L, (f) poloxamer 188 at a concentration of about 0.001% (volume / volume), (g) sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and (h) sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L. 3.1 ILLUSTRATIVE EMBODIMENTS 3.1.1. Setl 1. A method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising delivering to the cerebrospinal fluid (CSF) of the human subject a therapeutically effective amount of a glycosylated recombinant human iduronate-2-sulfatase (IDS) precursor produced by human neuronal or human glial cells, wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain magnetic resonance imaging (MRI) of the human subject’s brain. 2. The method of paragraph 1, wherein the glycosylated recombinant human IDS precursor is secreted at a detectable level. 3. The method of paragraph 1 or 2, wherein the human neuronal or human glial cells carry at least one mutation in the endogenous gene encoding human IDS precursor. 4. The method of any one of paragraphs 1-3, wherein the human neuronal or human glial cells are transduced with a recombinant adeno-associated virus vector (rAAV). 5. The method of any one of paragraphs 1-4, wherein the glycosylated recombinant human IDS precursor is expressed under the control of a CB7 promoter. 6. The method of any one of paragraphs 1-5, wherein the glycosylated recombinant human IDS precursor is expressed from a cDNA encoding human IDS precursor. 7. The method of any one of paragraphs 1-6, wherein the glycosylated recombinant human IDS precursor is about 90 kDa as measured by polyacrylamide gel electrophoresis. 8. The method of any one of paragraphs 1-7, wherein the glycosylated recombinant human IDS precursor contains a formylglycine. 9. The method of any one of paragraphs 1-8, wherein the glycosylated recombinant human IDS precursor (a) is a2,6-sialylated; (b) does not contain detectable NeuGc; (c) does not contain detectable a-Gal antigen; (d) contains tyrosine-sulfation; and / or (e) is mannose-6-phosphorylated. 10. The method of any one of paragraphs 1-9, in which the glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO. 1. 11. The method of any one of paragraphs 1-10, wherein the recombinant nucleotide expression vector is an AAV9 or AAVrhlO vector. 12. The method of any one of paragraphs 1-11, wherein the human subject’s brain mass is converted from the human subject’s brain volume by multiplying the human subject’s brain volume in cm3 by a factor of 1.046 g / cm3, wherein the human subject’s brain volume is obtained from the human subject’s brain MRI. 13. The method of any one of paragraphs 1-12, wherein the recombinant nucleotide expression vector is administered at a dose of about 1.3 x 1010 GC / g brain mass as determined by MRI, or about 6.5 * 1010 GC / g brain mass as determined by MRI. 14. The method of any one of paragraphs 1-13, wherein the recombinant nucleotide expression vector is administered via intraci sternal (IC) administration. 15. The method of any one of paragraphs 1-13, wherein the recombinant nucleotide expression vector is administered via intracerebroventricular (ICV) administration. 16. The method of any one of paragraphs 1-15, wherein the recombinant nucleotide expression vector is administered at a volume that does not exceed 10% of the total cerebrospinal fluid volume of the human subject. 17. The method of any one of paragraphs 1-16, wherein the glycosylated recombinant human IDS precursor is delivered to lysosomes of cells in the CNS of the human subject. 18. The method of any one of paragraphs 1-17, further comprising administering an immune suppression therapy to the human subject before or concurrently with the human IDS precursor treatment and optionally continuing immune suppression therapy thereafter. 19. The method of paragraph 18, wherein the immune suppression therapy comprises administering one or more corticosteroids, sirolimus, and / or tacrolimus. 20. The method of paragraph 19, wherein the one or more corticosteroids are methylprednisolone and / or prednisone. 21. The method of any one of paragraphs 18-20, further comprising administering one or more antibiotics to the human subject before or concurrently with the immune suppression therapy. 22. The method of paragraph 21, wherein the one or more antibiotics are trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atovaquone. 23. The method of any one of paragraphs 18-22, further comprising administering one or more antifungal therapies to the human subject before or concurrently with the immune suppression therapy. 24. The method of any one of paragraphs 1-23, further comprising a step of measuring one or more of the following biomarkers after administration of the recombinant nucleotide expression vector: (a) level of glycosaminoglycans (GAGs) in CSF; (b) level of iduronate-2-sulfatase (I2S) in CSF; (c) level of GAGs in plasma; (d) level of I2S in plasma; (e) level of leukocyte I2S enzyme activity; and (f) level of GAGs in urine. 25. The method of paragraph 24, wherein the GAGs in CSF comprise heparin sulfate in CSF. 26. The method of paragraph 24, wherein the GAGs in CSF are heparin sulfate in CSF. 27. The method of any one of paragraphs 24-26, wherein the GAGs in plasma comprise heparin sulfate in plasma. 28. The method of any one of paragraphs 24-26, wherein the GAGs in plasma are heparin sulfate in plasma. 29. The method of any one of paragraphs 24-28, wherein the GAGs in urine comprise heparin sulfate in urine. 30. The method of any one of paragraphs 24-28, wherein the GAGs in urine are heparin sulfate in urine. 31. The method of any one of paragraphs 24-30, wherein the step of measuring comprises mearing level of heparin sulfate in CSF. 32. The method of any one of paragraphs 24-31, wherein the step of measuring comprises measuring level of leukocyte I2S enzyme activity. 3.1.2. Set 2 1. A method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising delivering to the cerebrospinal fluid (CSF) of the human subject a therapeutically effective amount of a glycosylated recombinant human iduronate-2-sulfatase (IDS) precursor produced by human neuronal or human glial cells, wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain magnetic resonance imaging (MRI) of the human subject’s brain. 2. The method of paragraph 1, wherein the glycosylated recombinant human IDS precursor is secreted at a detectable level. 3. The method of paragraph 1 or 2, wherein the human neuronal or human glial cells carry at least one mutation in the endogenous gene encoding human IDS precursor. 4. The method of any one of paragraphs 1-3, wherein the human neuronal or human glial cells are transduced with a recombinant adeno-associated virus vector (rAAV). 5. The method of any one of paragraphs 1-4, wherein the glycosylated recombinant human IDS precursor is expressed under the control of a CB7 promoter. 6. The method of any one of paragraphs 1-5, wherein the glycosylated recombinant human IDS precursor is expressed from a cDNA encoding human IDS precursor. 7.   The method of any one of paragraphs 1-6, wherein the glycosylated recombinant human IDS precursor is about 90 kDa as measured by polyacrylamide gel electrophoresis. 8. The method of any one of paragraphs 1-7, wherein the glycosylated recombinant human IDS precursor contains a formylglycine. 9. The method of any one of paragraphs 1-8, wherein the glycosylated recombinant human IDS precursor (a) is a2,6-sialylated; (b) does not contain detectable NeuGc; (c) does not contain detectable a-Gal antigen; (d) contains tyrosine-sulfation; and / or (e) is mannose-6-phosphorylated. 10. The method of any one of paragraphs 1-9, in which the glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO. 1. 11. The method of any one of paragraphs 1-10, wherein the recombinant nucleotide expression vector is an AAV9 or AAVrhlO vector. 12. The method of any one of paragraphs 1-11, wherein the human subject’s brain mass is converted from the human subject’s brain volume by multiplying the human subject’s brain volume in cm3 by a factor of 1.046 g / cm3, wherein the human subject’s brain volume is obtained from the human subject’s brain MRI. 13. The method of any one of paragraphs 1-12, wherein the recombinant nucleotide expression vector is administered at a dose of about 1.3 x 1010 GC / g brain mass as determined by MRI, or about 6.5 * 1010 GC / g brain mass as determined by MRI. 14. The method of any one of paragraphs 1-12, wherein the recombinant nucleotide expression vector is administered at a dose of about 2.0 x io11 GC / g brain mass or about 2.9 x 1011 GC / g brain mass as determined by MRI. 15. The method of any one of paragraphs 1-12, wherein the human subject is 5 years old or older and less than 18 years old. 16. The method of paragraph 15, wherein the recombinant nucleotide expression vector is administered at a dose of about 6.5 x io10 GC / g brain mass as determined by MRI. 17. The method of paragraph 15, wherein the recombinant nucleotide expression vector is administered at a dose according to the table below: Brain Mass (in g) Target Brain Mass (in g) Dose: Total GC by Poly-A-specific PCR assay (6.5 x 1010 GC / g brain mass) Min Max 801 900 850 5.5 x 1013 901 1050 975 6.3 x 1013 1051 1200 1125 7.3 x 1013 1201 - 1300 8.5 x 1013 18. The method of any one of paragraphs 1-12, wherein the human subject is 4 months old or older and less than 5 years old. 19. The method of paragraph 18, wherein the recombinant nucleotide expression vector is administered at a dose chosen from Dose 1 or Dose 2 according to the table below: Brain Mass (in g) Target Dose Levels Min Max Dose 1 Total GC by Poly-A specific PCR assay (1.3 x io10 GC / g brain mass) Dose 2 Total GC by Poly-A specific PCR assay (6.5 x 1010 GC / g brain mass) - 700 650 8.5 x 1012 4.2 x 1013 701 800 750 9.8 x 1012 4.9 x 1013 801 900 850 1.1 x 1013 5.5 x 1013 901 1050 975 1.3 x 1013 6.3 x 1013 1051 1200 1125 1.5x 1013 7.3 x 1013 1201 - 1300 1.7 x 1013 8.5 x 1013 20. The method of paragraph 18, wherein the recombinant nucleotide expression vector is administered at a dose according to the tables below: Brain Mass (in g) Target Dose 3 Total GC by Poly-A-specific PCR assay (2.0 x 1011 GC / g brain mass) Min Max - 474 450 9.0 x 1013 475 524 500 1.0 x 1014 525 574 550 1.1 x 1014 575 624 600 1.2 x 1014 625 674 650 1.3 x 1014 675 724 700 1.4 x 1014 725 774 750 1.5 x 1014 775 824 800 1.6 x 1014 825 874 850 1.7 x 1014 875 924 900 1.8 x 1014 925 974 950 1.9 x 1014 975 1024 1000 2.0 x 1014 1025 1074 1050 2.1 x 1014 1075 1124 1100 2.2 x 1014 1125 1174 1150 2.3 x 1014 1175 1224 1200 2.4 x 1014 1225 1274 1250 2.5 x 1014 1275 >1300 1300 2.6 x 1014 Total Dose Administered by Brain Mass Dose 3 EC Brain Mass (in g) Target Dose 3 EC Total GC by Transgene PCR assay (2.9 x 1011 GC / g brain mass) Min Max - 474 450 1.3 x 1014 475 524 500 1.5 x 1014 525 574 550 1.6 x 1014 575 624 600 1.8 x 1014 625 674 650 1.9 x 1014 675 724 700 2.1 x 1014 725 774 750 2.2 x 1014 775 824 800 2.4 x 1014 825 874 850 2.4 x 1014 875 924 900 2.6 x 1014 925 974 950 2.8 x 1014 975 1024 1000 2.9 x 1014 1025 1074 1050 3.1 x 1014 1075 1124 1100 3.2 x 1014 1125 1174 1150 3.4 x 1014 1175 1224 1200 3.5 x 1014 1225 1274 1250 3.7 x 1014 1275 >1300 1300 3.8 x 1014 21. The method of any one of paragraphs 1-20, wherein the recombinant nucleotide expression vector is administered via intraci sternal (IC) administration. 22. The method of any one of paragraphs 1-20, wherein the recombinant nucleotide expression vector is administered via intracerebroventricular (ICV) administration. 23. The method of any one of paragraphs 1-22, wherein the recombinant nucleotide expression vector is administered at a volume that does not exceed 10% of the total cerebrospinal fluid volume of the human subject. 24. The method of any one of paragraphs 1-23, wherein the glycosylated recombinant human IDS precursor is delivered to lysosomes of cells in the CNS of the human subject. 25. The method of any one of paragraphs 1-24, further comprising administering an immune suppression therapy to the human subject before or concurrently with the human IDS precursor treatment and optionally continuing immune suppression therapy thereafter. 26. The method of paragraph 25, wherein the immune suppression therapy comprises administering one or more corticosteroids, sirolimus, and / or tacrolimus. 27. The method of paragraph 26, wherein the one or more corticosteroids are methylprednisolone and / or prednisone. 28. The method of any one of paragraphs 25-27, further comprising administering one or more antibiotics to the human subject before or concurrently with the immune suppression therapy. 29. The method of paragraph 28, wherein the one or more antibiotics are trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atovaquone. 30. The method of any one of paragraphs 25-29, further comprising administering one or more antifungal therapies to the human subject before or concurrently with the immune suppression therapy. 31. The method of any one of paragraphs 1-30, further comprising a step of measuring one or more of the following biomarkers after administration of the recombinant nucleotide expression vector: (a) level of glycosaminoglycans (GAGs) in CSF; (b) level of iduronate-2-sulfatase (I2S) in CSF; (c) level of GAGs in plasma; (d) level of I2S in plasma; (e) level of leukocyte I2S enzyme activity; and (f) level of GAGs in urine. 32. The method of paragraph 31, wherein the GAGs in CSF comprise heparin sulfate in CSF. 33. The method of paragraph 31, wherein the GAGs in CSF are heparin sulfate in CSF. 34. The method of any one of paragraphs 31-33, wherein the GAGs in plasma comprise heparin sulfate in plasma. 35. The method of any one of paragraphs 31-33, wherein the GAGs in plasma are heparin sulfate in plasma. 36. The method of any one of paragraphs 31-35, wherein the GAGs in urine comprise heparin sulfate in urine. 37. The method of any one of paragraphs 31-35, wherein the GAGs in urine are heparin sulfate in urine. 38. The method of any one of paragraphs 31-37, wherein the step of measuring comprises mearing level of heparin sulfate in CSF. 39. The method of any one of paragraphs 31-38, wherein the step of measuring comprises measuring level of leukocyte I2S enzyme activity. 3.1.3. Set 3 1. A method for treating hepatosplenomegaly in a human subject diagnosed with mucopolysaccharidosis type II (MPS II), the method comprising administering to the cerebrospinal fluid (CSF) of the human subject in need of treatment a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS). 2. A method for treating hepatosplenomegaly and central nervous system (CNS) symptoms in a human subject diagnosed with MPS II, the method comprising administering to the CSF of the human subject in need of treatment a single dose of an rAAV encoding hIDS, wherein no additional therapy for MPS II is administered outside the CNS. 3. The method of paragraph 2, wherein the CNS symptom is cognitive ability as measured by the Bayley Scale of Infant and Toddler Development, 3rd Edition (BSID-III). 4. The method of paragraph 2, wherein the CNS symptom is language as measured by the language domain of the BSID III. 5. The method of paragraph 2, wherein the CNS symptom is motor function as determined by the motor domain of the BSID-III. 6. A method of delivering to the liver and / or spleen of a human subject diagnosed with MPS II an rAAV encoding hIDS, the method comprising administering a single dose of the rAAV to the CSF of the human subject. 7.   The method of any one of paragraphs 1-6, wherein the methods results in the size of the liver decreasing by about 10%, about 20%, or about 30% compared to the size of the liver prior to administration of the rAAV. 8. The method of any one of paragraphs 1-7, wherein the method results in the size of the spleen decreasing by about 10%, about 20%, or about 30% compared to the size of the spleen prior to administration of the rAAV. 9. A method for treating a human subject diagnosed with MPS II, the method comprising (i) administering to the CSF of the human subject a therapeutically effective amount of an rAAV encoding hIDS and (ii) measuring the levels of heparin sulfate (HS) D2S6 in the CSF of the subject. 10. The method of paragraph 9, wherein the reduction in HS D2S6 correlates with improved neurocognitive parameters in the human subject. 11. The method of paragraph 9 or 10, further comprising a step of determining whether an additional treatment is required. 12. The method of paragraph 11, further comprising a step of administering an additional treatment. 13. The method of paragraph 12, wherein the additional treatment is a second administration of the same rAAV as in step (i). 14. The method of paragraph 12, wherein the additional treatment is a second administration of the same rAAV as in step (i) at a higher dose. 15. The method of paragraph 12, wherein the additional treatment is enzyme replacement therapy with recombinant idursulfase. 16. The method of anyone of paragraphs 1-15, wherein the method results in a decrease in the levels of HS D2S6 in the CSF of the subject of about 10%, about 20%, about 30, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than 95% compared to the levels of HS D2S6 in the CSF of the subject prior to administration of the rAAV. 17. The method of paragraph 16, wherein the levels of HS D2S6 are determined about 2 months, about 4 months, about 6 months, about 8 months, about 10 months, about 12 months, about 18 months, about 24 months, about 3 years, about 4 years, or about 5 years after administration of the rAAV. 18. The method of paragraph 16 or 17, wherein the decrease in levels of HS D2S6 is sustained for at least 6 months, at least 9 months, at last 12 months, at least 15 months, at least 18 months, at least 21 months, or at least 24 months after administration of the rAAV. 19. The method of any one of paragraphs 1-18, wherein the rAAV is administered at a dose of about 1.3 * io10 GC / g brain mass, about 6.5 x 1010 GC / g brain mass, or about 2 x 1011 GC / g brain mass, as determined by MRI. 20. The method of any one of paragraphs 1-19, wherein the human subject is 5 years old or older and less than 18 years old. 21. The method of any one of paragraphs 1-19, wherein the human subject is 4 months old or older and less than 5 years old. 22. The method of any one of paragraphs 1-21, wherein the human subject is receiving enzyme replacement therapy (ERT) at the time of the administration of the rAAV. 23. The method of paragraph 22, wherein the human subject is not responsive to ERT. 24. The method of paragraph 22 or 23, wherein the ERT is recombinant idursulfase. 25. The method of any one of paragraph 22-24, wherein the ERT is discontinued about 6 months, about 90 months, about 12 months, about 15 months, about 18 months, about 21 months, or about 24 months after the administration of the rAAV. 26. The method of any one of paragraphs 1-25, wherein the rAAV is administered via intraci sternal (IC) administration. 27. The method of any one of paragraphs 1-25, wherein the rAAV is administered via intracerebroventricular (ICV) administration. 28. The method of any one of paragraphs 1-27, wherein the rAAV is administered at a volume that does not exceed 10% of the total cerebrospinal fluid volume of the human subject. 29. The method of any one of paragraphs 1-28, wherein the rAAV is a recombinant adeno-associated virus serotype 9 30. The method of paragraph 29, wherein the rAAV contains a human IDS expression cassette, wherein expression is driven by a hybrid of the cytomegalovirus (CMV) enhancer and the chicken beta actin promoter (CB7), 31. The method of paragraph 30, wherein the human IDS expression cassette comprises (i) an IDS transgene flanked by inverted terminal repeats (ITRs), (ii) the chicken beta actin intron, and (iii) a rabbit beta-globin polyadenylation (polyA) signal. 32. The method of paragraph 31, wherein the ITRs are AAV2 ITRs. 33. The method of any one of paragraphs 30-32, wherein the human IDS expression cassette comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO:45. 34. The method of any one of paragraphs 1-33, further comprising administering an immune suppression therapy to the human subject before or concurrently with the rAAV and optionally continuing immune suppression therapy thereafter. 35. The method of paragraph 34, wherein the immune suppression therapy comprises administering one or more corticosteroids, sirolimus, and / or tacrolimus. 36. The method of paragraph 35, wherein the one or more corticosteroids are methylprednisolone and / or prednisone. 37. The method of any one of paragraphs 1-36, further comprising administering one or more antibiotics to the human subject before or concurrently with the rAAV. 38. The method of paragraph 37, wherein the one or more antibiotics are trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atovaquone. 39. The method of any one of paragraphs 1-38, further comprising administering one or more antifungal therapies to the human subject before or concurrently with rAAV 40. A method of treating a human subject diagnosed with MPS II, comprising: (a) administering a therapeutically effective amount of an rAAV encoding hIDS to the human subject, wherein the human subject was treated with ERT or is being treated with ERT; and (b) discontinuing ERT treatment in the human subject if the level of at least one biomarker in a biological sample from the human subject is lower than a reference, wherein the biological sample was obtained from the human subject after the administering, and wherein the at least one biomarker comprises D2S6, HS, and / or total GAG. 41. A method of treating a human subject diagnosed with MPS II, comprising: (a) administering a therapeutically effective amount of an rAAV encoding hIDS to the human subject, wherein the human subject was treated with ERT or is being treated with ERT; and (b) discontinuing ERT treatment in the human subject if the level of at least one biomarker in a biological sample from the human subject is higher than a reference, wherein the biological sample was obtained from the human subject before the administering, and wherein the at least one biomarker is an anti-IDS antibody. 42. The method of paragraph 40, wherein the at least one biomarker is D2S6. 43. The method of any one of paragraphs 40 or 42, wherein the at least one biomarker is about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% lower than the reference before ERT is discontinued. 44. The method of paragraph 41, wherein the at least one biomarker is about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% higher than the reference before ERT is discontinued. 45. The method of any one of paragraphs 40-44, wherein the reference is a predetermined value. 46. The method of any one of paragraphs 40-44, wherein the reference is the level of the at least one biomarker in a biological sample obtained from the human subject prior to administration of the rAAV to the human subject. 47. The method of any one of paragraphs 40-44, wherein the reference is the level of the at least one biomarker in a biological sample obtained from a subject diagnosed with MPS II but not receiving ERT. 48. The method of any one of paragraphs 40-47, wherein the ERT treatment is discontinued on the same day that the rAAV is administered to the human subject. 49. The method of any one of paragraphs 40 or 42-47, wherein the ERT treatment is discontinued 52 weeks after the rAAV is administered to the human subject. 50. The method of any one of paragraphs 40-49, wherein the biological sample is CSF, urine, plasma, or serum. 51. A method of treating a human subject diagnosed with MPS II comprising: (a) discontinuing ERT treatment in the human subject, wherein the human subject was treated with ERT or is being treated with ERT; and (b) administering a therapeutically effective amount of an rAAV encoding hIDS to the human subject, wherein the administering is after ERT treatment is discontinued in the human subject. 52. The method of paragraph 51, wherein the ERT treatment is discontinued about or at least about 1 year, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 3 weeks, 2 weeks, 1 week, 10 days, 5 days, or one day before the administering. 53. The method of any one of paragraphs 51-52, wherein the level of at least one biomarker is not detected in a biological sample from the human subject prior to the discontinuing. 54. The method of paragraph 53, wherein the at least one biomarker is an anti-AAV antibody. 55. The method of paragraph 54, wherein the anti-AAV antibody is an anti-AAV9 antibody. 56. The method of any one of paragraphs 51-55, wherein the biological sample is serum. 57. A method of determining efficacy or monitoring efficacy of MPS II treatment in a human subject, comprising administering a therapeutically effective amount of an rAAV encoding hIDS to the human subject, wherein a decrease in the level of D2S6 in a biological sample from the human subject as compared to a reference is indicative of efficacy of the MPS II treatment in the human subject, wherein the biological sample was obtained from the human subject after the administering. 58. The method of any one of paragraphs 40-57, wherein the human subject has hepatosplenomegaly. 59. The method of paragraph 58, wherein the human subject was treated with ERT prior to the administering and / or received ERT treatment after the administering. 60. The method of any one of paragraphs 40-59, wherein the ERT is enzyme replacement therapy with recombinant idursulfase. 61. The method of any one of paragraphs 57-60, wherein the biological sample is CSF. 62. The method of any one of paragraphs 57-61, wherein the decrease in the level of D2S6 is a decrease of about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% as compared to the reference. 63. The method of any one of paragraphs 57-62, wherein the efficacy of MPS II treatment is an improvement in at least one subtest of the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III) as compared to a reference. 64. The method of paragraph 63, wherein the at least one subtest is age equivalence score, cognitive developmental quotient (DQ), expressive language DQ, receptive language DQ, gross motor DQ, and / or fine motor DQ. 65. The method of paragraph 63 or 64, wherein the improvement is an improvement in DQ of about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100. 66. The method of paragraph 64, wherein the age equivalence score is increased by about or at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 monts, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, or more than 24 months. 67. The method of any one of paragraphs 63-66, wherein the reference is the score of the at least one subtest of the BSID-III obtained from the human subject prior to the administering. 68. The method of any one of paragraphs 63-66, wherein the reference is an average score of the at least one subtest of the BSID-III obtained from human subjects with MPS II of the same age as the human subject. 69. The method of any one of paragraphs 57-68, wherein the reference is the level of D2S6 in a biological sample obtained from the human subject prior to the administering. 70. The method of any one of paragraphs 57-69, wherein the biological sample was obtained from the human subject at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 days, or 1, 2, 3, 4, 5, 6, 7, 8, 10, 16, 20, 24, 30, 35, 40, 45, 48, 50, 52, 56, 104 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years after the administering. 71. The method of any one of paragraphs 51-70, wherein the human subject is a pediatric subject. 72. The method of any one of paragraphs 57-71, wherein the efficacy of MPS II treatment is demonstrated by a decrease in the level of D2S6 of about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% as compared to the level of D2S6 in the human subject prior to the administering. 73. The method of any one of paragraphs 1-72, wherein the rAAV is administered intrathecally to the human subject. 74. The method of any one of paragraphs 1-73, wherein the rAAV is administered to the human subject in a solution comprising: (a) sodium chloride at a concentration of about 8.77 g / L, (b) magnesium chloride, at a concentration of about 0.244 g / L, (c) potassium chloride at a concentration of about 0.224 g / L, (d) calcium chloride at a concentration of about 0.206 g / L, (e) dextrose at a concentration of about 0.793 g / L, (f) poloxamer 188 at a concentration of about 0.001% (volume / volume), (g) sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and (h) sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L. 75. A method of identifying or diagnosing a subject as having neuronopathic MPS II, wherein the method comprises: (a) determining the level of one or more heparan sulfate disaccharide(s) in a biological sample from the subject; (b) identifying or diagnosing the subject as having neuronopathic MPS II if the level of the one or more heparan sulfate disaccharide(s) is elevated as compared to a reference level; and (c) administering a therapeutically effective amount of an rAAV encoding hIDS to the subject identified or diagnosed as having neuronopathic MPS II. 76. The method of paragraph 75, wherein the one or more heparan sulfate disaccharide(s) comprises one or more of D0A0, D0S0, D0A6, D2S6, or a combination thereof. 77. The method of paragraph 76, wherein the one or more heparan sulfate disaccharide(s) is D2S6. 78. A method of identifying or diagnosing a subject as having neuronopathic MPS II, wherein the subject is identified or diagnosed as having neuronopathic MPS II if the level of D2S6 in a biological sample from the subject is elevated as compared to a reference level, and wherein a therapeutically effective amount of an rAAV encoding hIDS is administerd to the subject identified or diagnosed as having neuronopathic MPS II. 79. The method of any one of paragraphs 75-78, wherein the biological sample is cerebrospinal fluid. 80. The method of any one of paragraphs 75-79, wherein the subject is presymptomatic or has no visible or detectable MPS II symptom. 81. The method of any one of paragraphs 75-80, wherein the subject has MPS II. 82. The method of any one of paragraphs 75-77 and 79-81, wherein the reference level is the level of the at least one or more heparan sulfate disaccharide(s) in a biological sample from one or more healthy subjects and / or from one or more non-neuronopathic subjects. 83. The method of any one of paragraphs 77-81, wherein the reference level is the level of D2S6 in a biological sample from one or more healthy subjects, and / or from one or more non-neuronopathic subjects. 84. The method of paragraph 82 or 83, wherein the biological sample from one or more healthy subjects and / or from one or more non-neuronopathic subjects is a CSF sample. 85. The method of any one of paragraphs 75-84, wherein the reference level is a predetermined level. 86. The method of any one of paragraphs 77-85, wherein the level of D2S6 is about or at least about 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, or higher than 40% of the total heparan sulfate disaccharides (HS) in the biological sample from the subject. 87. The method of paragraph 86, wherein the level of D2S6 is about or at least about 20% of the total heparan sulfate disaccharides (HS) in the biological sample from the subject. 88. The method of any one of paragraphs 75-87, wherein the level of the one or more heparan sulfate disaccharide(s) or the level of D2S6 in the biological sample from the subject is about or at least about 50 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 270 ng / mL, 280 ng / mL, 290 ng / mL, 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, or more than 400 ng / mL. 89. The method of any one of paragraphs 75-88, wherein the level of the one or more heparan sulfate disaccharide(s) or the level of D2S6 in the biological sample from the subject is about or at least about 100 ng / mL, 110 ng / / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, or more than 200 ng / mL. 90. The method of any one of paragraphs 75-89, wherein the level of the one or more heparan sulfate disaccharide(s) or the level of D2S6 in the biological sample from the subject is elevated by about or at least about 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 270 ng / mL, 280 ng / mL, 290 ng / mL, 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, 410 ng / mL, 420 ng / mL, 430 ng / mL, 440 ng / mL, 450 ng / mL, 460 ng / mL, 470 ng / mL, 480 ng / mL, 490 ng / mL, 500 ng / mL, or more than 500 ng / mL as compared to the reference level. 91. A method of determining efficacy or monitoring efficacy of MPS I treatment in a human subject, comprising administering a therapeutically effective amount of an rAAV encoding human IDUA to the human subject, wherein a decrease in the level of I0S6 in a biological sample from the human subject as compared to a reference is indicative of efficacy of the MPS I treatment in the human subject, wherein the biological sample was obtained from the human subject after the administering. 92. The method of paragraph 91, wherein the biological sample is plasma. 93. The method of paragraph 90 or 91, wherein the human subject was treated with ERT prior to the administering and / or received ERT treatment after the administering. 94. The method of any one of paragraphs 91-93, wherein the ERT is enzyme replacement therapy with recombinant idursulfase. 95. The method of any one of paragraphs 91-94, wherein the decrease in the level of I0S6 is a decrease of about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% as compared to the reference. 96. The method of any one of paragraphs 91-95, wherein the reference is the level of I0S6 in a biological sample obtained from the human subject prior to the administering. 97. The method of any one of paragraphs 91-95, wherein the reference is a predetermined value. 98. The method of any one of paragraphs 91-95, wherein the reference is the level of I0S6 in a biological sample obtained from another human subject diagnosed with MPS I or a population of human subjects diagnosed with MPS I. 99. The method of any one of paragraphs 91-98, wherein the efficacy of MPS I treatment is an improvement in at least one subtest of the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III) as compared to a reference. 100. The method of paragraph 99, wherein the at least one subtest is age equivalence score, cognitive developmental quotient (DQ), expressive language DQ, receptive language DQ, gross motor DQ, and / or fine motor DQ. 101. The method of paragraph 99 or 100, wherein the reference is the score of the at least one subtest of the BSID-III obtained from the human subject prior to the administering. 102. The method of any one of paragraphs 99-101, wherein the reference is an average score of the at least one subtest of the BSID-III obtained from human subjects with MPS I of the same age as the human subject. 103. The method of any one of paragraphs 91-102, wherein the rAAV is administered to the human subject in a solution comprising: (a) sodium chloride at a concentration of about 8.77 g / L, (b) magnesium chloride, at a concentration of about 0.244 g / L, (c) potassium chloride at a concentration of about 0.224 g / L, (d) calcium chloride at a concentration of about 0.206 g / L, (e) dextrose at a concentration of about 0.793 g / L, (f) poloxamer 188 at a concentration of about 0.001% (volume / volume), (g) sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and (h) sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L. 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1. The amino acid sequence of human IDS. A post-translational formylglycine modification of C84 (shown in bold in FIG. 1) is required for enzyme activity. Eight N linked glycosylation sites (N31, N115, N144, N246, N280, N325, N513 and N537) are bold and boxed. One tyrosine-O-sulfation site (Y) is bold and the full sulfation site sequence (PSSEKY165ENTKTCRGPD) is boxed. The N-terminus of the mature 42 kDa and mature 14 kDa polypeptides are indicated by horizontal arrows. In the brain, the N-terminus of the mature 42 kDa form starts at positions 34 or 36 as follows: T34DALNVLLI; and A36LNVLLIIV as indicated in FIG. 1. (See, Sleat, 2005, Proteomics 5: 1520-1532, Table S2). Two of the eight N-linked glycosylation sites, namely N280 and N116, are mannose-6-phophorylated in IDS obtained from human brain. (Sleat et al., 2006, Mol & Cell Proeomics 5.4: 686-701, reported at Table V).

[0008] FIG. 2. Multiple sequence alignment of hIDS with known orthologs. The names of the species and protein IDs are as follows: SP|P22304|IDS_HUMAN [Homo sapiens]; TR|K6ZGI9_PANTR [Pan troglodytes (Chimpanzee)]; TR|K7BKV4_PANTR [Pan troglodytes (Chimpanzee)]; TR|H9FTX2_MACMU [Macaca mulatta (Rhesus macaque)]; TRF7EJG2 CALJA [Callithrix jacchus (White-tufted-ear marmoset)]; TR|U3DTL8_CALJA [Callithrix jacchus (White-tufted-ear marmoset)]; TR|G7NRX7_MACMU [Macaca mulatta (Rhesus macaque)]; TR|G7Q1 V9 MACFA [Macaca fascicularis (Crab-eating macaque; Cynomologous monkey)]; TR|H2PX10_PONAB [Pongo abelii (Sumatran orangutan)]; TR|A0A0D9R4Dl_CHLSB [Chlorocebus sabaeus (Green monkey)]; TR|G1RST8|G1RST8_NOMLE [Nomascus leucogenys (Northern white-cheeked gibbon)]; UPI0000D9F625 [Macaca mulatta (Rhesus macaque)]; UPI000274358B [Pan paniscus (Pygmy chimpanzee; Bonobo)]; UPI00027F6FC5 [Papio Anubis (Olive baboon)]; UPI00027FAE03 [Saimiri boliviensis (Bolivian squirrel monkey)]; UPI0003ABBF28 [Macaca fascicularis (Crabeating macaque; Cynomologous monkey)]; UPI000533297F [Rhinopithecus roxellana (Golden snub-nosed monkey; Pygathrix roxellana)]; UPI0005F40BD2 [Colobus angolensis palliates (Peters’ Angolan colobus)] (SEQ ID NOs: 27-44).

[0009] FIG. 3. MPS II mutations in hIDS and corresponding disease phenotypes, mild, intermediate or severe, (from Uniprot).

[0010] FIG. 4. Human IDS processing as reported in Millat et al., 1997, Exp. Cell. Res. 230: 362-367, atFig.7.

[0011] FIG. 5 Schematic Representation of Construct 1.

[0012] FIG. 6. Clustal Multiple Sequence Alignment of AAV capsids 1-9 (SEQ ID NOs: 16-26). Amino acid substitutions (shown in bold in the bottom rows) can be made to AAV9 and AAV8 capsids by “recruiting” amino acid residues from the corresponding position of other aligned AAV capsids. Sequence regions designated by “HVR” = hypervariable regions.

[0013] FIG. 7. Heparan Sulfate (HS) digestion with Heparinase.

[0014] FIG. 8. Graph showing cerebral spinal fluid (CSF) heparan sulfate biomarker (ng / mL). Graph showed consistent HS decrease in the CSF after Construct 1 dosing. The median change from baseline at week 8 (N=6) was -30.3% (p-value = 0.03 as measured by Wilcoxon signed rank test). The median change from baseline at the last available timepoint (N=6) was -35.8% (p-value = 0.03 as measured by Wilcoxon signed rank test). The graph showed measurable CSF I2S enzyme concentration in cohort 2 after Construct 1 administration with a range of 1170-1940 pg / mL.

[0015] FIG. 9. Graph showing cerebral spinal fluid (CSF) D2S6 biomarker (ng / mL). The median change from baseline at week 8 (N=6) was -44.2% (p-value = 0.03 as measured by Wilcoxon signed rank test). The median change from baseline at the last available timepoint (N=6) was -39.2% (p-value = 0.03 as measured by Wilcoxon signed rank test).

[0016] FIGs. 10A-10C. Graphs showing neurodevelopment function comparing age equivalence (months) and age (months). The Graph in FIG. 10A showed continued cognitive development in 4 of 5 patients with > 6 months of follow-up. Patients 1, 3, and 5 demonstrated continued cognitive development within a normal range. Patients 2 and 4 presented with significant cognitive delay at baseline. Patient 2 has continued cognitive development. Patient 4 acquired expressive and receptive language skills. FIGs. 10B and 10C show cognitive developmental quotient and age equivalence quotient, respectively, of patients in Construct 1 gene therapy clinical trial. The graphs in FIGs. 10B and 10C show that 3 out of 4 patients who entered the study with cognitive skill above the -2SD of the normal range continued to stay above -2SD as of > 6-month follow-up. One patient entered the study with significant delay in neurocognitive development at baseline but demonstrated relative stabilization following Construct 1 administration at an older age (59 months) and continued to acquire expressive and receptive language skills.

[0017] FIGs. 11A-11D. Graphs showing neurodevelopment function in terms of language and motor skills. FIG. 11A displays expressive communication; FIG. 11B displays receptive communication; FIG. 11C displays gross motor skills; and FIG. HD displays fine motor skills. Graphs showed continued language and / or motor skills acquisition in patients with > 6 months of follow-up.

[0018] FIG. 12. Graph showing systemic efficacy by measuring plasma I2S protein concentration (pg / mL). The graph shows a general increase in plasma I2S enzyme levels in 5 out of 6 patients after Construct 1 administration (normal range (14,706 pg / mL ~ < 100,000 pg / mL)).

[0019] FIG. 13. Graph showing systemic efficacy by measuring urine total GAGs level (g / mol CK) in ERT-treated patients. Graph showed sustained decrease in urine GAG levels across all patients receiving ERT.

[0020] FIG. 14. Graph showing systemic efficacy by measuring urine total GAGs level (g / mol CK) in ERT naive and ERT discontinued patients. Graphs showed a rapid decrease in urine GAGs in ERT-naive patients after Construct 1 administration (absence of urine GAG rebound post ERT withdrawal).

[0021] FIG. 15. Ultrasound of liver or spleen in ERT naive patient. Liver and spleen dimensions in ERT-naive patient decreased 24 weeks after Construct 1 administration.

[0022] FIG. 16. Proof-of-concept Study Activities.

[0023] FIG. 17. Dose Rationale for the First-in-Human Clinical Trial.

[0024] FIG. 18. Total heparan sulfate in CSF samples from healthy subjects, MPS I (neuronopathic and non-neuronopathic) subjects, and MPS II (neuronopathic and non-neuronopathic) subjects.

[0025] FIGs. 19A-19C. Graphs showing the level of D0S0 disaccharide (FIG. 19A), D0A6 disaccharide (FIG. 19B), and D0A0 (FIG. 19C) in healthy subjects, MPS I (neuronopathic and non-neuronopathic) subjects, and MPS II (neuronopathic and non-neuronopathic) subjects.

[0026] FIG. 20. Graph showing the level of D2S6 disaccharide in healthy subjects, MPS I (neuronopathic and non-neuronopathic) subjects, and MPS II (neuronopathic and non-neuronopathic) subjects.

[0027] FIG. 21. Graph showing the percent composition of heparan sulfate disaccharides in healthy subjects, MPS I (neuronopathic and non-neuronopathic) subjects, and MPS II (neuronopathic and non-neuronopathic) subjects.

[0028] FIG. 22. Diagram showing MPS II phase 1 / 2 clinical study summary.

[0029] FIG. 23. Graphs showing widespread CNS and systemic biodistribution after Construct 1 IC administration in non-human. The term “LD” is low dose, “IS” is immunosuppression, and “HD” is high dose.

[0030] FIGS. 24A-24B. Graphs showing cerebral spinal fluid (CSF) Biomarker Heparan Sulfate (HS) in subjects in the Phase 1 / 2 study. CSF HS measurements showed dose-dependent reductions in Cohorts 1-3 at Weeks 8 and 24.

[0031] FIGS. 25A-25B. Graphs showing concentration of D2S6 in patients in Phase 1 / 2 study. CSF D2S6 measurement showed dose-dependent reductions in Cohorts 1-3 at Week 8 and 24, with Cohort 3 participants approaching normal levels.

[0032] FIGS. 26A-26C. Graphs showing cognition, expressive language, and fine motor neurodevelopmental function for cohort 1 patients in Phase 1 / 2 study.

[0033] FIGS. 27A-27C. Graphs showing cognition, expressive language, and fine motor neurodevelopmental function for cohort 2 patients in Phase 1 / 2 study.

[0034] FIGS. 28A-28B. Graphs showing maladaptive behavior index and toileting skills for patients in Phase 1 / 2 study.

[0035] FIGS. 29A-29B. Graphs showing plasma I2S protein levels and urine GAG levels in patients in Phase 1 / 2 study.

[0036] FIG. 30. Diagram showing MPS I phase 1 / 2 clinical study summary.

[0037] FIGS. 31A-31B. Graphs showing concentration of cerebral spinal fluid (CSF) biomarker and heparin sulfate in participants in the MPS I Phase 1 / 2 study (FIG. 31 A) and in a single participant (FIG. 3 IB). Graphs show a decrease in CSF heparin sulfate in all participants through last time point available (e.g., week 24 for Phase 1 / 2 trial (FIG. 31 A) and week 59 for the single participant (FIG. 3 IB). Study showed a measurable CSF IDUA enzyme activity in the majority of participants in the Phase 1 / 2 trial and in the single participant.

[0038] FIGS. 32A-32F. Graphs showing neurodevelopment function BSID-III as cognition, expressive language, and fine motor in MPS I Phase 1 / 2 study and the single participant study.

[0039] FIG. 33. Graph showing neurodevelopmental function BSID cognition in the MPS I single participant study.

[0040] FIG. 34. Tables showing the neurodevelopmental function (WASI-II and VABS-III) for a 13 year-old MPS I Phase 1 / 2 study participant.

[0041] FIGS. 35A-35B. Graphs showing level of I0S6 in MPS I Phase 1 / 2 participants (FIG. 3 5 A) and in the single participant study (FIG. 35B).

[0042] FIGS. 36A-36B. Graphs showing level of total urine GAGs in MPS I Phase 1 / 2 participants (FIG. 36A) and in the single participant study (FIG. 36B). 5. DETAILED DESCRIPTION OF THE INVENTION

[0043] The invention involves the delivery of recombinant human iduronate-2-sulfatase (rhIDS) produced by human neuronal or glial cells to the cerebrospinal fluid (CSF) of the central nervous system (CNS) of a human subject diagnosed with mucopolysaccharidosis II (MPS II), including, but not limited to patients diagnosed with Hunter syndrome. See, also, International Patent Application No. PCT / US2017 / 027770, filed April 14, 2017 (published as WO / 2017 / 181113 on October 19, 2017), which is incorporated by reference herein in its entirety, for compositions and methods that can be used according to the invention described herein.

[0044] In a preferred embodiment, the treatment is accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding human IDS (hIDS), or a derivative of hIDS, to the CSF of a patient (human subject) diagnosed with MPS II, so that a permanent depot of transduced neuronal and / or glial cells is generated that continuously supplies the transgene product to the CNS. The rhIDS secreted from the neuronal / glial cell depot into the CSF will be endocytosed by cells in the CNS, resulting in “cross-correction” of the enzymatic defect in the recipient cells. Moreover, it has been found, unexpectedly, that the depot of transduced neural and glial cells in the CNS can deliver the recombinant enzyme to both the CNS and systemically, which may reduce or eliminate the need for systemic treatment, e.g., weekly i.v. injections of the enzyme. Also provided herein is the treatment of MPS I by administering a viral vector or other DNA expression construct encoding human IDUA to a subject (e.g., non-replicating recombinant AAV of serotype 9 capsid containing an hIDUA expression cassette; Construct 2; refer to PCT / US2021 / 014129; PCT / US2018 / 015910; and PCT / US2019 / 042205, each of which is incorporated by reference herein in its entirety).

[0045] In an alternative embodiment, the hIDS can be produced by human neuronal or glial cells in cell culture (e.g., bioreactors) and administered as an enzyme replacement therapy (“ERT”), e.g., by injecting the enzyme - into the CSF, directly into the CNS, and / or systemically. However, the gene therapy approach offers several advantages over ERT since systemic delivery of the enzyme will not result in treating the CNS because the enzyme cannot cross the blood brain barrier; and, unlike the gene therapy approach of the invention, direct delivery of the enzyme to the CSF and / or CNS would require repeat injections which are not only burdensome, but pose a risk of infection.

[0046] The hIDS encoded by the transgene can include, but is not limited to human IDS (hIDS) having the amino acid sequence of SEQ ID NO. 1 (as shown in FIG. 1), and derivatives of hIDS having amino acid substitutions, deletions, or additions, e.g., including but not limited to amino acid substitutions selected from corresponding non-conserved residues in orthologs of IDS shown in FIG. 2, with the proviso that such mutations do not include replacement of the cysteine residue at position 84 (C84) which is required for enzyme activity (Millat et al., 1997, Biochem J 326: 243-247); or a mutation that has been identified in severe, severe-intermediate, intermediate, or attenuated MPS II phenotypes e.g., as shown in FIG. 3, or as reported by Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-761 (reporting “attenuated” mutants R48P, A85T, W337R, and the truncated mutant Q531X; and “severe” mutants P86L, S333L, S349I, R468Q, R468L); Millat et al., 1998, BBA 1406: 214-218 (reporting “attenuated” mutants P480L and P480Q; and “severe” mutant P86L); and Bonucelli et al., 2001, BBA 1537:233-238, each of which is incorporated by reference herein in its entirety.

[0047] For example, amino acid substitutions at a particular position of hIDS can be selected from among corresponding non-conserved amino acid residues found at that position in the IDS orthologs aligned in FIG. 2, with the proviso that such substitutions do not include any of the deleterious mutations shown in FIG. 3 or as reported by Sukegawa-Hayasaka et al., 2006, supra, Millat et al., 1998, supra, or Bonucelli et al., 2001, supra, each of which is incorporated by reference herein in its entirety. The resulting transgene product can be tested using conventional assays in vitro, in cell culture or test animals to ensure that the mutation does not disrupt IDS function. Preferred amino acid substitutions, deletions or additions selected should be those that maintain or increase enzyme activity, stability or half-life of IDS, as tested by conventional assays in vitro, in cell culture or animal models for MPS II. For example, the enzyme activity of the transgene product can be assessed using a conventional enzyme assay with, for example, 4-Methylumbelliferyl a-L-idopyranosiduronic acid 2-sulfate or 4-methylumbelliferyl sulfate as the substrate (see, e.g., Lee et al., 2015, Clin. Biochem. 48(18):1350-1353, Dean et al., 2006, Clin. Chern. 52(4):643-649 for exemplary IDS enzyme assays that can be used, each of which is incorporated by reference herein in its entirety). The ability of the transgene product to correct MPS II phenotype can be assessed in cell culture; e.g., by transducing MPS II cells in culture with a viral vector or other DNA expression construct encoding hIDS or a derivative; by adding the transgene product or a derivative to MPS II cells in culture; or by co-culturing MPS II cells with human neuronal / glial host cells engineered to express and secrete rhIDS or a derivative, and determining correction of the defect in the MPS II cultured cells, e.g., by detecting IDS enzyme activity and / or reduction in GAG storage in the MPS II cells in culture (see, e.g., Stroncek et al., 1999, Transfusion 39(4):343-350, which is incorporated by reference herein in its entirety).

[0048] Animal models for MPS II have been described that can be used to assess the therapeutics described herein. For example, a knockout mouse model (IDS-knockout) of MPS II was engineered by replacing exons 4 and 5 of the IDS gene with the neomycin resistance gene. (Garcia et al., 2007, J Inherit Metab Dis 30: 924-34). This IDS-knockout mouse exhibits many of the characteristics of MPS II, including skeletal abnormalities, hepatosplenomegaly, elevated urinary and tissue GAG, and brain storage lesions (Muenzer et al., 2001, Acta Paediatr Suppl 91:98-99) and was used to assess the effect of enzyme replacement therapy in MPS II in support of clinical trials for ERT. This mouse model, therefore, is a relevant model for studying the effects of gene therapy delivering rIDS produced by neuronal or glial cells as a treatment for MPS II (see, e.g., Polito and Cosma, 2009, Am. J. Hum. Genet. 85(2):296-301, which is incorporated by reference herein in its entirety).

[0049] Preferably, the hIDS transgene produced by the human neuronal / glial cells should be controlled by expression control elements that function in neurons and / or glial cells, e.g., the CB7 promoter (a chicken P-actin promoter and CMV enhancer), and can include other expression control elements that enhance expression of the transgene driven by the vector (e.g., chicken P-actin intron and rabbit P-globin poly A signal). The cDNA construct for the hIDS transgene should include a coding sequence for a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced CNS cells. Such signal peptides used by CNS cells may include but are not limited to: Oligodendrocyte-myelin glycoprotein (hOMG) signal peptide: MEYQILKMSLCLFILLFLTPGILC (SEQ ID NO:2) Cellular repressor of ElA-stimulated genes 2 (hCREG2) signal peptide: MSVRRGRRPARPGTRLSWLLCCSALLSPAAG (SEQ ID NO:3) V-set and transmembrane domain containing 2B (hVSTM2B) signal peptide: MEQRNRLGALGYLPPLLLHALLLFVADA (SEQ ID NO:4) Protocadherin alpha-1 (hPCADHAl) signal peptide: MVFSRRGGLGARDLLLWLLLLAAWEVGSG (SEQ ID NO:5) FAM19A1 (TAFA1) signal peptide: MAMVSAMSWVLYLWISACA (SEQ ID NO:6) Interleukin-2 signal peptide: MYRMQLLSCIALILALVTNS (SEQ ID NO: 14) Signal peptides may also be referred to herein as leader sequences or leader peptides.

[0050] The recombinant vector used for delivering the transgene should have a tropism for cells in the CNS, including but limited to neurons and / or glial cells. Such vectors can include non-replicating recombinant adeno-associated virus vectors (“rAAV”), particularly those bearing an AAV9 or AAVrhlO capsid are preferred. AAV variant capsids can be used, including but not limited to those described by Wilson in US Patent No. 7,906,111 which is incorporated by reference herein in its entirety, with AAV / hu.31 and AAV / hu.32 being particularly preferred; as well as AAV variant capsids described by Chatterjee in US Patent No. 8,628,966, US Patent No. 8,927,514 and Smith et al., 2014, Mol Ther 22: 1625-1634, each of which is incorporated by reference herein in its entirety. However, other viral vectors may be used, including but not limited to lentiviral vectors, vaccinia viral vectors, or non-viral expression vectors referred to as “naked DNA” constructs.

[0051] Pharmaceutical compositions suitable for administration to the CSF comprise a suspension of the rhIDS vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients. In certain embodiments, the pharmaceutical compositions are suitable for intrathecal administration. In certain embodiments, the pharmaceutical compositions are suitable for intraci sternal administration (injection into the cistema magna). In certain embodiments, the pharmaceutical compositions are suitable for injection into the subarachnoid space via a Cl-2 puncture. In certain embodiments, the pharmaceutical compositions are suitable for intracerebroventricular administration. In certain embodiments, the pharmaceutical compositions are suitable for administration via lumbar puncture. In some embodiments, the pharmaceutical composition comprising the rAAV of the present disclosure comprises sodium chloride at a concentration of about 8.77 g / L, magnesium chloride 6-hydrate, at a concentration of about 0.244 g / L, potassium chloride at a concentration of about 0.224 g / L, calcium chloride dihydrate at a concentration of about 0.206 g / L, dextrose anhydrous at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.001% (volume / volume), sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L.

[0052] Therapeutically effective doses of the recombinant vector should be administered to the CSF via intrathecal administration (i.e., injection into the subarachnoid space so that the recombinant vectors distribute through the CSF and transduce cells in the CNS). In some embodiments, the recombinant vector is administered in a solution comprising sodium chloride at a concentration of about 8.77 g / L, magnesium chloride 6-hydrate, at a concentration of about 0.244 g / L, potassium chloride at a concentration of about 0.224 g / L, calcium chloride dihydrate at a concentration of about 0.206 g / L, dextrose anhydrous at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.001% (volume / volume), sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L. This can be accomplished in a number of ways - e.g., by intracranial (cisternal or ventricular) injection , or injection into the lumbar cistern. For example intraci sternal (IC) injection (into the cistema magna) can be performed by CT-guided suboccipital puncture; or injection into the subarachnoid space can be performed via a Cl-2 puncture when feasible for the patient; or lumbar puncture (typically diagnostic procedures performed in order to collect a sample of CSF) can be used to access the CSF. Alternatively, intracerebroventricular (ICV) administration (a more invasive technique used for the introduction of antiinfective or anticancer drugs that do not penetrate the blood-brain barrier) can be used to instill the recombinant vectors directly into the ventricles of the brain. Alternatively, intranasal administration may be used to deliver the recombinant vector to the CNS.

[0053] Because of the relatively rapid brain growth that occurs early in a developing child, the total dose of AAV9.hIDS administered IC depends on the assumed brain mass across different age strata, see, e.g.. Table 2 below. For brain mass by age for the study subjects see, e.g., AS Dekaban, Ann Neurol, 1978 Oct; 4(4): 345-56. Table 2. Total dose administered by age Subject Age Assumed Dose 1 Dose 2 brain mass (g) (total GC*) (total GC*) > 4 to < 9 months 600 7.8 x 1012 3.9 x 1013 > 9 to < 18 months 1000 1.3 x 1013 6.5 x 1013 >18 months to < 3 years 1100 1.4 x 1013 7.2 x 1013 > 3 years 1300 1.7 x 1013 8.5 x 1013 *GC was determined using a Poly-A-specific PCR assay

[0054] CSF concentrations can be monitored by directly measuring the concentration of rhIDS in the CSF fluid obtained from occipital or lumbar punctures, or estimated by extrapolation from concentrations of the rhIDS detected in the patient’s serum.

[0055] By way of background, human IDS is translated as a 550 amino acid polypeptide that contains eight potential N-glycosylation sites (N31, N115, N144, N246, N280, N325, N513 and N537) depicted in FIG.l and includes a 25 amino acid signal sequence which is cleaved during processing. An initial 76 kDa intracellular precursor is converted into a phosphorylated 90 kDa precursor after modification of its oligosaccharide chains in the Golgi apparatus. This precursor is processed by glycosylation modifications and proteolytic cleavage through various intracellular intermediates to a major 55 kDa form. To summarize, after removal of the 25 aa signal sequence, proteolytic processing involves N-terminal proteolytic cleavage downstream of N31 removing a propeptide of eight amino acids (residues 26-33), and C-terminal proteolytic cleavage upstream of N513 which releases an 18 kDa polypeptide and produces a 62 kDa intermediate that is converted to a 55 kDa mature form. Further proteolytic cleavage yields a 45 kDa mature form located in the lysosomal compartment. (See FIG. 4 for diagram reproduced from Millat et al., 1997, Exp Cell Res 230: 362-367 (“Millat 1997”); Millat et al. 1997, Biochem J. 326: 243-247 (“Millat 1997a”); and Froissart et al., 1995, Biochem J. 309:425-430, each of which is incorporated by reference herein in its entirety).

[0056] A formylglycine modification of C84 (shown in bold in FIG. 1) required for enzyme activity probably occurs as an early post-translational or co-translational event, most probably in the endoplasmic reticulum. (See, Millat 1997a, citing Schmidt et al., 1995, Cell 82: 271-278). Post-translational processing continues in the Golgi to include addition of complex sialic acidcontaining glycans and acquisition of mannose-6-phosphate residues which tag the enzyme for delivery to the lysosomal compartment. (See, Clarke, 2008, Expert Opin Pharmacother 9: 311317 for a concise review which is incorporated by reference herein in its entirety). While no single glycosylation site is essential for IDS stability, glycosylation at position N280 is important for cellular internalization and lysosomal targeting via the mannose-6-phosphate (M6P) receptor. (Chung et al., 2014, Glycoconj J 31:309-315 at p. 310, first column). In the normal physiologic state, IDS is produced at very low levels and very little, if any, enzyme is secreted from the cell. (Clarke, 2008, supra).

[0057] The invention is based, in part, on the following principles: (i)     Neuronal and glial cells in the CNS are secretory cells that possess the cellular machinery for post-translational processing of secreted proteins - including glycosylation, mannose-6-phosphorylation, and tyrosine-O-sulfation - robust processes in the CNS. See, e.g., Sleat et al., 2005, Proteomics 5: 1520-1532, and Sleat 1996, J Biol Chern 271: 19191-98 which describes the human brain mannose-6-phosphate glycoproteome and notes that the brain contains more proteins with a much greater number of individual isoforms and mannose-6-phosphorylated proteins than found in other tissues; and Kanan et al., 2009, Exp. Eye Res. 89: 559-567 and Kanan & Al-Ubaidi, 2015, Exp. Eye Res. 133: 126-131 reporting the production of tyrosinesulfated glycoproteins secreted by neuronal cells, each of which is incorporated by reference in its entirety for post-translational modifications made by human CNS cells. (ii) The human brain produces multiple isoforms of natural / native IDS. In particular, N-terminal sequencing of human brain mannose-6-phosphorylated glycoproteins revealed that the N-terminal sequence of the mature 42 kDa chain of hIDS varies in the brain, starting at positions 34 or 36 as follows: T34DALNVLLI; and A36LNVLLIIV. (Sleat, 2005, Proteomics 5: 1520-1532, Table S2). Two of the eight N-linked glycosylation sites, namely N280 and N116, were found to be mannose-6-phophorylated in IDS obtained from human brain. (Sleat et al., 2006, Mol & Cell Proeomics 5.4: 686-701, reported at Table V). (iii) During processing of hIDS, two polypeptides, 76 kDa and 90 kDa, are secreted by neural and glial cells, but only the 90 kDa polypeptide is mannose-6-phosphorylated, which is necessary for secreted forms of the enzyme to achieve cross correction. (See, Millat, 1997, Fig. 1 results for transduced lymphoblastoid cells, and Froissart 1995, Fig. 4 showing similar results for transduced fibroblasts - in culture medium, only the 90 kDa form is phosphorylated). Interestingly, it has been demonstrated that recombinant IDS produced by neuronal and glial cells may be endocytosed by recipient CNS cells more avidly than recombinant IDS produced by other cells such as kidney. Daniele 2002 demonstrated M6P-receptor mediated endocytosis of recombinant IDS from conditioned media of transduced neuronal and glial cell cultures by a recipient population of non-transduced neuronal and glial cells which properly processed the precursor to the 45 kDa mature active form. Uptake of the recombinant IDS produced by the neuronal and glial cell lines (74% endocytosis) far exceeded uptake of the enzyme produced by a kidney cell line (5.6% endocytosis). In each case, uptake was inhibited by M6P, indicating that recombinant IDS uptake was M6P-receptor mediated. (See Daniele 2002, Tables 2 and 4 and accompanying description in Results at pp. 205-206 summarized in Table 3 below). Table 3. Summary of Results Reported in Daniele 2002 Cell Line Source of rIDS Media Enzyme Units Recipient Cells: Units Recovered % Endocytosis (mean value) Neuronal Glial Kidney (transfected’ 35 U 1.7U 2.2 U 5.6% Neuronal 12 U 8.8 U 8.8 U 74% Glial ^d-transduced) 14 U 10.5 U 10.5 U 74% (iv) The gene therapy approach described herein should result in the continuous secretion of an hIDS glycoprotein precursor of about 90 kDa as measured by polyacrylamide gel electrophoresis (depending on the assay used) that is enzymatically active. First, the enzyme responsible for the formylglycine modification of C84 which is required for IDS activity — the FGly-Generating Enzyme (FGE, aka SUMF1) — is expressed in the cerebral cortex of the human brain (gene expression data for SUMF1 may be found, for example, at GeneCards, accessible at http: / / www.genecards.org). Second, the secreted glycosylated / phosphorylated rIDS produced by transduced neurons and glial cells in situ should be taken up and correctly processed by untransduced neural and glial cells in the CNS. Without being bound to any theory, it appears that the secreted rhIDS precursor produced in situ by gene therapy may be more avidly endocytosed by recipient cells in the CNS than would traditional recombinant enzymes used for ERT if administered to the CNS. For example, Elaprase® (made in HT1080, a fibrosarcoma cell line) is a purified protein reported to have a molecular weight of about 76 kDa - not the 90 kDa species secreted by neuronal and glial cells that appears to be more heavily phosphorylated. While the eight N-linked glycosylation sites are reported to be fully occupied in Elaprase® and contain two bis-mannose-6-phosphate terminated glycans as well as complex highly sialylated glycans, the post-translational modification of C84 to FGly, which is an absolute requirement for enzyme activity, is only about 50%. (Clarke, 2008, Expert Opin Pharmacother 9:311-317; Elaprase® Full Prescribing Information and EMA filing). Another recombinant product, Hunterase® is made in CHO cells. While reported to have higher FGly and activity than Elaprase®, mannose-6-phosphorylation and uptake did not differ. (Chung, 2014, Glycoconj J 31:309-315). (v) The extracellular IDS efficacy in vivo depends on uptake (cell and lysosome internalization) through mannose-6-phosphate (M6P) and its active site formylglycine (FGly), which is converted from C84 through post-translational modification by formylglycine-generating enzyme. As shown above in Table 3, brain cells (neuronal and glial cells) show higher enzyme activities when incubated with IDS precursor media secreted by transduced neuronal and glial cells than with IDS precursor media secreted by genetically engineered kidney cells. The resultant five-fold increase in activity can likely be attributed to the efficient uptake of IDS (See Daniele 2002, Tables 2 and 4). Commercial forms of IDS, which are generated by CHO cells or HT-1080 cells, have a FGly content of about 50% to 70%, which determines the enzyme activity. However, neuronal and glial cells may improve upon this activity, due to improvement of IDS uptake. (vi) The cellular and subcellular trafficking / uptake of lysosomal proteins, including IDS, is through M6P. IDS from brain cells may contain higher M6P content, as reported in Daniele 2002, and in Sleat, Proteomics, 2005 (indicating that the human brain contains more (in both a quantitative and qualitative sense) Man6-P glycoproteins than other tissues.). It is possible to measure the M6P content of an IDS precursor, as done in Daniele 2002. In the presence of inhibitory M6P (e.g., 5 mM), the uptake of IDS precursor generated by non-neuronal or non-glial cells, such as the genetically engineered kidney cells of Daniele 2002, is predicted to decrease to levels close to that of the control cells, as was shown in Daniele 2002. While in the presence of inhibitory M6P, the uptake of IDS precursor generated by brain cells, such as neuronal and glial cells, is predicted to remain at a high level, as was shown in Daniele 2002, where the uptake was four times higher than control cells and comparable to the level of IDS activity (or uptake) of IDS precursor generated by genetically engineered kidney cells without the presence of inhibitory M6P. This assay allows for a way to predict the M6P content in IDS precursor generated by brain cells, and, in particular, to compare the M6P content in IDS precursors generated by different types of cells. The gene therapy approach described herein should result in the continuous secretion of an hIDS precursor that may be taken up into neuronal and glial cells at a high level in the presence of inhibitory M6P in such an assay. (vii) The M6P content and uptake of IDS precursor may also be demonstrated by 90 kDa and 76 kDa gel bands (e.g., SDS-PAGE gel bands). The 90 kDa is reported to be highly glycosylated / phosphorylated and contains M6P, while 76 kDa is not. A very broad gel band with a range from 76 kDa to 95 kDa and with an average MW of SO-85 kDa, similar to the IDS precursor gel band generated from genetically engineered kidney cells (Daniele 2002, Figure 1), may be contrasted with a gel band of IDS precursor generated from brain cells. In Daniele 2002, the gel band cannot be obtained due to unsuccessful immunoprecipitation of the IDS precursor. The gene therapy approach described herein should result in the continuous secretion of an hIDS precursor that differs from the IDS precursor gel band generated from genetically engineered kidney cells. (viii) The M6P content of commercial IDS precursor is 2 to 2.5 mol / mol, majority of which is present in a form of di-phosphorylated glycans. Although in average, every IDS precursor is phosphorylated, a normal distribution of glycans will have some IDS precursor with 2, 1 and 0 of di-phosphorylated M6P glycans assuming multiple phosphorylation sites. Uptake rate should be significant higher with multiple phosphorylation. (ix) The glycosylation of hIDS by human cells of the CNS will result in the addition of glycans that can improve stability, half-life and reduce unwanted aggregation of the transgene product. Significantly, the glycans that are added to hIDS of the invention include 2,6-sialic acid, incorporating Neu5 Ac (“NANA”) but not its hydroxylated derivative, NeuGc (N-Glycolylneuraminic acid, i.e., “NGNA” or “Neu5Gc”). Such glycans are not present in recombinant IDS products, such as Hunterase®, made in CHO cells because CHO cells do not have the 2,6-sialyltransferase required to make this post-translational modification; nor do CHO cells produce bisecting GlcNAc, although they do add Neu5Gc (NGNA) as sialic acid not typical (and potentially immunogenic) to humans instead of Neu5Ac (NANA). See, e.g., Dumont et al., 2016, Critical Rev in Biotech 36(6):1110-1122 (Early Online pp. 1-13 atp. 5); and Hague et al., 1998 Electrophor 19:2612-2630 (“[t]he CHO cell line is considered ‘phenotypically restricted,’ in terms of glycosylation, due to the lack of an a2,6-sialyl-transferase”). Moreover, CHO cells can also produce an immunogenic glycan, the a-Gal antigen, which reacts with anti-a-Gal antibodies present in most individuals, and at high concentrations can trigger anaphylaxis. See, e.g., Bosques, 2010, Nat Biotech 28: 1153-1156. The human glycosylation pattern of the rhIDS of the invention should reduce immunogenicity of the transgene product and improve efficacy. (x) Immunogenicity of a transgene product could be induced by various factors, including the immune condition of the patient, the structure and characteristics of the infused protein drug, the administration route, and the duration of treatment. Process-related impurities, such as host cell protein (HCP), host cell DNA, and chemical residuals, and product-related impurities, such as protein degradants and structural characteristics, such as glycosylation, oxidation and aggregation (sub-visible particles), may also increase immunogenicity by serving as an adjuvant that enhances the immune response. The amounts of process-related and product-related impurities can be affected by the manufacturing process: cell culture, purification, formulation, storage and handling, which can affect commercially manufactured IDS products. In gene therapy, proteins are produced in vivo, such that process-related impurities are not present and protein products are not likely to contain product-related impurities / degradants associated with proteins produced by recombinant technologies, such as protein aggregation and protein oxidation. Aggregation, for example, is associated with protein production and storage due to high protein concentration, surface interaction with manufacturing equipment and containers, and the purification process with certain buffer systems. But these conditions that promote aggregation are not present when a transgene is expressed in vivo. Oxidation, such as methionine, tryptophan and histidine oxidation, is also associated with protein production and storage, caused, for example, by stressed cell culture conditions, metal and air contact, and impurities in buffers and excipients. The proteins expressed in vivo may also oxidize in a stressed condition, but humans, like many organisms, are equipped with an antioxidation defense system, which not only reduces the oxidation stress, but can also repairs and / or reverses the oxidation. Thus, proteins produced in vivo are not likely to be in an oxidized form. Both aggregation and oxidation could affect the potency, pharmacokinetics (clearance) and can increase immunogenicity concerns. The gene therapy approach described herein should result in the continuous secretion of an hIDS precursor with a reduced immunogenicity compared to commercially manufactured products. (xi) In addition to the N-linked glycosylation sites, hIDS contains a tyrosine (“Y”) sulfation site (PSSEKY165ENTKTCRGPD). (See, e.g., Yang et al., 2015, Molecules 20:2138-2164, esp. at p. 2154 which is incorporated by reference in its entirety for the analysis of amino acids surrounding tyrosine residues subjected to protein tyrosine sulfation. The “rules” can be summarized as follows: Y residues with E or D within +5 to -5 position of Y, and where position -1 of Y is a neutral or acidic charged amino acid - but not a basic amino acid, e.g., R, K, or H that abolishes sulfation). While not intending to be bound by any theory, sulfation of this site in hIDS may improve stability of the enzyme and binding affinity for substrate. Tyrosine-sulfation of hIDS - a robust post-translational process in human CNS cells - should result in improved processing and activity of transgene products. The significance of tyrosine-sulfation of lysosomal proteins has not been elucidated; but in other proteins it has been shown to increase avidity of protein-protein interactions (antibodies and receptors), and to promote proteolytic processing (peptide hormone). (See, Moore, 2003, J Biol. Chern. 278: 24243-46; and Bundegaard et al., 1995, The EMBO J 14: 3073-79). The tyrosylprotein sulfotransferase (TPST1) responsible for tyrosine-sulfation (which may occur as a final step in IDS processing) is apparently expressed at higher levels (based on mRNA) in the brain (gene expression data for TPST1 may be found, for example, at the EMBL-EBI Expression Atlas, accessible at http: / / www.ebi.ac.uk / gxa / home). Such post-translational modification, at best, is under-represented in CHO cell products. Unlike human CNS cells, CHO cells are not secretory cells and have a limited capacity for post-translational tyrosine-sulfation. (See, e.g., Mikkelsen & Ezban, 1991, Biochemistry 30: 1533-1537, esp. discussion atp. 1537).

[0058] For the foregoing reasons, the production of rhIDS by human neuronal and / or glial cells should result in a “biobetter” molecule for the treatment of MPS II accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding rhIDS to the CSF of a patient (human subject) diagnosed with an MPS II disease (including but not limited to Hunter) to create a permanent depot in the CNS that continuously supplies a fully human-glycosylated, mannose-6-phosphorylated, sulfated transgene product secreted by the transduced CNS cells. The hIDS transgene product secreted from the depot into the CSF will be endocytosed by cells in the CNS, resulting in “cross-correction” of the enzymatic defect in the MPS II recipient cells.

[0059] It is not essential that every rhIDS molecule produced either in the gene therapy or protein therapy approach be fully glycosylated, phosphorylated, and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation (including 2,6-sialylation and mannose-6-phosphorylation) and sulfation to demonstrate efficacy. The goal of gene therapy treatment of the invention is to slow or arrest the progression of disease. Efficacy may be monitored by measuring cognitive function (e.g., prevention or decrease in neurocognitive decline); reductions in biomarkers of disease (such as GAG) in CSF and or serum; and / or increase in IDS enzyme activity in CSF and / or serum. Signs of inflammation and other safety events may also be monitored.

[0060] As an alternative, or an additional treatment to gene therapy, the rhIDS glycoprotein can be produced in human neural or glial cell lines by recombinant DNA technology and the glycoprotein can be administered to patients diagnosed with MPS II systemically and / or into the CSF for ERT). Human cell lines that can be used for such recombinant glycoprotein production include but are not limited to HT-22, SK-N-MC, HCN-1 A, HCN-2, NT2, SH-SY5y, hNSCl 1, or ReNcell VM (see, e.g., Dumont et al., 2016, Critical Rev in Biotech 36(6): 1110-1122 “Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives” which is incorporated by reference in its entirety for a review of the human cell lines that could be used for the recombinant production of the rHuGlylDS glycoprotein). To ensure complete glycosylation, especially sialylation, and tyrosine-sulfation, the cell line used for production can be enhanced by engineering the host cells to co-express a-2,6-sialyltransferase (or both a-2,3-and a-2,6-sialyltransferases) and / or TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation.

[0061] While the delivery of rhIDS should minimize immune reactions, the clearest potential source of toxicity related to CNS-directed gene therapy is generating immunity against the expressed rhIDS protein in human subjects who are genetically deficient for IDS and, therefore, potentially not tolerant of the protein and / or the vector used to deliver the transgene.

[0062] Thus, in a preferred embodiment, it is advisable to co-treat the patient with immune suppression therapy — especially when treating patients with severe disease who have close to zero levels of IDS. Immune suppression therapies involving a regimen of tacrolimus or rapamycin (sirolimus) in combination with mycophenolic acid, or other immune suppression regimens used in tissue transplantation procedures can be employed. Such immune suppression treatment may be administered during the course of gene therapy, and in certain embodiments, pre-treatment with immune suppression therapy may be preferred. Immune suppression therapy can be continued subsequent to the gene therapy treatment, based on the judgment of the treating physician, and may thereafter be withdrawn when immune tolerance is induced; e.g., after 180 days.

[0063] Combinations of delivery of the rhIDS to the CSF accompanied by delivery of other available treatments are encompassed by the methods of the invention. The additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment. Available treatments for MPS II that could be combined with the gene therapy of the invention include but are not limited to enzyme replacement therapy using Elaprase® administered systemically or to the CSF; and / or HSCT therapy.

[0064] In certain embodiments, described herein is a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising delivering to the cerebrospinal fluid (CSF) of said human subject a therapeutically effective amount of a recombinant human iduronate-2-sulfatase (IDS) precursor produced by human neuronal or human glial cells.

[0065] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising delivering to the cerebrospinal fluid (CSF) of said human subject, a therapeutically effective amount of a recombinant human iduronate-2-sulfatase (IDS) glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, has a formylglycine residue at C84 (Fig. 1), is a2,6-sialylated, does not contain detectable NeuGc, and is mannose-6-phosphorylated.

[0066] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising delivering to the cerebrospinal fluid (CSF) of said human subject, a therapeutically effective amount of a recombinant human iduronate-2-sulfatase (IDS) glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, has a formylglycine residue at C84 (Fig. 1), is a2,6-sialylated, does not contain detectable NeuGc and / or a-Gal antigen, and is mannose-6-phosphorylated.

[0067] In certain embodiments, the human IDS precursor is delivered to the CSF from a depot of cells in the central nervous system genetically engineered to secrete said IDS precursor into the CSF. In certain embodiments, the depot is formed in the subject’s brain. In certain embodiments, the human subject is deficient in IDS activity. In certain embodiments, the human IDS comprises the amino acid sequence of SEQ ID NO. 1.

[0068] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering to the cerebrospinal fluid (CSF) of said human subject a recombinant nucleotide expression vector encoding human iduronate-2-sulfatase (IDS), wherein said expression vector when used to transduce a primary human neuronal cell in culture directs the expression of a secreted human IDS glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, has a formylglycine residue at C84 (Fig. 1), is a2,6-sialylated and mannose-6-phosphorylated.

[0069] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering to the cerebrospinal fluid of the brain of said human subject, a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS, so that a depot is formed in the subject’s central nervous system that secretes a recombinant human IDS glycoprotein precursor that is a2,6-sialylated and mannose-6-phosphorylated.

[0070] In certain embodiments, secretion of said recombinant human IDS glycoprotein precursor that is a2,6-sialylated is confirmed by transducing a human neuronal cell line with said recombinant nucleotide expression vector in cell culture. In certain embodiments, secretion of said recombinant human IDS glycoprotein precursor that is mannose-6-phosphorylated is confirmed by transducing a human neuronal cell line with said recombinant nucleotide expression vector in cell culture. In certain embodiments, the secretion is confirmed in the presence and absence of mannose-6-phosphate.

[0071] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering to the cerebrospinal fluid of the brain of said human subject, a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS, so that a depot is formed that secretes a glycosylated IDS precursor containing a a2,6-sialylated glycan; wherein said recombinant vector, when used to transduce human neuronal cells in culture results in secretion of said glycosylated IDS precursor containing a a2,6-sialylated glycan in said cell culture.

[0072] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering to the cerebrospinal fluid of the brain of said human subject, a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS, so that a depot is formed that secretes a glycosylated IDS precursor that contains a mannose-6-phosphate; wherein said recombinant vector, when used to transduce human neuronal cells in culture results in secretion of said glycosylated IDS precursor that is mannose-6-phosphorylated in said cell culture.

[0073] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering to the cerebrospinal fluid of the brain of said human subject, a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS, so that a depot is formed that secretes a glycosylated IDS precursor that contains a formylglycine; wherein said recombinant vector, when used to transduce human neuronal cells in culture results in secretion of said glycosylated IDS precursor that contains a formylglycine in said cell culture.

[0074] In certain embodiments, the human IDS comprises the amino acid sequence of SEQ ID NO. 1. In certain embodiments, the IDS transgene encodes a leader peptide. In certain embodiments, the expression vector is a replication defective AAV vector. In certain embodiments, the expression vector is delivered to the CSF of the subject by intrathecal (e.g., intracisternal, Cl-2 puncture if feasible for the patient, or lumbar puncture), intracerebroventricular, or intranasal administration. In certain embodiments, the human subject is deficient in IDS activity. In some embodiments, the expression vector is delivered to the CSF of the subject by intrathecal administration in a solution comprising sodium chloride at a concentration of about 8.77 g / L, magnesium chloride 6-hydrate, at a concentration of about 0.244 g / L, potassium chloride at a concentration of about 0.224 g / L, calcium chloride dihydrate at a concentration of about 0.206 g / L, dextrose anhydrous at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.001% (volume / volume), sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L.

[0075] In preferred embodiments, the glycosylated IDS does not contain detectable NeuGc and / or a-Gal. The phrase “detectable NeuGc and / or a-Gal” used herein means NeuGc and / or a-Gal moieties detectable by standard assay methods known in the art. For example, NeuGc may be detected by HPLC according to Hara etaL, 1989, “Highly Sensitive Determination of N-Acetyl-and N-Glycolylneuraminic Acids in Human Serum and Urine and Rat Serum by Reversed-Phase Liquid Chromatography with Fluorescence Detection.” J. Chromatogr., B: Biomed. 377: 111-119, which is hereby incorporated by reference for the method of detecting NeuGc. Alternatively, NeuGc may be detected by mass spectrometry. The a-Gal may be detected using an ELISA, see, for example, Galili et al., 1998, “A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody.” Transplantation. 65(8): 1129-32, or by mass spectrometry, see, for example, Ayoub et al., 2013, “Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques.” Landes Bioscience. 5(5): 699-710. See also the references cited in Platts-Mills etal., 2015, “Anaphylaxis to the Carbohydrate Side-Chain Alpha-gal” Immunol Allergy Clin North Am. 35(2): 247-260.

[0076] In one aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising delivering to the CSF of the human subject a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or human glial cells, wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain MRI of the human subject’s brain.

[0077] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising determining the human subject’s brain mass from the human subject’s brain MRI, and subsequently delivering to the CSF of the human subject a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal cells or human glial cells, wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, and wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass.

[0078] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising (a) determining the human subject’s brain mass from the human subject’s brain MRI, (b) calculating the dose based on the human subject’s brain mass, and (c) subsequently administrating to the CSF of the subject the dose of recombinant nucleotide expression vector encoding human IDS.

[0079] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising, in the following order: (a) delivering to the CSF of the human subject a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or human glial cells; (b) measuring level of heparan sulfate in the CSF of the human subject; and (c) comparing the level of heparan sulfate in the CSF of the human subject with level of heapran sulfatae in a reference population; wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain magnetic resonance imaging (MRI) of the human subject’s brain. In certain embodiments, the reference population consists of: (a) at least 1, 2, 3, 4, 5, 10, 25, 50, 75, 100, 200, 250, 300, 400, 500, or 1000 individual healthy people without MPS II, preferably of similar age, weight, and / or of the same gender as the human subject.

[0080] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising, in the following order: (a) taking a first measurement of the level of heparan sulfate in the CSF of the human subject; (b) delivering to the CSF of the human subject a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or human glial cells; and (c) after a period of time, taking a second measurement of the level of heparan sulfate; wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain magnetic resonance imaging (MRI) of the human subject’s brain. In certain embodiments, the period of time is about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 moths, 4 months, 5 months, 6 months, 7 months, 8 months, 11 months, or 1 year.

[0081] In a preferred embodiment, the glycosylated recombinant human IDS precursor will be endocytosed by cells in the CNS. In a preferred embodiment, the glycosylated recombinant human IDS precursor is delivered to lysosomes of cells in the CNS of the human subject.

[0082] In certain embodiments of the method for treating described herein, the human subject’s brain mass is converted from the human subject’s brain volume by multiplying the human subject’s brain volume in cm3 by a factor of 1.046 g / cm3, wherein the human subject’s brain volume is determined by brain MRI of the subject’s brain.

[0083] In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x 1010 GC / g brain mass as determined by MRI, or about 6.5 x 1010 GC / g brain mass as determined by MRI. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x 1010 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.9 * 1010 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 6.5 * 1010 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x 1011 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x 1011 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay).

[0084] In various embodiments of the method for treating described herein, the human subject is 5 years old or older and less than 18 years old. In specific embodiments, the human subject is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 years old. In specific embodiments, the human subject is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 years old. In specific embodiments, the human subject is 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18 or 18-19 years old. In specific embodiments, the human subject is about 56, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18 or 18-19 years old. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x io10 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose according to Table 7.

[0085] In various embodiments of the method for treating described herein, the human subject is 4 months old or older and less than 5 years old. In specific embodiments, the human subject is 4, 5, 6, 7, 8, 9, 10, or 11 months old. In specific embodiments, the human subject is about 4, 5, 6, 7, 8, 9, 10, or 11 months old. In specific embodiments, the human subject is 4-5, 56, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months old. In specific embodiments, the human subject is about 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months old. In specific embodiments, the human subject is 1, 2, 3, 4, or 5 years old. In specific embodiments, the human subject is about 1, 2, 3, 4, or 5 years old. In specific embodiments, the human subject is 1-2, 2-3, 3-4, 4-5, or 5-6 years old. In specific embodiments, the human subject is about 1-2, 2-3, 3-4, 4-5, or 5-6 years old. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x 1010 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 * 1010 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x io11 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x io11 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x io10 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x io10 GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x io11 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x io11 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x io10 GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose chosen from Dose 1 or Dose 2 according to Table 5. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose according to Table 6.

[0086] In some embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered via intraci sternal (IC) administration. In other embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered via intracerebroventricular (ICV) administration.

[0087] In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a volume that does not exceed 10% of the total cerebrospinal fluid volume of the human subject.

[0088] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is secreted at a detectable level.

[0089] In certain embodiments of the method for treating described herein, the human neuronal or human glial cells carry at least one mutation in the endogenous gene encoding human IDS precursor.

[0090] In certain embodiments of the method for treating described herein, the human neuronal or human glial cells are transduced with a recombinant adeno-associated virus vector (rAAV).

[0091] In a preferred embodiment, the recombinant nucleotide expression vector is an AAV9 or AAVrhlO vector.

[0092] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is expressed under the control of a CB7 promoter.

[0093] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is expressed from a cDNA encoding human IDS precursor.

[0094] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is about 90 kDa as measured by polyacrylamide gel electrophoresis.

[0095] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor contains a formylglycine.

[0096] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor (a) is a2,6-sialylated; (b) does not contain detectable NeuGc; (c) does not contain detectable a-Gal antigen; (d) contains tyrosine-sulfation; and / or (e) is mannose-6-phosphorylated.

[0097] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO. 1.

[0098] In certain embodiments provided herein, the method further comprising administering an immune suppression therapy to the human subject before or concurrently with the human IDS precursor treatment and optionally continuing immune suppression therapy thereafter.

[0099] In some embodiments, the immune suppression therapy comprises administering one or more corticosteroids, sirolimus, and / or tacrolimus. In a specific embodiment, the one or more corticosteroids are methylprednisolone and / or prednisone.

[00100] In a specific embodiment, the immune suppression therapy comprises administering prednisone at a dose of about 0.10 mg / kg, 0.11 mg / kg, 0.12 mg / kg, 0.13 mg / kg, 0.14 mg / kg, 0.15 mg / kg, 0.16 mg / kg, 0.17 mg / kg, 0.18 mg / kg, 0.19 mg / kg, 0.20 mg / kg, 0.21 mg / kg, 0.22 mg / kg, 0.23 mg / kg, 0.24 mg / kg, 0.25 mg / kg, 0.26 mg / kg, 0.27 mg / kg, 0.28 mg / kg, 0.29 mg / kg, 0.30 mg / kg, 0.31 mg / kg, 0.32 mg / kg, 0.33 mg / kg, 0.34 mg / kg, 0.35 mg / kg, 0.36 mg / kg, 0.37 mg / kg, 0.38 mg / kg, 0.39 mg / kg, 0.40 mg / kg, 0.41 mg / kg, 0.42 mg / kg, 0.43 mg / kg, 0.44 mg / kg, 0.45 mg / kg, 0.46 mg / kg, 0.47 mg / kg, 0.48 mg / kg, 0.49 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or 1 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering prednisone at a dose ranging from about 0.10 mg / kg to about 0.20 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering prednisone at a dose ranging from about 0.20 mg / kg to about 0.30 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering prednisone at a dose ranging from about 0.30 mg / kg to about 0.40 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering prednisone at a dose ranging from about 0.40 mg / kg to about 0.50 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering prednisone at a dose ranging from about 0.50 mg / kg to about 1 mg / kg. In a particular embodiment, the dose is administered daily. In a particular embodiment, the immune suppression therapy comprises administering prednisone at a dose of 0.5 mg / kg daily. In another particular embodiment, the immune suppression therapy comprises administering prednisone at a dose of 0.5 mg / kg daily with gradual tapering and discontinuation.

[00101] In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose of about 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4mg / kg, 4.5 mg / kg, 5mg / kg, 5.5 mg / kg, 6mg / kg, 6.5 mg / kg, 7mg / kg, 7.5 mg / kg, 8mg / kg, 8.5 mg / kg, 9mg / kg, 9.5 mg / kg, lOmg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 0.50 mg / kg to about 1.0 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 1.0 mg / kg to about 2.0 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 2.0 mg / kg to about 3.0 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 3.0 mg / kg to about 5.0 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 5.0 mg / kg to about 10.0 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 10.0 mg / kg to about 15.0 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 15.0 mg / kg to about 20.0 mg / kg. In a particular embodiment, the methylprednisolone is administered once. In a particular embodiment, the methylprednisolone is administered intravenously. In a particular embodiment, the methylprednisolone is administered for a maximum of 500 mg, In a particular embodiment, the methylprednisolone s administered over at least 30 minutes. In a particular embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose of 10 mg / kg IV for maximum of 500 mg over at least 30 minutes.

[00102] In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose to maintain a target blood level of 1-3 ng / mL. In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose of about 0.25 mg / m2 / day, 0.3 mg / m2 / day, 0.4 mg / m2 / day, 0.5 mg / m2 / day, 0.6 mg / m2 / day, 0.7 mg / m2 / day, 0.8 mg / m2 / day, 0.9 mg / m2 / day, 1 mg / m2 / day, 1.25 mg / m2 / day, 1.5 mg / m2 / day, 1.75 mg / m2 / day, 2 mg / m2 / day, 2.25 mg / m2 / day, 2.5 mg / m2 / day, 2.75 mg / m2 / day, 3 mg / m2 / day, 3.25 mg / m2 / day, 3.5 mg / m2 / day, 3.75 mg / m2 / day, 4 mg / m2 / day, 4.25 mg / m2 / day, 4.5 mg / m2 / day, 4.75 mg / m2 / day, or 5 mg / m2 / day. In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose ranging from about 0.25 mg / m2 / day to about 0.5 mg / m2 / day. In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose ranging from about 0.50 mg / m2 / day to about 1.0 mg / m2 / day. In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose ranging from about 1.0 mg / m2 / day to about 1.5 mg / m2 / day. In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose ranging from about 1.5 mg / m2 / day to about 2 mg / m2 / day. In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose ranging from about 2 mg / m2 / day to about 5mg / m2 / day. In a particular embodiment, the dose is divided in BID dosing. In a particular embodiment, the immune suppression therapy comprises administering sirolimus at a dose of about 1 mg / m2 / day every 4 hours. In a particular embodiment, the immune suppression therapy comprises administering sirolimus at a dose of about 0.5 mg / m2 / day divided in BID dosing.

[00103] In a specific embodiment, the immune suppression therapy comprises administering tacrolimus at a dose to maintain a target blood level of 2-4 ng / mL. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose of about 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg 0.04 mg / kg 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, or 0.10 mg / kg. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose ranging from 0.01 mg / kg to 0.02 mg / kg. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose ranging from 0.02 mg / kg to 0.03 mg / kg. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose ranging from 0.03 mg / kg to 0.05mg / kg. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose ranging from 0.05mg / kg to 0.07mg / kg. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose ranging from 0.07mg / kg to O.lOmg / kg. In a particular embodiment, the dose is administered twice daily. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose of about 0.05mg / kg twice daily.

[00104] In some embodiments, the method further comprises administering one or more antibiotics to the human subject before or concurrently with the immune suppression therapy. In a specific embodiment, the one or more antibiotics are trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atovaquone. In another specific embodiment, the one or more antibiotics are trimethoprim and / or sulfamethoxazole. In another specific embodiment, the one or more antibiotics are pentamidine, dapsone, and / or atovaquone. In specific embodiments, the one or more antibiotics are administered at a dose of about 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg. In specific embodiments, the one or more antibiotics are administered at a dose ranging from about 1 mg / kg to 2 mg / kg. In specific embodiments, the one or more antibiotics are administered at a dose ranging from about 2 mg / kg to 3 mg / kg. In specific embodiments, the one or more antibiotics are administered at a dose ranging from about 3 mg / kg to 5 mg / kg. In specific embodiments, the one or more antibiotics are administered at a dose ranging from about 5 mg / kg to 7 mg / kg. In specific embodiments, the one or more antibiotics are administered at a dose ranging from about 7 mg / kg to 10 mg / kg. In a specific embodiment, the one or more antibiotics are administered at a dose of about three times a week. In certain embodiments, the one or more antibiotics are administered to prevent Pneumocystis carinii pneumonia.

[00105] In some embodiments, the method further comprises administering one or more antifungal therapies to the human subject before or concurrently with the immune suppression therapy. In certain embodiments, the one or more antifungal therapies are initiated if the absolute neutrophil count is < 500 mm3.

[00106] In some embodiments, the method further comprises a step of measuring one or more of the following biomarkers after administration of the recombinant nucleotide expression vector: (a) level of glycosaminoglycans (GAGs) in CSF; (b) level of iduronate-2-sulfatase (I2S) in CSF; (c) level of GAGs in plasma; (d) level of I2S in plasma; (e) level of leukocyte I2S enzyme activity; and (f) level of GAGs in urine. In a specific embodiment, the GAGs in CSF comprise heparin sulfate in CSF. In another specific embodiment, the GAGs in CSF are heparin sulfate in CSF. In another specific embodiment, the GAGs in plasma comprise heparin sulfate in plasma. In another specific embodiment, the GAGs in plasma are heparin sulfate in plasma. In another specific embodiment, the GAGs in urine comprise heparin sulfate in urine. In another specific embodiment, the GAGs in urine are heparin sulfate in urine. In a specific embodiment, the step of measuring comprises mearing level of heparin sulfate in CSF. In another specific embodiment, the step of measuring comprises measuring level of leukocyte I2S enzyme activity.

[00107] In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is a liquid composition. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is a frozen composition. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is a lyophilized composition or a reconstituted lyophilized composition. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector provided herein may be formulated in various dosage forms for IC or ICV administration. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector provided herein may be provided in a unit-dosage form or multiple-dosage form. A unit-dosage form, as used herein, refers to a physically discrete unit suitable for administration to human and animal subjects, and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the said recombinant nucleotide expression vector and / or other ingredient(s) sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carriers or excipients. Examples of a unit-dosage form include an ampoule, a vial, a prefilled syringe, or a cartridge.

[00108] In certain embodiments of the method for treating described herein, a unit-dosage form may be administered in fractions or multiples thereof. In certain embodiments of the method for treating described herein, a multiple-dosage form is a plurality of identical unitdosage forms packaged in a single container to be administered in segregated unit-dosage form. Examples of a multiple-dosage form include a vial, a prefilled syringe, or a cartridge. In certain embodiments, the prefilled syringe comprises 8.5* 1012 GC of the recombinant nucleotide expression vector In certain embodiments, the prefilled syringe comprises 9.8* 1012 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.1 * 1013 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.3x 1013 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.5x 1013 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.7x 1013 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 4.2x 1013 GC of the recombinant nucleotide expression vector In certain embodiments, the prefilled syringe comprises 4.9x 1013 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 5.5* 1013 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 6.3* 1013 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 7.3 x 1013 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 8.5* 1013 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 9.Ox 1013 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises l.Ox 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.1 x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.2x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.3x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.4x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.5x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.6x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.7x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.8x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.9x 1014 of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.Ox 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.1 x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.2x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.3 x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.4x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.5x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.6x 1014 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.3x 1010 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.9x 1010 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 6.5* 1010 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 9.6* 1010 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.Ox 1011 GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.9x 1011 GC of the recombinant nucleotide expression vector.

[00109] As used herein, the term “about” means within plus or minus 10% of a given value or range. In certain embodiments, the term “about” means within plus or minus 1% of a given value or range, wherein the value is a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain MRI of the human subject’s brain. In certain embodiments, the term “about” means within plus or minus 2% of a given value or range, wherein the value is a dose that is determined by brain MRI of the subject’s brain, and wherein the brain mass is determined by brain MRI of the human subject’s brain. In certain embodiments, the term “about” means within plus or minus 5% of a given value or range, wherein the value is a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain MRI of the human subject’s brain. In certain embodiments, the term “about” means within plus or minus 7% of a given value or range, wherein the value is a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain MRI of the human subject’s brain. In certain embodiments, the term “about” means within plus or minus 10% of a given value or range, wherein the value is a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain MRI of the human subject’s brain. However, it is to be understood that in this specification, the term “about” also affords support for recitation of the exact value with which the term is connected. For example, “about 10” also provides support for the number “10” exactly. 5.1 PROCESSING, N-GLYCOSYLATION AND TYROSINE SULFATION 5.1.1. Processing

[00110] Human IDS includes a 25 amino acid signal sequence which is cleaved during processing. An initial 76 kDa intracellular IDS precursor is converted into a phosphorylated 90 kDa IDS precursor after modification of its oligosaccharide chains in the Golgi apparatus. This precursor is processed by glycosylation modifications and proteolytic cleavage through various intracellular intermediates to a major 55 kDa form. To summarize, after removal of the 25 aa signal sequence, proteolytic processing involves N-terminal proteolytic cleavage downstream of N31 removing a propeptide of eight amino acids (residues 26-33), and C-terminal proteolytic cleavage upstream of N513 which releases an 18 kDa polypeptide and produces a 62 kDa intermediate that is converted to a 55 kDa mature form. Further proteolytic cleavage yields a 45 kDa mature form located in the lysosomal compartment. (See FIG. 4 for diagram reproduced from Millat et al., 1997, Exp Cell Res 230: 362-367 (“Millat 1997”); Millat et al. 1997, Biochem J. 326: 243-247 (“Millat 1997a”); and Froissart et al., 1995, Biochem J. 309:425-430, each of which is incorporated by reference herein in its entirety).

[00111] A formylglycine modification of C84 (shown in bold in FIG. 1) required for enzyme activity probably occurs as an early post-translational or co-translational event, most probably in the endoplasmic reticulum. (See, Millat 1997a, citing Schmidt et al., 1995, Cell 82: 271-278). Post-translational processing continues in the Golgi to include addition of complex sialic acidcontaining glycans and acquisition of mannose-6-phosphate residues which tag the enzyme for delivery to the lysosomal compartment. (See, Clarke, 2008, Expert Opin Pharmacother 9: 311317 for a concise review which is incorporated by reference herein in its entirety).

[00112] In a specific embodiment, HuGlylDS used in accordance with the methods described herein, when expressed in a neuronal or glial cell, in vivo or in vitro, can be the 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) mannose-6-phosphorylated form of the enzyme. IDS produced from neuronal and glial cells may contain higher M6P content, as reported in Daniele 2002, and in Sleat, Proteomics, 2005 (indicating that the human brain contains more (in both a quantitative and qualitative sense) M6P glycoproteins than other tissues.). It is possible to measure the M6P content of an IDS precursor, as done in Daniele 2002. Accordingly, in certain embodiments, HuGlylDS used in accordance with the methods described herein, when expressed in a neuronal or glial cell, in vivo or in vitro, is mannose-6-phosphorylated at a higher level than IDS expressed in a non-neuronal or glial cell. In particular, HuGlylDS used in accordance with the methods described herein, when expressed in a neuronal or glial cell, in vivo or in vitro, is mannose-6-phosphorylated at a higher level than IDS expressed in a HT1080 or CHO cell. In certain embodiments, the mannose-6-phosphorylation level of the expresssed IDS is measured by uptake of the IDS by a human neuronal cell in the presence of M6P (e.g., 5 mM M6P). In certain embodiments, when expressed in a neuronal or glial cell, in vivo or in vitro, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% -80%, 80% - 90%, or 90% - 100% of HuGlylDS molecules used in accordance with the methods described herein are mannose-6-phosphorylated. 5.1.2. N-Glycosylation

[00113] Neuronal and glial cells in the CNS are secretory cells that possess the cellular machinery for post-translational processing of secreted proteins - including glycosylation and tyrosine-O-sulfation. hIDS has eight asparaginal (“N”) glycosylation sites identified in FIG. 1 (N31ST; N115FS; N144HT; N246IT; N280IS; N325ST; N513FS; N537DS). Two of the eight N-linked glycosylation sites, namely N280 and N116, are mannose-6-phophorylated in IDS obtained from human brain. (Sleat et al., 2006, Mol & Cell Proeomics 5.4: 686-701, reported at Table V). While no single glycosylation site is essential for IDS stability, glycosylation at position N280 is important for cellular internalization and lysosomal targeting via the mannose-6-phosphate (M6P) receptor. (Chung et al., 2014, Glycoconj J 31:309-315 at p. 310, first column). In the normal physiologic state, IDS is produced at very low levels and very little, if any, enzyme is secreted from the cell. (Clarke, 2008, supra).

[00114] It is not essential that every molecule produced either in the gene therapy or protein therapy approach be fully glycosylated and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation and sulfation to demonstrate efficacy.

[00115] In a specific embodiment, HuGlylDS used in accordance with the methods described herein, when expressed in a neuronal or glial cell, in vivo or in vitro, could be glycosylated at 100% of its N-glycosylation sites. However, one of skill in the art will appreciate that not every N-glycosylation site of HuGlylDS need be N-glycosylated in order for benefits of glycosylation to be attained. Rather, benefits of glycosylation can be realized when only a percentage of N-glycosylation sites are glycosylated, and / or when only a percentage of expressed IDS molecules are glycosylated. Accordingly, in certain embodiments, HuGlylDS used in accordance with the methods described herein, when expressed in a neuronal or glial cell, in vivo or in vitro, is glycosylated at 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% -80%, 80% - 90%, or 90% - 100% of its available N-glycosylation sites. In certain embodiments, when expressed in a neuronal or glial cell, in vivo or in vitro, 10% - 20%, 20% - 30%, 30% -40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% of HuGlylDS molecules used in accordance with the methods described herein are glycosylated at least one of their available N-glycosylation sites.

[00116] In a specific embodiment, at least 10%, 20% 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites present in HuGlylDS used in accordance with the methods described herein are glycosylated at an Asn residue (or other relevant residue) present in an N-glycosylation site, when the HuGlylDS is expressed in a neuronal or glial cell, in vivo or in vitro. That is, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites of the resultant HuGlylDS are glycosylated.

[00117] In another specific embodiment, at least 10%, 20% 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites present in a HuGlylDS molecule used in accordance with the methods described herein are glycosylated with an identical attached glycan linked to the Asn residue (or other relevant residue) present in an N-glycosylation site, when the HuGlylDS is expressed in a neuronal or glial cell, in vivo or in vitro. That is, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites of the resultant HuGlylDS have an identical attached glycan.

[00118] Importantly, when the IDS proteins used in accordance with the methods described herein are expressed in neuronal or glial cells, the need for in vitro production in prokaryotic host cells (e.g., E. coli) or eukaryotic host cells (e.g., CHO cells) is circumvented. Instead, as a result of the methods described herein (e.g., use of neuronal or glial cells to express IDS), N-glycosylation sites of the IDS proteins are advantageously decorated with glycans relevant to and beneficial to treatment of humans, and, in particular, at the target location of treatment. Such an advantage is unattainable when CHO cells or E. coli are utilized in protein production, because e.g., CHO cells (1) do not express 2,6 sialyltransferase and thus cannot add 2,6 sialic acid during N-glycosylation and (2) can add Neu5Gc as sialic acid instead of Neu5Ac; and because E. coli does not naturally contain components needed for N-glycosylation. Furthermore, such an advantage may be unattainable when human cells that are not neuronal or glial cells are utilized in protein production. Accordingly, in one embodiment, an IDS protein expressed in a neuronal or glial cell to give rise to a HuGlylDS used in the methods of treatment described herein is glycosylated in the manner in which a protein is N-glycosylated in human neuronal or glial cells, but is not glycosylated in the manner in which proteins are glycosylated in CHO cells. In another embodiment, an IDS protein expressed in a neuronal or glial cell to give rise to a HuGlylDS used in the methods of treatment described herein is glycosylated in the manner in which a protein is N-glycosylated in a neuronal or glial cells, wherein such glycosylation is not naturally possible using a prokaryotic host cell, e.g., using E. coli. In one embodiment, an IDS protein expressed in a human neuronal or glial cell to give rise to a HuGlylDS used in the methods of treatment described herein is glycosylated in the manner in which a protein is N-glycosylated in human neuronal or glial cells, but is not glycosylated in the manner in which proteins are glycosylated in human cells which are not neuronal or glial cells.

[00119] Assays for determining the glycosylation pattern of proteins are known in the art. For example, hydrazinolysis can be used to analyze glycans. First, polysaccharides are released from their associated protein by incubation with hydrazine (the Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK can be used). The nucleophile hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein and allows release of the attached glycans. N-acetyl groups are lost during this treatment and have to be reconstituted by re-N-acetylation. The free glycans can be purified on carbon columns and subsequently labeled at the reducing end with the fluorophor 2-amino benzamide. The labeled polysaccharides can be separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al, Anal Biochem 2002, 304(1):70-90. The resulting fluorescence chromatogram indicates the polysaccharide length and number of repeating units. Structural information can be gathered by collecting individual peaks and subsequently performing MS / MS analysis. Thereby the monosaccharide composition and sequence of the repeating unit can be confirmed and additionally in homogeneity of the polysaccharide composition can be identified. Specific peaks of low molecular weight can be analyzed by MALDLMS / MS and the result used to confirm the glycan sequence. Each peak corresponds to a polymer consisting of a certain number of repeat units and fragments thereof. The chromatogram thus allows measurement of the polymer length distribution. The elution time is an indication for polymer length, while fluorescence intensity correlates with molar abundance for the respective polymer.

[00120] Homogeneity of the glycan patterns associated with proteins, as it relates to both glycan length and numbers glycans present across glycosylation sites, can be assessed using methods known in the art, e.g., methods that measure glycan length and hydrodynamic radius. Size exclusion-HPLC allows the measurement of the hydrodynamic radius. Higher numbers of glycosylation sites in a protein lead to higher variation in hydrodynamic radius compared to a carrier with less glycosylation sites. However, when single glycan chains are analyzed, they may be more homogenous due to the more controlled length. Glycan length can measured by hydrazinolysis, SDS PAGE, and capillary gel electrophoresis. In addition, homogeneity can also mean that certain glycosylation site usage patterns change to a broader / narrower range. These factors can be measured by Glycopeptide LC-MS / MS.

[00121] N-glycosylation confers numerous benefits on the HuGlylDS used in the methods described herein. Such benefits are unattainable by production of proteins in E. coli, because E. coll does not naturally possess components needed for N-glycosylation. Further, some benefits are unattainable through protein production in, e.g., CHO cells, because CHO cells lack components needed for addition of certain glycans (e.g., 2,6 sialic acid) and because CHO cells can add glycans, e.g., Neu5Gc not typical to humans, and the a-Gal antigen which is immunogenic in most individuals and at high concentrations can trigger anaphylaxis. Even further, some benefits are unattainable through protein production in human cells that are not neuronal or glial cells. Thus, the expression of IDS in human neuronal or glial cells results in the production of HuGlylDS comprising beneficial glycans that otherwise would not be associated with the protein if produced in CHO cells, in E. coli, or in human cells which are not neuronal or glial cells. 5.1.3. Tyrosine Sulfation

[00122] In addition to the N-linked glycosylation sites, hIDS contains a tyrosine (“Y”) sulfation site (PSSEKY165ENTKTCRGPD). (See, e.g., Yang et al., 2015, Molecules 20:21382164, esp. at p. 2154 which is incorporated by reference in its entirety for the analysis of amino acids surrounding tyrosine residues subjected to protein tyrosine sulfation. The “rules” can be summarized as follows: Y residues with E or D within +5 to -5 position of Y, and where position -1 of Y is a neutral or acidic charged amino acid - but not a basic amino acid, e.g., R, K, or H that abolishes sulfation).

[00123] Importantly, tyrosine-sulfated proteins cannot be produced in E. coll, which naturally does not possess the enzymes required for tyrosine-sulfation. Further, CHO cells are deficient for tyrosine sulfation-they are not secretory cells and have a limited capacity for post-translational tyrosine-sulfation. See, e.g., Mikkelsen & Ezban, 1991, Biochemistry 30: 1533- 1537. Advantageously, the methods provided herein call for expression of IDS, e.g., HuGlylDS, in neurons or glial cells, which are secretory and do have capacity for tyrosine sulfation. Assays for detection tyrosine sulfation are known in the art. See, e.g., Yang et al., 2015, Molecules 20:2138-2164.

[00124] Tyrosine-sulfation of hIDS - a robust post-translational process in human CNS cells -should result in improved processing and activity of transgene products. The significance of tyrosine-sulfation of lysosomal proteins has not been elucidated; but in other proteins it has been shown to increase avidity of protein-protein interactions (antibodies and receptors), and to promote proteolytic processing (peptide hormone). (See, Moore, 2003, J Biol. Chern. 278:2424346; and Bundegaard et al., 1995, The EMBO J 14: 3073-79). The tyrosylprotein sulfotransferase (TPST1) responsible for tyrosine-sulfation (which may occur as a final step in IDS processing) is apparently expressed at higher levels (based on mRNA) in the brain (gene expression data for TPST1 may be found, for example, at the EMBL-EBI Expression Atlas, accessible at http: / / www.ebi.ac.uk / gxa / home). 5.2 CONSTRUCTS AND FORMULATIONS

[00125] For use in the methods provided herein are viral vectors or other DNA expression constructs encoding iduronate-2-sulfatase (IDS), e.g., human IDS (hIDS). For use in the methods provided herein are viral vectors or other DNA expression constructs encoding glycosylated (HuGly) a-L-iduronidase (IDUA), e.g., human IDUA (hIDUA). The viral vectors and other DNA expression constructs provided herein include any suitable method for delivery of a transgene to the cerebrospinal fluid (CSF). The means of delivery of a transgene include viral vectors, liposomes, other lipid-containing complexes, other macromolecular complexes, synthetic modified mRNA, unmodified mRNA, small molecules, non-biologically active molecules (e.g., gold particles), polymerized molecules (e.g., dendrimers), naked DNA, plasmids, phages, transposons, cosmids, or episomes. In some embodiments, the vector is a targeted vector, e.g., a vector targeted to neuronal cells.

[00126] In some aspects, the disclosure provides for a nucleic acid for use, wherein the nucleic acid encodes an IDS, e.g., hIDS, operatively linked to a promoter selected from the group consisting of: cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65 promoter and opsin promoter.

[00127] In certain embodiments, provided herein are recombinant vectors that comprise one or more nucleic acids (e.g. polynucleotides). The nucleic acids may comprise DNA, RNA, or a combination of DNA and RNA. In certain embodiments, the DNA comprises one or more of the sequences selected from the group consisting of promoter sequences, the sequence of the gene of interest (the transgene, e.g., IDS), untranslated regions, and termination sequences. In certain embodiments, viral vectors provided herein comprise a promoter operably linked to the gene of interest.

[00128] In certain embodiments, nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59:149-161).

[00129] In another aspect, the disclosure provides for a formulation comprising a recombinant nucleotide expression vector encoding human IDS, wherein the formulation is suitable for administration to the cerebrospinal fluid of human brain, so that a depot is formed in the human central nervous system that secretes a recombinant human IDS glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains a formylglycine, is a2,6-sialylated, does not contain detectable NeuGc, does not contain a-Gal antigen, and / or is mannose-6-phosphorylated. For example, the formulation may contain buffer (such as, a buffer having a particular pH, or a buffer containing a particular ingredient) that makes it suitable for administration to the cerebrospinal fluid of human brain, so that a depot is formed in the human central nervous system that secretes a recombinant human IDS glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains a formylglycine, is a2,6-sialylated, does not contain detectable NeuGc, does not contain a-Gal antigen, and / or is mannose-6-phosphorylated. In a specific embodiment, the buffer comprises a physiologically compatible aqueous buffer, a surfactant and optional excipients.

[00130] In another aspect, the disclosure provides for a kit comprising a recombinant nucleotide expression vector encoding human IDS and a pharmaceutically acceptable carrier, wherein the recombinant nucleotide expression vector is suitable for administration to the cerebrospinal fluid (CSF) of human brain, so that a depot is formed in the human central nervous system that secretes a recombinant human IDS glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains a formylglycine, is a2,6-sialylated, does not contain detectable NeuGc, does not contain detectable a-Gal antigen, and / or is mannose-6-phosphorylated. In another aspect, the disclosure provides for a kit comprising a formulation comprising a recombinant nucleotide expression vector encoding human IDS, wherein the formulation is suitable for administration to the CSF of human brain, so that a depot is formed in the human central nervous system that secretes a recombinant human IDS glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains a formylglycine, is a2,6-sialylated, does not contain detectable NeuGc, does not contain detectable a-Gal antigen, and / or is mannose-6-phosphorylated. A kit described herein comprises the recombinant nucleotide expression vector or the formulation in one or more containers. Optionally associated with such one or more containers can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[00131] The formulations and kits encompassed herein can be used in accordance with the methods for treating a human patient as provided in this disclosure. 5.2.1. mRNA

[00132] In certain embodiments, the vectors provided herein are modified mRNA encoding for the gene of interest (e.g., the transgene, for example, IDS). The synthesis of modified and unmodified mRNA for delivery of a transgene to the CSF is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is incorporated by reference herein in its entirety. In certain embodiments, provided herein is a modified mRNA encoding for IDS, e.g, hIDS. 5.2.2. Viral vectors

[00133] Viral vectors include adenovirus, adeno-associated virus (AAV, e.g, AAV9, AAVrhlO), lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus, hemagglutinin virus of Japan (HVJ), alphavirus, vaccinia virus, and retrovirus vectors. Retroviral vectors include murine leukemia virus (MLV)- and human immunodeficiency virus (HlV)-based vectors. Alphavirus vectors include semliki forest virus (SFV) and sindbis virus (SIN). In certain embodiments, the viral vectors provided herein are recombinant viral vectors. In certain embodiments, the viral vectors provided herein are altered such that they are replication-deficient in humans. In certain embodiments, the viral vectors are hybrid vectors, e.g., an AAV vector placed into a “helpless” adenoviral vector. In certain embodiments, provided herein are viral vectors comprising a viral capsid from a first virus and viral envelope proteins from a second virus. In specific embodiments, the second virus is vesicular stomatitus virus (VSV). In more specific embodiments, the envelope protein is VSV-G protein.

[00134] In certain embodiments, the viral vectors provided herein are HIV based viral vectors. In certain embodiments, HIV-based vectors provided herein comprise at least two polynucleotides, wherein the gag and pol genes are from an HIV genome and the env gene is from another virus.

[00135] In certain embodiments, the viral vectors provided herein are herpes simplex virusbased viral vectors. In certain embodiments, herpes simplex virus-based vectors provided herein are modified such that they do not comprise one or more immediately early (IE) genes, rendering them non-cytotoxic.

[00136] In certain embodiments, the viral vectors provided herein are MLV based viral vectors. In certain embodiments, MLV-based vectors provided herein comprise up to 8 kb of heterologous DNA in place of the viral genes.

[00137] In certain embodiments, the viral vectors provided herein are lentivirus-based viral vectors. In certain embodiments, lentiviral vectors provided herein are derived from human lentiviruses. In certain embodiments, lentiviral vectors provided herein are derived from nonhuman lentiviruses. In certain embodiments, lentiviral vectors provided herein are packaged into a lentiviral capsid. In certain embodiments, lentiviral vectors provided herein comprise one or more of the following elements: long terminal repeats, a primer binding site, a polypurine tract, att sites, and an encapsidation site.

[00138] In certain embodiments, the viral vectors provided herein are alphavirus-based viral vectors. In certain embodiments, alphavirus vectors provided herein are recombinant, replication-defective alphaviruses. In certain embodiments, alphavirus replicons in the alphavirus vectors provided herein are targeted to specific cell types by displaying a functional heterologous ligand on their virion surface.

[00139] In certain embodiments, the viral vectors provided herein are AAV based viral vectors. In preferred embodiments, the viral vectors provided herein are AAV9 or AAVrhlO based viral vectors. In certain embodiments, the AAV9 or AAVrhlO based viral vectors provided herein retain tropism for CNS cells. Multiple AAV serotypes have been identified. In certain embodiments, AAV-based vectors provided herein comprise components from one or more serotypes of AAV. In certain embodiments, AAV based vectors provided herein comprise components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAV10 or AAV11. In preferred embodiments, AAV based vectors provided herein comprise components from one or more of AAV8, AAV9, AAVrhlO, AAV10, or AAV11 serotypes. AAV9-based viral vectors are used in the methods described herein. Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety. In one aspect, provided herein are AAV (e.g., AAV9 or AAVrhl0)-based viral vectors encoding a transgene (e.g., IDS). In specific embodiments, provided herein are AAV9-based viral vectors encoding IDS. In more specific embodiments, provided herein are AAV9-based viral vectors encoding hIDS.

[00140] Provided in particular embodiments are AAV9 vectors comprising an artificial genome comprising (i) an expression cassette containing the transgene under the control of regulatory elements and flanked by ITRs; and (ii) a viral capsid that has the amino acid sequence of the AAV9 capsid protein or is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAV9 capsid protein (SEQ ID NO: 26) while retaining the biological function of the AAV9 capsid. In certain embodiments, the encoded AAV9 capsid has the sequence of SEQ ID NO: 26 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions and retaining the biological function of the AAV9 capsid. FIG. 6 provides a comparative alignment of the amino acid sequences of the capsid proteins of different AAV serotypes with potential amino acids that may be substituted at certain positions in the aligned sequences based upon the comparison in the row labeled SUBS. Accordingly, in specific embodiments, the AAV9 vector comprises an AAV9 capsid variant that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions identified in the SUBS row of FIG. 6 that are not present at that position in the native AAV9 sequence.

[00141] In certain embodiments, the AAV that is used in the methods described herein is Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety. In certain embodiments, the AAV that is used in the methods described herein comprises one of the following amino acid insertions: LGETTRP or LALGETTRP, as described in United States Patent Nos. 9,193,956; 9458517; and 9,587,282 and US patent application publication no. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV that is used in the methods described herein is AAV.7m8, as described in United States Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and US patent application publication no. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV that is used in the methods described herein is any AAV disclosed in United States Patent No. 9,585,971, such as AAV-PHP.B. In certain embodiments, the AAV that is used in the methods described herein is an AAV disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282 US patent application publication nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335.

[00142] In certain embodiments, a single-stranded AAV (ssAAV) may be used supra. In certain embodiments, a self-complementary vector, e.g., scAAV, may be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82, McCarty et al, 2001, Gene Therapy, Vol 8, Number 16, Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).

[00143] In certain embodiments, the viral vectors used in the methods described herein are adenovirus based viral vectors. A recombinant adenovirus vector may be used to transfer in the IDS. The recombinant adenovirus can be a first generation vector, with an El deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region. The recombinant adenovirus can be a second generation vector, which contains full or partial deletions of the E2 and E4 regions. A helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi). The transgene is inserted between the packaging signal and the 3’ITR, with or without stuff er sequences to keep the artificial genome close to wild-type size of approx. 36 kb. An exemplary protocol for production of adenoviral vectors may be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,” Gene Therapy 12:S18-S27, which is incorporated by reference herein in its entirety.

[00144] In certain embodiments, the viral vectors used in the methods described herein are lentivirus based viral vectors. A recombinant lentivirus vector may be used to transfer in the IDS. Four plasmids are used to make the construct: Gag / pol sequence containing plasmid, Rev sequence containing plasmids, Envelope protein containing plasmid (i.e. VSV-G), and Cis plasmid with the packaging elements and the IDS gene.

[00145] For lentiviral vector production, the four plasmids are co-transfected into cells (i.e., HEK293 based cells), whereby polyethylenimine or calcium phosphate can be used as transfection agents, among others. The lentivirus is then harvested in the supernatant (lentiviruses need to bud from the cells to be active, so no cell harvest needs / should be done). The supernatant is filtered (0.45 pm) and then magnesium chloride and benzonase added. Further downstream processes can vary widely, with using TFF and column chromatography being the most GMP compatible ones. Others use ultracentrifugation with / without column chromatography. Exemplary protocols for production of lentiviral vectors may be found in Lesch et al., 2011, “Production and purification of lentiviral vector generated in 293T suspension cells with baculoviral vectors,” Gene Therapy 18:531-538, and Ausubel et al., 2012, “Production of CGMP-Grade Lentiviral Vectors,” Bioprocess Int. 10(2):32-43, both of which are incorporated by reference herein in their entireties.

[00146] In a specific embodiment, a vector for use in the methods described herein is one that encodes an IDS (e.g., hIDS) such that, upon transduction of cells in the CNS, or a relevant cell (e.g., a neuronal cell in vivo or in vitro), a glycosylated variant of IDS is expressed by the transduced cell. In a specific embodiment, a vector for use in the methods described herein is one that encodes an IDS (e.g., hIDS) such that, upon transduction of a cell in the CNS, or a relevant cell (e.g., a neuronal cell in vivo or in vitro), a sulfated variant of IDS is expressed by the cell. 5.2.3. Promoters and Modifiers of Gene Expression

[00147] In certain embodiments, the vectors provided herein comprise components that modulate gene delivery or gene expression (e.g, “expression control elements”). In certain embodiments, the vectors provided herein comprise components that modulate gene expression. In certain embodiments, the vectors provided herein comprise components that influence binding or targeting to cells. In certain embodiments, the vectors provided herein comprise components that influence the localization of the polynucleotide (e.g., the transgene) within the cell after uptake. In certain embodiments, the vectors provided herein comprise components that can be used as detectable or selectable markers, e.g, to detect or select for cells that have taken up the polynucleotide.

[00148] In certain embodiments, the viral vectors provided herein comprise one or more promoters. In certain embodiments, the promoter is a constitutive promoter. In alternate embodiments, the promoter is an inducible promoter. The native IDS gene, like most housekeeping genes, primarily uses a GC-rich promoter. In a preferred embodiment, strong constitutive promoters that provide for sustained expression of hIDS are used. Such promoters include “CAG” synthetic promoters that contain: “C” - the cytomegalovirus (CMV) early enhancer element; “A” - the promoter as well as the first exon and intron of the chicken betaactin gene; and “G” - the splice acceptor of the rabbit beta-globin gene (see, Miyazaki et al., 1989, Gene 79: 269-277; and Niwa et al., Gene 108: 193-199).

[00149] In certain embodiments, the promoter is a CB7 promoter (see Dinculescu et al., 2005, Hum Gene Ther 16: 649-663, incorporated by reference herein in its entirety). In some embodiments, the CB7 promoter includes other expression control elements that enhance expression of the transgene driven by the vector. In certain embodiments, the other expression control elements include chicken P-actin intron and / or rabbit P-globin polA signal. In certain embodiments, the promoter comprises a TATA box. In certain embodiments, the promoter comprises one or more elements. In certain embodiments, the one or more promoter elements may be inverted or moved relative to one another. In certain embodiments, the elements of the promoter are positioned to function cooperatively. In certain embodiments, the elements of the promoter are positioned to function independently. In certain embodiments, the viral vectors provided herein comprise one or more promoters selected from the group consisting of the human CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus (RS) long terminal repeat, and rat insulin promoter. In certain embodiments, the vectors provided herein comprise one or more long terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2 LTRs. In certain embodiments, the vectors provided herein comprise one or more tissue specific promoters (e.g., a neuronal cell-specific promoter).

[00150] In certain embodiments, the viral vectors provided herein comprise one or more regulatory elements other than a promoter. In certain embodiments, the viral vectors provided herein comprise an enhancer. In certain embodiments, the viral vectors provided herein comprise a repressor. In certain embodiments, the viral vectors provided herein comprise an intron or a chimeric intron. In certain embodiments, the viral vectors provided herein comprise a polyadenylation sequence. 5.2.4. Signal Peptides

[00151] In certain embodiments, the vectors provided herein comprise components that modulate protein delivery. In certain embodiments, the viral vectors provided herein comprise one or more signal peptides. In certain embodiments, the signal peptides allow for the transgene product (e.g., IDS) to achieve the proper packaging (e.g. glycosylation) in the cell. In certain embodiments, the signal peptides allow for the transgene product (e.g., IDS) to achieve the proper localization in the cell. In certain embodiments, the signal peptides allow for the transgene product (e.g., IDS) to achieve secretion from the cell. Examples of signal peptides to be used in connection with the vectors and transgenes provided herein may be found in Table 4. Signal peptides may also be referred to herein as leader sequences or leader peptides. Table 4. Signal peptides for use with the vectors provided herein. SEQ ID NO. Signal Peptide Sequence 2 Oligodendrocyte-myelin glycoprotein (hOMG) signal peptide MEYQILKMSLCLFILLFLTPGILC 3 Cellular repressor of E1A-stimulated genes 2 (hCREG2) signal peptide MSVRRGRRPARPGTRLSWLLCCSALLSP AAG SEQ ID NO. Signal Peptide Sequence 4 V-set and transmembrane domain containing 2B (hVSTM2B) signal peptide MEQRNRLGALGYLPPLLLHALLLFVADA 5 Protocadherin alpha-1 (hPCADHAl) signal peptide M VF SRRGGLGARDLLLWLLLL AAWE VG SG 6 FAM19A1 (TAFA1) signal peptide MAMVSAMSWVLYLWISACA 7 VEGF-A signal peptide MNFLLSWVHW SLALLLYLHH AKWSQA 8 Fibulin-1 signal peptide MERAAPSRRV PLPLLLLGGL ALLAAGVDA 9 Vitronectin signal peptide MAPLRPLLIL ALLAWVALA 10 Complement Factor H signal peptide MRLLAKIICLMLWAICVA 11 Opticin signal peptide MRLLAFLSLL ALVLQETGT 12 Albumin signal peptide MKW VTFISLLFLF S S AYS 13 Chymotrypsinogen signal peptide MAFLWLLSCWALLGTTFG 14 Interleukin-2 signal peptide MYRMQLLSCIALILALVTNS 15 Trypsinogen-2 signal peptide MNLLLILTFVAAAVA 5.2.5. Untranslated regions

[00152] In certain embodiments, the viral vectors provided herein comprise one or more untranslated regions (UTRs), e.g., 3’ and / or 5’ UTRs. In certain embodiments, the UTRs are optimized for the desired level of protein expression. In certain embodiments, the UTRs are optimized for the mRNA half life of the transgene. In certain embodiments, the UTRs are optimized for the stability of the mRNA of the transgene. In certain embodiments, the UTRs are optimized for the secondary structure of the mRNA of the transgene. 5.2.6. Inverted terminal repeats

[00153] In certain embodiments, the viral vectors provided herein comprise one or more inverted terminal repeat (ITR) sequences. ITR sequences may be used for packaging the recombinant gene expression cassette into the virion of the viral vector. In certain embodiments, the ITR is from an AAV, e.g., AAV9 (see, e.g., Yan et al., 2005, J. Virol., 79(l):364-379; United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety). 5.2.7. Transgenes

[00154] In certain embodiments, the vectors provided herein encode an IDS transgene. In specific embodiments, the IDS is controlled by appropriate expression control elements for expression in neuronal cells: In certain embodiments, the IDS (e.g., hIDS) transgene comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the IDS (e.g, hIDS) transgene comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 1.

[00155] The HuGlylDS encoded by the transgene can include, but is not limited to human IDS (hIDS) having the amino acid sequence of SEQ ID NO. 1 (as shown in FIG. 1), and derivatives of hIDS having amino acid substitutions, deletions, or additions, e.g., including but not limited to amino acid substitutions selected from corresponding non-conserved residues in orthologs of IDS shown in FIG. 2, with the proviso with the proviso that such mutations do not include replacement of the cysteine residue at position 84 (C84) which is required for enzyme activity (Millat et al., 1997, Biochem J 326: 243-247); or a mutation that has been identified in severe, severe-intermediate, intermediate, or attenuated MPS II phenotypes e.g., as shown in FIG. 3, or as reported by Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-761 (reporting “attenuated” mutants R48P, A85T, W337R, and the truncated mutant Q531X; and “severe” mutants P86L, S333L, S349I, R468Q, R468L); Millat et al., 1998, BBA 1406: 214-218 (reporting “attenuated” mutants P480L and P480Q; and “severe” mutant P86L); and Bonucelli et al., 2001, BBA 1537:233-238, each of which is incorporated by reference herein in its entirety.

[00156] For example, amino acid substitutions at a particular position of hIDS can be selected from among corresponding non-conserved amino acid residues found at that position in the IDS orthologs aligned in FIG. 2, with the proviso that such substitutions do not include any of the deleterious mutations shown in FIG. 3 or as reported by Sukegawa-Hayasaka et al., 2006, supra, Millat et al., 1998, supra, or Bonucelli et al., 2001, supra, each of which is incorporated by reference herein in its entirety. The resulting transgene product can be tested using conventional assays in vitro, in cell culture or test animals to ensure that the mutation does not disrupt IDS function. Preferred amino acid substitutions, deletions or additions selected should be those that maintain or increase enzyme activity, stability or half-life of IDS, as tested by conventional assays in vitro, in cell culture or animal models for MPS II. For example, the enzyme activity of the transgene product can be assessed using a conventional enzyme assay with, for example, 4-Methylumbelliferyl a-L-idopyranosiduronic acid 2-sulfate or 4-methylumbelliferyl sulfate as the substrate (see, e.g., Lee et al., 2015, Clin. Biochem. 48(18):1350-1353, Dean et al., 2006, Clin. Chern. 52(4):643-649 for exemplary IDS enzyme assays that can be used, each of which is incorporated by reference herein in its entirety). The ability of the transgene product to correct MPS II phenotype can be assessed in cell culture; e.g., by transducing MPS II cells in culture with a viral vector or other DNA expression construct encoding hIDS or a derivative; by adding the transgene product or a derivative to MPS II cells in culture; or by co-culturing MPS II cells with human neuronal / glial host cells engineered to express and secrete rhIDS or a derivative, and determining correction of the defect in the MPS II cultured cells, e.g., by detecting IDS enzyme activity and / or reduction in GAG storage in the MPS II cells in culture (see, e.g., Stroncek et al., 1999, Transfusion 39(4):343-350, which is incorporated by reference herein in its entirety).

[00157] In some embodiments, a dose of a recombinant AAV of the disclosure is determined using a PCR assay. In some embodiments, the PCR assay is a Poly-A PCR assay. In some embodiments, the PCR assay is a transgene-specific PCR assay. In some embodiments, the dose determined in one assay is different (e.g., higher or lower) than a dose determined in another assay. For example, a dose determined using a transgene-specific PCR assay is higher (e.g., about 50% higher) than a dose determined using a Poly-A-specific PCR assay. In some embodiments, a dose determined by a transgene-specific PCR assay is higher by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or higher than about 75% higher than a dose determined by a Poly-A-specific PCR assay. In some embodiments, a dose of 2.0 x io11 GC / g brain mass, where the number of genome copies is determined using a Poly-A-specific PCR assay is equivalent to a dose of 2.9 x 1011 GC / g brain mass, where the number of genome copies is determined using a transgene-specific PCR assay. In some embodiments, the total dose administered to a subject accounts for the estimated brain mass of the subject, which can be determined using a magnetic resonance imaging (MRI) screening. 5.2.8. Constructs

[00158] In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a sequence encoding the transgene (e.g., IDS), h) a fourth linker sequence, i) a poly A sequence, j) a fifth linker sequence, and k) a second ITR sequence.

[00159] In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a promoter sequence, and b) a sequence encoding the transgene (e.g., IDS). In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a promoter sequence, and b) a sequence encoding the transgene (e.g., IDS), wherein the transgene comprises a signal peptide.

[00160] In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding the transgene (e.g., IDS), i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, 1) a fifth linker sequence, and m) a second ITR sequence.

[00161] In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding the transgene (e.g., IDS), i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, 1) a fifth linker sequence, and m) a second ITR sequence, wherein the transgene comprises a signal peptide, and wherein the transgene encodes hIDS.

[00162] In a specific embodiment, the viral vector described herein comprises the elements and in the order as illustrated in FIG. 5. 5.2.9. Manufacture and testing of vectors

[00163] The viral vectors provided herein may be manufactured using host cells. The viral vectors provided herein may be manufactured using mammalian host cells, for example, A549 , WEHI, 10T1 / 2, BHK, MDCK, C0S1, C0S7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells. The viral vectors provided herein may be manufactured using host cells from human, monkey, mouse, rat, rabbit, or hamster.

[00164] The host cells are stably transformed with the sequences encoding the transgene and associated elements (i.e., the vector genome), and the means of producing viruses in the host cells, for example, the replication and capsid genes (e.g., the rep and cap genes of AAV). For a method of producing recombinant AAV vectors with AAV8 capsids, see Section IV of the Detailed Description of U.S. Patent No. 7,282,199 B2, which is incorporated herein by reference in its entirety. Genome copy titers of said vectors may be determined, for example, by TAQMAN® analysis. Virions may be recovered, for example, by CsCh sedimentation.

[00165] In vitro assays, e.g., cell culture assays, can be used to measure transgene expression from a vector described herein, thus indicating, e.g., potency of the vector. For example, the HT-22, SK-N-MC, HCN-1 A, HCN-2, NT2, SH-SY5y, hNSCl 1, or ReNcell VM cell lines, or other cell lines that are derived from neuronal or glial cells or progenitors of neuronal or glial cells can be used to assess transgene expression. Once expressed, characteristics of the expressed product (i.e., HuGlylDS) can be determined, including determination of the glycosylation and tyrosine sulfation patterns associated with the HuGlylDS. 5.2.10. Compositions

[00166] Compositions are described comprising a vector encoding a transgene described herein and a suitable carrier. A suitable carrier (e.g., for administration to the CSF, and, for example, to neuronal cells) would be readily selected by one of skill in the art. 5.3 GENE THERAPY

[00167] Methods are described for the administration of a therapeutically effective amount of a transgene construct to human subjects having MPS II. More particularly, methods for administration of a therapeutically effective amount of a transgene construct to patients having MPS II, in particular, for administration to the CSF are described. In particular embodiments, such methods for administration to the CSF of a therapeutically effective amount of a transgene construct can be used to treat to patients having Hunter’s syndrome. 5.3.1. Target Patient Populations

[00168] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients diagnosed with MPS II. In specific embodiments, the patients have been diagnosed with mild MPS II. In specific embodiments, the patients have been diagnosed with severe MPS II. In specific embodiments, the patients have been diagnosed with Hunter’s syndrome. In specific embodiments, the patients have been diagnosed with neuronopathic MPS II. In some embodiments, a patient has been diagnosed with hepatosplenomegaly, has a symptom associated with hepatosplenomegaly, is suspected of having hepatosplenomegaly, and / or has a predisposition to suffer from hepatosplenomegaly. Examples of symptoms associated with hepatosplenomegaly include, but are not limited to, brown urine, clay-colored bowel movements, enlarged or swollen abdomen, fever, itching, jaundice or yellowing of the eyes and skin, nausea, pain (e.g., I the upper right portion of the stomach), fatigue, and / or vomiting. In some embodiments, a patient diagnosed with MPS II has hepatosplenomegaly. In some embodiments, a patient is suffering from hepatosplenomegaly associated with MPS II. In some embodiments, a patient is being treated or has been treated with ERT.

[00169] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients diagnosed with MPS II who have been identified as responsive to treatment with IDS, e.g., hIDS.

[00170] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to pediatric patients. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are less than three years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are aged 2 to 4 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 4 months old or older and less than 5 years old. In a specific embodiment, therapeutically effective doses of the recombinant vector are administered to patients that have severe MPS II and are 4 months old or older and less than 5 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 5 years old or older and less than 18 years old. In a specific embodiment, therapeutically effective doses of the recombinant vector are administered to patients that have neuronopathic MPS II and are 5 years old or older and less than 18 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 18 months old or older and 8 years old or younger. In a specific embodiment, therapeutically effective doses of the recombinant vector are administered to patients that are pediatric male patients and are 18 months old or older and 8 years old or younger. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are aged 3 to 8 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are aged 8 to 16 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are aged 5 to 18 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 10 years old or younger. In a specific embodiment, therapeutically effective doses of the recombinant vector are administered to patients that have severe MPS II and are 10 years old or younger. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 18 years old or younger. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are more than 5 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are more than 10 years old.

[00171] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 4, 5, 6, 7, 8, 9, 10, or 11 months old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are about 4, 5, 6, 7, 8, 9, 10, or 11 months old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are about 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 1011, or 11-12 months old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 1, 2, 3, 4, or 5 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are about 1, 2, 3, 4, or 5 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 1-2, 2-3, 3-4, 4-5, or 5-6 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are about 1-2, 2-3, 3-4, 4-5, or 5-6 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, or 18-19 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are about 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, or 18-19 years old.

[00172] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to adolescent patients. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to adult patients. In some embodiments, therapeutically effective doses of the recombinant vector are administered to male patients. In other embodiments, therapeutically effective doses of the recombinant vector are administered to female patients.

[00173] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients diagnosed with MPS II who have been identified as responsive to treatment with IDS, e.g., hIDS, injected into the CSF prior to treatment with gene therapy. 5.3.2. Dosage and Mode of Administration

[00174] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to the CSF via intrathecal administration (i.e., injection into the subarachnoid space so that the recombinant vectors distribute through the CSF and transduce cells in the CNS). This can be accomplished in a number of ways - e.g., by intracranial (cisternal or ventricular) injection , or injection into the lumbar cistern. In certain embodiments, intrathecal administration is performed via intraci sternal (IC) injection (e.g., into the cisterna magna). In specific embodiments, intraci sternal injection is performed by CT-guided suboccipital puncture. In specific embodiments, intrathecal injection is performed by lumbar puncture. In specific embodiments, injection into the subarachnoid space is performed by Cl-2 puncture if feasible for the patient. Alternatively, intracerebroventricular (ICV) administration (a more invasive technique used for the introduction of antiinfective or anticancer drugs that do not penetrate the blood-brain barrier), for example, image-assisted ICV injection, can be used to instill the recombinant vectors directly into the ventricles of the brain. In a specific embodiment, the recombinant vector is administered via a single image-assisted ICV injection. In a further specific embodiment, the recombinant vector is administered via a single image-assisted ICV injection with immediate removal of the administration catheter. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to the CNS via intranasal administration. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to the CNS via intraparenchymal injection. In certain embodiments, intraparenchymal injection is targeted to the striatum. In certain embodiments, intraparenchymal injection is targeted to the white matter. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to the CSF by any means known to the art, for example, by any means disclosed in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is hereby incorporated by reference in its entirety.

[00175] In preferred embodiments, for intrathecal administration (including IC and ICV administration), therapeutically effective doses of the recombinant vector are administered to the CSF in an injection volume that does not exceed 10% of the total CSF volume, which total CSF volume is about 50 mL in infants and about 150 mL in adults. A carrier suitable for intrathecal injection, such as Elliott’s B Solution or a modified Elliott’s B Solution, should be used as a vehicle for the recombinant vectors. Elliott’s B Solution (generic name: sodium chloride, sodium bicarbonate, anhydrous dextrose, magnesium sulfate, potassium chloride, calcium chloride and sodium phosphate) is a sterile, nonpyrogenic, isotonic solution containing no bacteriostatic preservatives and is used as a diluent for intrathecal administration of chemotherapeutics. The modified Elliott’s B solution includes 8.77 g / L sodium chloride, 0.244 g / L magnesium chloride, 0.0278 g / L sodium phosphate monobasic monohydrate, 0.114 g / L sodium phosphate dibasic anhydrous, 0.224 g / L potassium chloride, 0.206 g / L calcium chloride, 0.793 g / L dextrose, 0.001% poloxamer 188, pH 7.26. In some embodiments, the AAV or a composition comprising the AAV of the present disclosure is provided in a modified Elliott’s B solution for intrathecal administration.

[00176] In one embodiment, a non-replicating recombinant AAV9 vector expressing human iduronate-2-sulfatase (IDS) is used for treatment. In certain embodiments, the IDS expression cassette is flanked by inverted terminal repeats (ITRs) and expression is driven by a hybrid of the cytomegalovirus (CMV) enhancer and the chicken beta actin promoter (CB7). In certain embodiments, the transgene includes the chicken beta actin intron and a rabbit beta-globin polyadenylation (polyA) signal.

[00177] In certain embodiments, the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass. In preferred embodiments, the brain mass is determined by brain magnetic resonance imaging (MRI) of the human subject’s brain. In certain embodiments, the human subject’s brain mass is converted from the human subject’s brain volume by multiplying the human subject’s brain volume in cm3 by a factor of 1.046 g / cm3, wherein the human subject’s brain volume is obtained from the human subject’s brain MRI. In some embodiments, a dose is the number of genome copies per weight of brain mass. In some embodiments, the number of genome copies in a dose (e.g., the dose of the recombinant nucleotide expression vector) is determined by a Poly-A-specific PCR assay. In some embodiments, the number genome copies in a dose (e.g., the dose of the recombinant nucleotide expression vector) is determined by a transgene-specific PCR assay. In some embodiments, the weight of brain mass is determined by MRI.

[00178] In certain embodiments, the rAAV9.hIDS is administered IC (by suboccipital injection) as a single flat dose ranging from 1.4 x 1013 GC (1.1 x 1010 GC / g brain mass) to 7.0 x 1013 GC (5.6 x 1010 GC / g brain mass) in a volume of about 5 to 20 ml. In the event the patient has neutralizing antibodies to AAV, doses at the high range may be used. In some embodiments, a single dose of an rAAV encoding hIDS is administered to the subject in the central nervous system (e.g., in the cerebrospinal fluid) and, surprisingly, treatment effects are observed outside of the CNS. For example, changes in organ sizes are observed outside of the CNS (e.g., spleen or liver) after the rAAV of the disclosure is administered to a subject in the CNS. In some embodiments, changes in biomarker levels (e.g., D2S6, HS, total GAG, and / or anti-IDS antibody) are detected outside of the CNS after administration of the rAAV of the disclosure in the cerebrospinal fluid (e.g., changes in biomarker levels detected in the liver, spleen, urine, plasma, or blood). In some embodiments, no additional therapy for MPS II is administered to the subject outside the CNS.

[00179] In certain embodiments, the recombinant vector described herein may be administered intrathecally as a single flat dose ranging from about 1.3 x io10 GC / g brain mass to about 6.5 x 1010 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 1.3 x io10 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 1.9 x io10 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 6.5 x 1010 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 9.6 x io10 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 2.0 x io11 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 2.9 x io11 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at Dose 1 or Dose 2 as listed in and according to Table 5 below (for example, when the human patient is 4 months old or older and less than 5 years old).

[00180] In certain embodiments, the recombinant vector described herein may be administered intrathecally as a single flat dose ranging from about 1.3 x io10 GC / g brain mass to about 2.0 x 1011 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In certain embodiments, the recombinant vector described herein may be administered intrathecally as a single flat dose ranging from about 1.3 x io10 GC / g brain mass to about 2.9 x 1011 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 2.0 x io11 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 2.9 x io11 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at Dose 3 as listed in and according to Table 6 below (for example, when the human patient is 4 months old or older and less than 5 years old).

[00181] In certain embodiments, the recombinant vector described herein may be administered by IC administration as a single flat dose ranging from about 1.3 x 1010 GC / g brain mass to about 6.5 x 1010 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 1.3 x 1010 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 1.9 * 1010 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 6.5 * 1010 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 9.6 x 1010 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at Dose 1 or Dose 2 as listed in and according to Table 5 below (for example, when the human patient is 4 months old or older and less than 5 years old).

[00182] In certain embodiments, the recombinant vector described herein may be administered by IC administration as a single flat dose ranging from about 1.3 * 1010 GC / g brain mass to about 2.0 x 1011 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In certain embodiments, the recombinant vector described herein may be administered by IC administration as a single flat dose ranging from about 1.3 x 1010 GC / g brain mass to about 2.9 x 1011 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 2.0 x 1011 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 2.9 x io11 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at Dose 3 as listed in and according to Table 6 below (for example, when the human patient is 4 months old or older and less than 5 years old).

[00183] In certain embodiments, the recombinant vector described herein may be administered by ICV administration as a single flat dose ranging from about 1.3 x 1010 GC / g brain mass to about 6.5 x 1010 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 1.3 x 1010 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 1.9 x io10 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 6.5 x io10 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 9.6 x io10 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at Dose 1 or Dose 2 as listed in and according to Table 5 below (for example, when the human patient is 4 months old or older and less than 5 years old).

[00184] In certain embodiments, the recombinant vector described herein may be administered by ICV administration as a single flat dose ranging from about 1.3 x 1010 GC / g brain mass to about 2.0 x 1011 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In certain embodiments, the recombinant vector described herein may be administered by ICV administration as a single flat dose ranging from about 1.3 x io10 GC / g brain mass to about 2.9 x io11 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 2.0 x io11 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 2.9 x io11 GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at Dose 3 as listed in and according to Table 6 below (for example, when the human patient is 4 months old or older and less than 5 years old). Table 5. Total Dose Administered by Brain Mass (Dose 1 or Dose 2) Brain Mass (in g) Target Dose Levels Min Max Dose 1 Total GC* (1.3 x io10 GC / g brain mass) Dose 2 Total GC* (6.5 x 1010 GC / g brain mass) - 700 650 8.5 x 1012 4.2 x 1013 701 800 750 9.8 x 1012 4.9 x 1013 801 900 850 1.1 x 1013 5.5 x 1013 901 1050 975 1.3 x 1013 6.3 x 1013 1051 1200 1125 1.5x 1013 7.3 x 1013 1201 - 1300 1.7 x 1013 8.5 x 1013 *GC determined based on a Poly-A-specific PCR assay Table 6. Total Dose Administered by Brain Mass (Dose 3) Brain Mass (in g) Target Dose 3 Total GC determined by Poly-A-specific PCR assay (2.0 x 1011 GC / g brain mass) Min Max - 474 450 9.0 x 1013 475 524 500 1.0 x 1014 525 574 550 1.1 x 1014 575 624 600 1.2 x 1014 625 674 650 1.3 x 1014 675 724 700 1.4 x 1014 725 774 750 1.5 x 1014 775 824 800 1.6 x 1014 825 874 850 1.7 x 1014 875 924 900 1.8 x 1014 925 974 950 1.9 x 1014 975 1024 1000 2.0 x 1014 1025 1074 1050 2.1 x 1014 1075 1124 1100 2.2 x 1014 1125 1174 1150 2.3 x 1014 Brain Mass (in g) Target Dose 3 Total GC determined by Poly-A-specific PCR assay (2.0 x 1011 GC / g brain mass) Min Max 1175 1224 1200 2.4 x 1014 1225 1274 1250 2.5 x 1014 1275 >1300 1300 2.6 x 1014 Total Dose Administered by Brain Mass Dose 3 EC Brain Mass (in g) Target Dose 3 EC Total GC determined by Transgene PCR assay (2.9 x 1011 GC / g brain mass) Min Max - 474 450 1.3 x 1014 475 524 500 1.5 x 1014 525 574 550 1.6 x 1014 575 624 600 1.8 x 1014 625 674 650 1.9 x 1014 675 724 700 2.1 x 1014 725 774 750 2.2 x 1014 775 824 800 2.4 x 1014 825 874 850 2.4 x 1014 875 924 900 2.6 x 1014 925 974 950 2.8 x 1014 975 1024 1000 2.9 x 1014 1025 1074 1050 3.1 x 1014 1075 1124 1100 3.2 x 1014 1125 1174 1150 3.4 x 1014 1175 1224 1200 3.5 x 1014 1225 1274 1250 3.7 x 1014 1275 >1300 1300 3.8 x 1014

[00185] In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 6.5 x io10 GC / g brain mass (for example, when the human patient is 5 years old or older and less than 18 years old). In another specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose as listed in and according to Table 7 below (for example, when the human patient is 5 years old or older and less than 18 years old).

[00186] In a specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 6.5 x io10 GC / g brain mass (for example, when the human patient is 5 years old or older and less than 18 years old). In another specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose as listed in and according to Table 7 below (for example, when the human patient is 5 years old or older and less than 18 years old).

[00187] In a specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 6.5 x io10 GC / g brain mass (for example, when the human patient is 5 years old or older and less than 18 years old). In another specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose as listed in and according to Table 7 below (for example, when the human patient is 5 years old or older and less than 18 years old). Table 7. Total Dose Administered by Brain Mass Brain Mass (in g) Target Brain Mass (in g) Dose: Total GC determined by Poly-A-specific PCR assay (6.5 x 1010 GC / g brain mass) Min Max 801 900 850 5.5 x 1013 901 1050 975 6.3 x 1013 1051 1200 1125 7.3 x 1013 1201 - 1300 8.5 x 1013 5.4 COMBINATION THERAPIES

[00188] Combinations of administration of the HuGlylDS to the CSF accompanied by administration of other available treatments are encompassed by the methods of the invention. The additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment. Available treatments for MPS II that could be combined with the gene therapy of the invention include but are not limited to enzyme replacement therapy (ERT) using idursulfase administered systemically or to the CSF; and / or HSCT therapy. In another embodiment, ERT can be administered using the rHuGlylDS glycoprotein produced in human neuronal and glial cell lines by recombinant DNA technology. Human neuronal and glial cell lines that can be used for such recombinant glycoprotein production include but are not limited to HT-22, SK-N-MC, HCN-1 A, HCN-2, NT2, SH-SY5y, hNSCl 1, or ReNcell VM to name a few. To ensure complete glycosylation, especially sialylation, and tyrosine-sulfation, the cell line used for production can be enhanced by engineering the host cells to co-express a-2,6-sialyltransferase (or both a-2,3- and a-2,6-sialyltransferases) and / or TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation. 5.5 BIOMARKERS / SAMPLING / MONITORING EFFICACY

[00189] Efficacy may be monitored by measuring cognitive function (e.g., prevention or decrease in neurocognitive decline); reductions in biomarkers of disease (such as GAG, including heparan sulfate and dermatan sulfate) in CSF and or serum; and / or increase in IDS enzyme activity in CSF and / or serum. Signs of inflammation and other safety events may also be monitored.

[00190] In one aspect, provided herein is a method of monitoring efficacy by determining if a subject undergoing ERT treatment or a subject who has received ERT treatment can discontinue ERT treatment after administration of a gene therapy (e.g., rAAV encoding hIDS) of the disclosure. For example, provided herein is a method of treating and / or identifying a subject diagnosed with MPS II (e.g., a subject who is likely to be responsive to discontinuing treatment with ERT), comprising: (a) administering a therapeutically effective amount of a gene therapy (e.g., an rAAV encoding hIDS) of the disclosure to the subject, wherein the subject was treated with ERT or is being treated with ERT; (b) identifying the subject as being likely to be responsive to discontinuing ERT treatment, comprising: i. obtaining or having obtained a biological sample from the subject; ii. determining the level of at least one biomarker in the biological sample; and iii. identifying the subject as being likely to be responsive to discontinuing ERT treatment if the level of the biomarker in the biological sample is different (e.g., higher or lower) than a reference (e.g., reference level of the at least one biomarker); and (c) discontinuing ERT treatment in the subject. In some embodiments, a biomarker is D2S6, HS, total GAG, and / or anti-IDS antibody. In some embodiments, ERT is recombinant idursulfase. In some embodiments, the subject diagnosed with MPS II has hepatosplenomegaly.

[00191] In another aspect, provided herein is a method of selectively treating a human subject with MPS II, comprising administering to the subject a therapeutically effective amount of an rAAV encoding hIDS, wherein the subject was treated with ERT or is being treated with ERT, and wherein the subject has been determined likely to be responsive to discontinuing treatment with ERT according to a method comprising: (a) obtaining a biological sample from the subject; and (b) determining the level of at least one biomarker in the biological sample; wherein the subject is determined likely to be responsive to discontinuing treatment with ERT when the level of the at least one biomarker is different (e.g., higher or lower) than a reference. In some embodiments, a biomarker is D2S6, HS, total GAG, and / or anti-IDS antibody. In some embodiments, ERT is recombinant idursulfase. In some embodiments, the subject diagnosed with MPS II has hepatosplenomegaly.

[00192] In another aspect provided herein is a method of identifying or diagnosing a subject as having neuronopathic or non-neuronopathic MPS II or MPS I. In some embodiments, the method comprises determining the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, D2S6) in a biological sample from a subject. In some embodiments, the method comprises determining the level of undegraded glycosaminoglycans (GAGs) in a biological sample from a subject. In some embodiments, the subject is identified or diagnosed as having neuronopathic MPS II or MPS I if the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) is elevated as compared to a reference level. In some embodiments, the subject is identified or diagnosed as having neuronopathic MPS II or MPS I if the level of GAG heparan sulfate (HS) is elevated (e.g., in the brain) as compared to a reference level. In some embodiments, the total amount of heparin sulfate (t-HS) is the sum of four disaccharides (D2S6, D0A0, D0S0, D0A6) in cerebrospinal fluid (CSF) following enzymatic digestion (e.g., as determined based on a bioanalytical mass spectrometry method). In some embodiments, an elevated level of D2S6 in e.g., CSF of a subject (e.g., presymptomatic subject) is indicative of neuronopathic MPS II or MPS I. In some embodiments, the level of D2S6 is indicative of iduronate-2-sulfatase enzyme activity and can be used for therapy monitoring. In some embodiments, the one or more disaccharides comprises one or more of D0A0, D0S0, D0A6, D2S6, or a combination thereof. In another aspect provided herein is a method of identifying or diagnosing a subject as having neuronopathic or non-neuronopathic MPS II or MPS I, wherein the subject is identified or diagnosed as having neuronopathic MPS II if the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample from the subject is elevated as compared to a reference level. In some embodiments, the subject is presymptomatic or has no visible or detectable MPS II or MPS I symptom. In some embodiments, the subject has or has been diagnosed with MPS I or MPS II. In some embodiments, the reference level is the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample from one or more healthy subjects and / or from one or more non-neuronopathic subjects. In some embodiments, the reference level is the level of D2S6 in a biological sample (e.g., CSF sample) from one or more healthy subjects and / or from one or more non-neuronopathic subjects. In some embodiments, the reference level is a pre-determined level. In some embodiments, the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, D2S6) is about or at least about 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, or higher than 40% of the total heparan sulfate disaccharides (HS) in a biological sample from a subject (e.g., a subject with MPS I or MPS II). In some embodiments, the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 20% the total heparan sulfate disaccharides (HS) in a biological sample from a subject (e.g., a subject with MPS I or MPS II). In some embodiments, the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample from a subject (e.g., a subject with MPS I or MPS II) is about or at least about 5%, 10%, 115, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more than 90% higher than the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample from a reference (e.g., a healthy subject). In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, D2S6) is about or at least about 250 ng / mL, 275 ng / mL, 300 ng / mL, 325 ng / mL, 350 ng / mL, 375 ng / mL, 400 ng / mL, 425 ng / mL, 450 ng / mL, 475 ng / mL, 500 ng / mL, 525 ng / mL, 550 ng / mL, 575 ng / mL, 600 ng / mL, 625 ng / mL, 650 ng / mL, 675 ng / mL, 700 ng / mL, 725 ng / mL, 750 ng / mL, 775 ng / mL, 800 ng / mL, 825 ng / mL, 850 ng / mL, 875 ng / mL, 900 ng / mL, 925 ng / mL, 950 ng / mL, 975 ng / mL, 1000 ng / mL, or more than 1000 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 500 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 600 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 700 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 800 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is between about 500 ng / mL to about 1000 ng / mL, about 300 ng / mL to about 1000 ng / mL, about 400 ng / mL to about 1000 ng / mL, about 550 ng / mL to about 1000 ng / mL about 600 ng / mL to about 1000 ng / mL, about 700 ng / mL to about 1000 ng / mL 500 ng / mL to about 900 ng / mL, about 300 ng / mL to about 900 ng / mL, about 400 ng / mL to about 900 ng / mL, about 550 ng / mL to about 900 ng / mL about 600 ng / mL to about 900 ng / mL, or about 700 ng / mL to about 900 ng / mL. In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 50 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 270 ng / mL, 280 ng / mL, 290 ng / mL, 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, or more than 400 ng / mL. In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, or more than 200 ng / mL. In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 80 ng / mL (e.g., for D0S0 or D0A6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 100 ng / mL (e.g., for D0S0, D0A6, or D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 90 ng / mL (e.g., for D0S0, D0A6, or D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 110 ng / mL (e.g., for D0S0, D0A6, or D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 120 ng / mL (e.g., for D0S0, D0A6, or D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 160 ng / mL (e.g., for D2S6 or D0A6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 250 ng / mL (e.g., for D2S6 or D0A0). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 300 ng / mL (e.g., for D2S6 or D0A0). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 350 ng / mL (e.g., for D2S6 or D0A0). ). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 150 ng / mL (e.g., for D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 170 ng / mL (e.g., for D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 180 ng / mL (e.g., for D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 200 ng / mL (e.g., for D2S6). In some embodiments, the level of one or more heparan sulfate disaccharide(s) (e.g., D0A0, DOSO, D0A6, and / or D2S6) or the level of D2S6 in a biological sample from a subject is elevated by about or at least about 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 270 ng / mL, 280 ng / mL, 290 ng / mL, 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, 410 ng / mL, 420 ng / mL, 430 ng / mL, 440 ng / mL, 450 ng / mL, 460 ng / mL, 470 ng / mL, 480 ng / mL, 490 ng / mL, 500 ng / mL, or more than 500 ng / mL as compared to a reference level (e.g., level of one or more heparan sulfate disaccharide(s) (e.g., DOA0, DOSO, D0A6, and / or D2S6) or the level of D2S6 in a biological sample from one or more healthy subjects and / or from one or more non-neuronopathic subjects, or a pre-determined value). In some embodiments, the total level of heparan sulfate disaccharide(s) (e.g., D0A0, DOSO, D0A6, and / or D2S6) in a biological sample from a subject is elevated by about or at least about 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 270 ng / mL, 280 ng / mL, 290 ng / mL, 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, 410 ng / mL, 420 ng / mL, 430 ng / mL, 440 ng / mL, 450 ng / mL, 460 ng / mL, 470 ng / mL, 480 ng / mL, 490 ng / mL, 500 ng / mL, 510 ng / mL, 520 ng / mL, 530 ng / mL, 540 ng / mL, 550 ng / mL, 560 ng / mL, 570 ng / mL, 580 ng / mL, 590 ng / mL, 600 ng / mL, 610 ng / mL, 620 ng / mL, 630 ng / mL, 640 ng / mL, 650 ng / mL, 660 ng / mL, 670 ng / mL, 680 ng / mL, 690 ng / mL, 700 ng / mL, 710 ng / mL, 720 ng / mL, 730 ng / mL, 740 ng / mL, 750 ng / mL, 760 ng / mL, 770 ng / mL, 780 ng / mL, 790 ng / mL, 800 ng / mL, 850 ng / mL, 900 ng / mL, 950 ng / mL, 1000 ng / mL, or more than 1000 ng / mL as compared to a reference level (e.g., the total level of heparan sulfate disaccharides (e.g., D0A0, D0S0, D0A6, and D2S6) in a biological sample from one or more healthy subjects and / or from one or more non-neuronopathic subjects, or a pre-determined value).

[00193] In some embodiments, a subject is determined to be responsive to a treatment of the disclosure (e.g, rAAV9 encoding hIDUA for treating MPS I; Construct 2) based on the level of I0S6 in a biological sample from the subject. In some embodiments, an elevated level of I0S6 in a biological sample from the subject (e.g., subject with MPS I) as compared to a reference, is indicative that the subject is responsive to a treatment of the disclosure (or treatment with rAAV9 encoding hIDUA for MPS I). In some embodiments, the reference is a level of I0S6 in a biological sample from a healthy subject or a population of healthy subjects. In some embodiments, the reference is a level of I0S6 in a biological sample from a subject with MPS I or a population of subjects with MPS I. In some embodiments, the reference is a level of I0S6 in a biological sample from a subject who does not have MPS I or is not diagnosed with MPS I or a population of subjects not diagnosed with MPS I. In some embodiments, the reference is a level of IS06 in a biological sample from the same subject but taken at a different time point (e.g., obtained at an earlier time point). In some embodiments, the reference is a predetermined value. 5.5.1. Disease Markers

[00194] In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring the level of a disease biomarker in the patient. In certain embodiments, the level of the disease biomarker is measured in the CSF of the patient. In certain embodiments, the level of the disease biomarker is measured in the serum of the patient. In certain embodiments, the level of the disease biomarker is measured in the plasma of the patient. In certain embodiments, the level of the disease biomarker is measured in the urine of the patient. In certain embodiments, the disease biomarker is GAG. In some embodiments, the disease biomarker is I0S6. In preferred embodiments, the disease biomarker is heparan sulfate. In certain embodiments, the disease biomarker is D2S6.12S enzyme cleaves sulfates from HS in the lysosome and absence of I2S causes long chains of fully sulfated D2S6 to accumulate. In some embodiments, quantitative measurement of D2S6 is reflective of I2S enzyme activity level and elevated levels of HS and D2S6 correlate closely with the neuronopathic phenotype of MPS II. In some embodiments, levels of D2S6 inversely correlate with neurocognitive development. In some embodiments, the disease biomarker is an anti-AAV antibody (e.g., anti-AAV9 antibody). In certain embodiments, the disease biomarker is dermatan sulfate. In certain embodiments, the disease biomarker is IDS enzyme activity. In certain embodiments, the disease biomarker is inflammation. In certain embodiments, the disease biomarker is a safety event.

[00195] In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring one or more of the following biomarkers in a sample from the patient: (a) level of GAGs in CSF; (b) level of I2S in CSF; (c) level of GAGs in plasma; (d) level of I2S in plasma; (e) level of leukocyte I2S enzyme activity; (f) level of GAGs in urine, (g) level of heparan sulfate in CSF, and (h) level of dermatan sulfate in CSF. In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring I2S and / or GAGs in CSF, urine, and / or plasma. In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring heparan sulfate in CSF, plasma, and / or urine. In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring nonreducing heparan sulfate. In some embodiments, heparan sulfate measured in CSF is the primary endpoint for determining efficacy of treatment. In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring D2S6 in CSF. In some embodiments, D2S6 is measured using any detectable / available assay or biological sample used for detecting D2S6 (e.g., CSF, urine, and / or plasma). In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring total urine GAGs, urine HS and / or plasma I2S enzyme activity. In some embodiments, urine GAG is indicative of systemic effect and / or is independent of ERT treatment. In some embodiments, efficacy of a treatment of the disclosure is determined based on the level of I2S protein concentration in a sample from a subject (e.g., an increase in the level of I2S protein concentration is indicative of efficacy). In some embodiments, Heparan sulfate (HS) and D2S6 (glycosaminoglycans (GAGs) are measured in the cerebrospinal fluid (CSF) at baseline and / or after administration of the recombinant vector of the present disclosure. In some embodiments, determining or monitoring efficacy of MPS II treatment in a subject is determined by detecting a level of at least one biomarker (e.g., D2S6) in a biological sample from a subject obtained at about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 days, or 1, 2, 3, 4, 5, 6, 7, 8, 10, 16, 20, 24, 30, 35, 40, 45, 48, 50, 52, 56, 104 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years after the the rAAV of the present disclosure is administered to the subject (e.g., and comparing the level with a reference). In some embodiments, determining or monitoring efficacy of MPS I treatment in a subject is determined by detecting a level of at least one biomarker (e.g., I0S6) in a biological sample from a subject obtained at about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 days, or 1, 2, 3, 4, 5, 6, 7, 8, 10, 16, 20, 24, 30, 35, 40, 45, 48, 50, 52, 56, 104 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years after the the rAAV of the present disclosure is administered to the subject (e.g., and comparing the level with a reference).

[00196] In some embodiments, the level of HS and / or D2S6 is decreased in a subject after administration of a recombinant vector of the present disclosure as compared to a reference (e.g., compared to the level of HS and / or D2S6 in the subject before administration of a recombinant vector of the present disclosure, or as compared to baseline, or a predetermined value). In some embodiments, the level of HS is decreased by about or at least about 3%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or more than 100% after administration of a recombinant vector of the...

Claims

1. A method of treating mucopolysaccharidosis II (MPS II) in a human subject, themethod comprising:a) measuring the level of D2S6 in a biological sample from the human subject; andb) delivering an effective amount of an active human iduronate-2-sulfatase (hIDS) to the central nervous system (CNS) of the human subject if the level of D2S6 in the biological sample is higher than a reference level,wherein the reference level is the level of D2S6 from one or more healthy subjects or one or more non-neuronopathic MPS II subjects.

2. A method of treating mucopolysaccharidosis II (MPS II) in a human subject, themethod comprising:a) measuring the levels of D2S6 and total heparan sulfate disaccharides (HS) in a biological sample from the human subject, wherein the total HS comprises disaccharides D2S6, D0A0, D0S0, and D0A6; andb) delivering an effective amount of an active human iduronate-2-sulfatase (hIDS) to the central nervous system (CNS) of the human subject if the ratio between the levels of D2S6 and total HS is higher than a reference ratio, or the ratio between the levels of D2S6 and total HS is at least about 20%,wherein the reference ratio is the ratio between the levels of D2S6 and total HS from one or more healthy subjects or one or more non-neuronopathic MPS II subjects.

3. Use of an active human iduronate-2-sulfatase (hIDS) in the manufacture of amedicament for treating mucopolysaccharidosis II (MPS II) in a human subject, wherein the level of D2S6 has been measured in a biological sample from the human subject and the level of D2S6 in the biological sample is higher than a reference level, wherein the hIDS is formulated to be delivered to the central nervous system (CNS) of the human subject, and wherein the reference level is the level of D2S6 from one or more healthy subjects or one or more non-neuronopathic MPS II subjects.

4. Use of an active human iduronate-2-sulfatase (hIDS) in the manufacture of amedicament for treating mucopolysaccharidosis II (MPS II) in a human subject, wherein the levels of D2S6 and total heparan sulfate disaccharides (HS) have been measured in a biological sample from the human subject and the ratio between the levels of D2S6 and total2022221284   28 Aug 2026HS in the biological sample is higher than a reference ratio or the ratio between the levels of D2S6 and total HS is at least about 20%, wherein the hIDS is formulated to be delivered to the central nervous system (CNS) of the human subject, and wherein the reference ratio is the ratio between the levels of D2S6 and total HS from one or more healthy subjects or one or more non-neuronopathic MPS II subjects.

5. The method of claim 1 or 2, wherein the step b) is performed via a viral vector, aliposome, a lipid-containing complex, a macromolecular complex, a synthetic modified mRNA, an unmodified mRNA, a small molecule, a non-biologically active molecule, a polymerized molecule, a naked DNA, a plasmid, a phage, a transposon, a cosmid, or an episome; orthe use of claim 3 or 4, wherein the hIDS is in the form of or encoded by a viral vector, a liposome, a lipid-containing complex, a macromolecular complex, a synthetic modified mRNA, an unmodified mRNA, a small molecule, a non-biologically active molecule, a polymerized molecule, a naked DNA, a plasmid, a phage, a transposon, a cosmid, or an episome.

6. The method or use of any one of claims 1-5, wherein the human subject hasneuronopathic MPS II.

7. The method of any one of claims 1, 5, and 6, wherein the step b) comprises deliveringan effective amount of an active hIDS to the CNS of the human subject if the level of D2S6 in the biological sample is at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% higher than the reference level;or the use of any one of claims 3, 5, and 6, wherein the level of D2S6 in the biological sample has been determined to be at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% higher than the reference level.

8. The method of any one of claims 2, 5, and 6, wherein the step b) comprises deliveringan effective amount of an active hIDS to the CNS of the human subject if the ratio between the levels of D2S6 and total HS is at least about 5%, at least about 10%, at least about 30%, at least about 50%, at least about 70%, at least about 100%, or at least about 150% higher than the reference ratio;or the use of any one of claims 4-6, wherein the ratio between the levels of D2S6 and2022221284   28 Aug 2026total HS has been determined to be at least about 5%, at least about 10%, at least about 30%, at least about 50%, at least about 70%, at least about 100%, or at least about 150% higher than the reference ratio.

9. A method of treating mucopolysaccharidosis II (MPS II) in a human subject in needthereof, the method comprising delivering an effective amount of an active human iduronate-2-sulfatase (hIDS) to the central nervous system (CNS) of the human subject, and measuring the level of D2S6 in a biological sample from the human subject after the active hIDS has been delivered to the CNS of the human subject.

10. Use of an active human iduronate-2-sulfatase (hIDS) in the manufacture of a medicament for treating mucopolysaccharidosis II (MPS II) in a human subject in need thereof, wherein the hIDS is formulated to be delivered to the central nervous system (CNS) of the human subject and wherein the level of D2S6 is to be measured in a biological sample from the human subject after the active hIDS has been delivered to the CNS of the human subject.

11. The method of claim 9 or the use of claim 10, wherein the human subject has neuronopathic MPS II.

12. The method of claim 11, wherein the level of D2S6 is in the attenuated range after delivering the active hIDS to the human subject; or the use of claim 11, wherein the level of D2S6 has been determined to be in the attenuated range after the active hIDS has been delivered to the human subject.

13. The method of any one of claims 9, 11, and 12, wherein the method further comprises monitoring the level of D2S6 in two or more biological samples from the human subject at two or more time points after delivering the active hIDS to the human subject, wherein a decrease in the level of D2S6 in the two or more biological samples over time is indicative of the efficacy of the active hIDS; or the use of any one of claims 10 to 12, wherein the level of D2S6 is to be measured in two or more biological samples from the human subject at two or more time points after the active hIDS has been delivered to the human subject, wherein a decrease in the level of D2S6 in the two or more biological samples over time is indicative of the efficacy of the active hIDS.2022221284   28 Aug 202614. The method or use of claim 13, wherein the level of D2S6 in at least one of the two or more biological samples is or is determined to be in the attenuated range after the active hIDS is or has been delivered to the CNS of the human subject.

15. The method of claim 13 or 14, wherein the two or more biological samples have been obtained from the human subject at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 10 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at least about 48 weeks, at least about 50 weeks, at least about 52 weeks, at least about 56 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 12 months, at least about 18 months, or at least about 24 months after delivering the active hIDS to the human subject; or the use of claim 13 or 14, wherein the two or more biological samples have been obtained from the human subject at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 10 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at least about 48 weeks, at least about 50 weeks, at least about 52 weeks, at least about 56 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 12 months, at least about 18 months, or at least about 24 months after the active hIDS has been delivered to the human subject.

16. The method or use of any one of claims 1-15, wherein the biological sample is a cerebrospinal fluid (CSF) sample of the human subject.

17. A method of treating mucopolysaccharidosis II (MPS II) in a human subject in need thereof, the method comprisingdelivering an effective amount of an active human iduronate-2-sulfatase (hIDS) to the central nervous system (CNS) of the human subject,measuring the level of D2S6 in a first cerebrospinal fluid (CSF) sample from the human subject before delivering the active hIDS to the human subject, and2022221284   28 Aug 2026measuring the level of D2S6 in a second CSF sample from the human subject after delivering the active hIDS to the human subject.

18. Use of an active human iduronate-2-sulfatase (hIDS) in the manufacture of a medicament for treating mucopolysaccharidosis II (MPS II) in a human subject in need thereof, wherein the hIDS is formulated to be delivered to the central nervous system (CNS) of the human subject, wherein the level of D2S6 has been measured in a first cerebrospinal fluid (CSF) sample from the human subject before the active hIDS has been delivered to the human subject and the level of D2S6 is to be measured in a second CSF sample from the human subject after the active hIDS has been delivered to the human subject.

19. The method of claim 17 or the use of claim 18, wherein the level of D2S6 in the second CSF sample is or has been determined to be decreased at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, as compared to the level of D2S6 in the first CSF sample.

20. The method of claim 17 or 19, or the use of claim 18 or 19, wherein the second CSF sample has been obtained from the human subject at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 10 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at least about 48 weeks, at least about 50 weeks, at least about 52 weeks, at least about 56 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 12 months, at least about 18 months, or at least about 24 months after delivering the active hIDS to the human subject or after the active hIDS has been delivered to the human subject.

21. The method or use of any one of claims 1-20, wherein the active hIDS is delivered or formulated to be delivered to the CNS by administering an enzyme replacement therapy comprising a recombinant hIDS to the human subject.2022221284   28 Aug 202622. The method or use of any one of claims 1-21, wherein the active hIDS is delivered or formulated to be delivered to the CNS via intrathecal administration, intracisternal (IC) administration, intracerebroventricular (ICV) administration, or intravenous administration.

23. The method or use of any one of claims 1-20, wherein the active hIDS is delivered or formulated to be delivered to the CNS by administering an adeno-associated virus (AAV) vector encoding the hIDS to the human subject.

24. The method or use of claim 23, wherein the AAV vector is an AAV9 vector or an AAVrh10 vector.

25. The method or use of claim 23 or 24, wherein the AAV vector comprises a CB7 promoter.

26. The method or use of any one of claims 23-25, wherein the AAV vector is administered or formulated to be administered as a single administration.

27. The method or use of any one of claims 23-26, wherein the AAV comprises the nucleotide sequence of SEQ ID NO: 45.

28. The method or use of any one of claims 23-27, wherein the active hIDS is delivered or formulated to be delivered to the CNS via intracisternal (IC) administration or intracerebroventricular (ICV) administration.

29. The method of any one of claims 23-28, further comprising administering an immunosuppressive therapy to the human subject starting before or concurrently with a first administration of the AAV; or the use of any one of claims 23-28, wherein the medicament is formulated to be administered in combination with an immunosuppressive therapy to the human subject starting before or concurrently with a first administration of the AAV.

30. The method or use of any one of claims 1-29, wherein the human subject is an adult human subject.

31. The method or use of any one of claims 1-29, wherein the human subject is 18 years old or younger than 18 years old, 5 years old or older and younger than 18 years old, or 4 months old or older and younger than 5 years old.

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