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

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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 entire contents of each of which are incorporated herein by reference.

[0002] Incorporation of Electronically Submitted Sequence Listings This application incorporates by reference the sequence listing in the text file entitled "Sequence_Listing_12656-146-228.TXT," created on January 27, 2022, and having a size of 172,391 bytes, which was submitted herewith.

[0003] 1. Introduction Compositions and methods are described for delivering 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 human subjects diagnosed with mucopolysaccharidosis type II (MPS II). [Background technology]

[0004] 2. Background of the invention Hunter syndrome / MPS II is a rare, X-linked recessive disorder affecting 0.5-1.3 per 100,000 live births. This progressive and devastating disease is caused by genetic mutations in the IDS gene, which leads to a deficiency of the lysosomal storage enzyme iduronate-2-sulfatase, an enzyme required for the lysosomal catabolism of heparan sulfate and dermatan sulfate. When I2S is absent or minimally present, this protein is unable to perform its normal lysosomal exohydrolase function, leading to the accumulation of these ubiquitous polysaccharides, termed glycosaminoglycans (GAGs), in the tissues and organs of MPS II patients, resulting in characteristic storage lesions and diverse disease sequelae. Morbidity and mortality are high in this patient population, with a reported life expectancy of 11.7 years for patients with a severe phenotype (characterized by neurocognitive deterioration) and 21.7 years for patients with a mild or attenuated phenotype (Young et al., 1982, "A clinical and genetic study of Hunter's syndrome. Differences between the mild and severe forms." J. Medical Genetics 19:408-411). The majority of patients (two-thirds) have been reported to have the severe form of the 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(1):E37-E46). The disease primarily affects boys, but affected females have been reported to be due to non-random X inactivation and / or mutations 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 the disease typically appear between 18 months and 4 years of age in severe cases and between 4 and 8 years of age in mild cases. Signs and symptoms common to all affected individuals include short stature, coarsening of the facial surface, macrocephaly, macroglossia, hearing loss, hepatomegaly and splenomegaly, dysostosis multiplex, joint contractures, spinal stenosis, and carpal tunnel syndrome. Upper respiratory tract and ear infections are common in most patients, and progressive airway obstruction is common, often leading to sleep apnea and death. Cardiac disease is the leading cause of death in this population and is characterized by biventricular hypertrophy and valvular dysfunction leading to heart failure. Obstructive airway disease or heart failure is the common cause of death.

[0006] In severe forms of the disease, developmental delays are readily apparent by 18–24 months of age, although early developmental milestones may be achieved. Some patients fail hearing screening at age 1 year and are delayed in other developmental milestones, such as the ability to sit unaided, walk, and speak. Developmental progress is reported to begin to stagnate between the ages of 3 and 5 years, with regression beginning at approximately 6.5 years of age. Of the approximately 50% of children with MPS II who are toilet-trained, most, if not all, become toilet-trained as the disease progresses (Wraith et al., 2007, supra; Martin et al., 2008, supra).

[0007] Patients with significant neurological disease exhibit severe behavioral disturbances, including hyperactivity, rigidity, and aggression, beginning at age 2 and continuing until age 8-9, when neurodegeneration weakens this behavior (Muenzer, et al., 2009, Mucopolysaccharidosis I: Management and Treatment Guidelines, Pediatrics 123(1):19-29).

[0008] Seizures are reported in more than half of severely affected individuals who reach the age of 10 years, and by the time of death, most individuals with CNS disease have severe mental disabilities and require constant care (Wraith et al., 2007, supra; Martin et al., 2008, supra). Patients with less severe disease have normal intellectual function, but MRI imaging reveals global brain abnormalities, including white matter lesions, ventriculomegaly, and brain atrophy, in all individuals with MPS II (Muenzer, et al., 2009, supra).

[0009] Enzyme replacement therapy (ERT) using recombinant idursulfase produced by HT1080 (fibrosarcoma) cells (Elaprase®, Shire Human Genetic Therapies) is the only product approved for the treatment of Hunter syndrome, and it is administered by 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 intravenous (IV) ERT (recombinant idursulfase) treatment has been shown to improve the systemic symptoms of MPS II, patients and their caregivers face the burden of living with such weekly ERT infusions for the rest of their lives, which impacts their quality of life.

[0010] Current ERTs do not cross the blood-brain barrier and therefore cannot address patients with severe forms of the disease, i.e., MPS II, which affects the CNS, neurocognition, and behavior. In a recent clinical trial designed to address this issue, pediatric patients received monthly intrathecally formulated idursulfase (Elaprase) via a spinally implanted intrathecal drug delivery device (a catheter inserted at the L4 / L5 level via an access port implanted through a lower rib incision). Patients also received weekly intravenous injections of idursulfase. See Muenzer et al., 2016, Genetics in Med 18:73-81, esp. p. 74. The abstract is available at https: / / www.ncbi.nlm.nih.gov / pubmed / 25834948?dopt=Abstract. Device malfunctions occurred, resulting in partial device revision, total surgical revision, or exclusion in 6 of 12 treated patients (50%). Notably, 12 of 14 SAEs were device-related (complications due to device insertion, device migration / connection problems, device breakage / malfunction / failure, implant site infection, procedural pain, and wound dehiscence) (Muenzer et al., 2016, p. 75 (paragraph 2 and Figure 1)). Device breakage and catheter migration from the spinal canal were exacerbated by the high activity level in this pediatric population (Muenzer et al., 2016 at p. 78 Discussion).

[0011] 3. Summary of the Invention The present 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 to cells of the liver for systemic distribution in human subjects diagnosed with Mucopolysaccharidosis Type II (MPS II), including but not limited to patients diagnosed with Hunter Syndrome.

[0012] In a preferred embodiment, treatment is achieved via gene therapy—for example, by administering a viral vector or other DNA expression construct encoding human IDS (hIDS) or a derivative of hIDS into the CSF of a patient (human subject) diagnosed with MPS II, thereby creating a durable depot of (a) transduced neurons that continuously deliver the transgene product to the CNS and (b) transduced hepatocytes that deliver the transgene product systemically. rhIDS secreted into the CSF from the neuronal / glial cell depot and systemically secreted from the liver depot are taken up by other CNS and hepatocyte cells, respectively, "cross-correcting" the enzyme deficiency in the recipient cells. Furthermore, it has been unexpectedly discovered that administration of a viral vector to the CSF results in systemic delivery of the vector, and that a depot of transduced neurons and glial cells in the CNS can deliver the recombinant enzyme both to the CNS and systemically, potentially reducing or eliminating the need for systemic therapy, e.g., weekly intravenous injections of the enzyme.

[0013] In an alternative embodiment, hIDS can be produced in cell cultures (e.g., bioreactors) of human neuronal or glial cells and administered as enzyme replacement therapy ("ERT"), for example, by injecting the enzyme into the CSF, directly into the CNS, and / or systemically. However, because enzymes cannot cross the blood-brain barrier, systemic delivery of enzymes does not result in treatment of the CNS, and unlike the gene therapy approaches of the present invention, direct delivery of enzymes to the CNS requires repeated injections, which are not only burdensome but also introduce the risk of infection. Gene therapy approaches offer several advantages over ERT.

[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 (shown in Figure 1), and derivatives of hIDS having amino acid substitutions, deletions, or additions, for example, but not limited to, amino acid substitutions selected from non-conserved residues corresponding to orthologs of the IDS shown in Figure 2. However, such mutations do not involve substitution of the cysteine ​​residue at position 84 (C84) required for enzymatic activity (Millat et al., 1997, Biochem J 326:243-247), or, for example, as shown in FIG. 3 or Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29:755-761 (reporting "weak" mutants R48P, A85T, W337R, and truncated mutant Q531X, and "severe" mutants P86L, S333L, S349I, R468Q, R468L), Millat et al., 1998, BBA, each of which is incorporated herein by reference in its entirety. 1406:214-218 (reporting the "weak" variants P480L and P480Q, and the "severe" variant P86L), and Bonucelli et al., 2001, BBA 1537:233-238 (reporting the "weak" variants P480L and P480Q, and the "severe" variant P86L).

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

[0016] It has been described that animal models for MPS II can be used to evaluate the therapeutic agents described herein. For example, a knockout mouse model of MPS II (IDS knockout) was developed by replacing exons 4 and 5 of the IDS gene with a neomycin resistance gene (Garcia et al., 2007, J Inherit Metab Dis 30:924-34). This IDS knockout mouse exhibits many characteristics of MPS II, such as skeletal abnormalities, hepatosplenomegaly, elevated urinary and tissue GAGs, and brain storage lesions (Muenzer et al., 2001, Acta Paediatr Suppl 91:98-99), and was used to evaluate the effects of enzyme replacement therapy on MPS II to support clinical trials of ERT. Therefore, this mouse model is suitable 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, the entire contents of which are incorporated herein by reference).

[0017] Preferably, the hIDS transgene produced in human neuronal / glial cells should be controlled by expression control elements that function in neuronal and / or glial cells, such as the CB7 promoter (chicken β-actin promoter and CMV enhancer), and may include other expression control elements that enhance vector-driven transgene expression (e.g., chicken β-actin intron and rabbit β-globin polyA signal). The cDNA construct for the hIDS transgene should include a coding sequence for a signal peptide that ensures appropriate co- and post-translational processing (glycosylation and protein sulfation) by the transduced CNS cells. Such signal peptides for use in CNS cells may include, but are not limited to, the following: Oligodendrocyte myelin glycoprotein (hOMG) signal peptide: MEYQILKMSLCLFILLFLTPGILC (SEQ ID NO: 2) Cellular repressor of E1A-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 (hPCADHA1) 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) A signal peptide may also be referred to herein as a leader sequence or leader peptide.

[0018] The recombinant vector used to deliver the transgene should have tropism for cells in the CNS, including, but not limited to, neurons and / or glial cells. Such vectors can include non-replicating recombinant adeno-associated viral vectors ("rAAV"), with viral vectors having AAV9 or AAVrh10 capsids being particularly preferred. AAV variant capsids, particularly preferred AAV / hu.31 and AAV / hu.32, described by Wilson in U.S. Pat. No. 7,906,111, the entire contents of which are incorporated herein by reference, as well as AAV variant capsids described by Chatterjee in U.S. Pat. Nos. 8,628,966, 8,927,514, and Smith et al., 2014, Mol Ther 22:1625-1634, the entire contents of which are incorporated herein by reference, can be used. However, other viral vectors can 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 to deliver a transgene. Construct 1 is a recombinant adeno-associated virus serotype 9 capsid containing a human iduronate-2-sulfatase expression cassette, expression of which is driven by a hybrid of the cytomegalovirus (CMV) enhancer and the chicken beta-actin promoter (CB7). The IDS expression cassette is flanked by inverted terminal repeats (ITRs), and the transgene contains a 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 contains a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 45.

[0020] Pharmaceutical compositions suitable for administration to the CSF include suspensions of rhIDS vectors in a formulation buffer containing a physiologically compatible aqueous buffer, a surfactant, and optional excipients. In certain embodiments, the pharmaceutical composition is suitable for intrathecal administration. In certain embodiments, the pharmaceutical composition is suitable for intracisternal administration (injection into the cisterna magna). In certain embodiments, the pharmaceutical composition is suitable for injection into the subarachnoid space via C1-2 puncture. In certain embodiments, the pharmaceutical composition is suitable for intraventricular administration. In certain embodiments, the pharmaceutical composition is suitable for administration via lumbar puncture. In some embodiments, a pharmaceutical composition comprising an rAAV of the disclosure comprises sodium chloride at a concentration of about 8.77 g / L, magnesium chloride hexahydrate 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 dehydrate at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.001% (vol / vol), sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and disodium phosphate anhydrous at a concentration of about 0.114 g / L.

[0021] A therapeutically effective dose of the recombinant vector should be administered into the CSF via intrathecal administration (i.e., injection into the subarachnoid space, allowing the recombinant vector to diffuse through the CSF and transduce cells of the CNS). In some embodiments, the recombinant vector is administered in a solution containing sodium chloride at a concentration of about 8.77 g / L, magnesium chloride hexahydrate 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 disodium phosphate anhydrous at a concentration of about 0.114 g / L. This can be accomplished in several ways, for example, by intracranial (cisternomagna or intraventricular) injection or injection into the lumbar cisterna. For example, intracisternal (IC) injection (into the cisterna magna) can be performed via CT-guided suboccipital puncture, or intrathecal injection can be performed via C1-2 puncture if feasible for the patient, or lumbar puncture (a diagnostic procedure commonly performed to collect a sample of CSF) can be used to access the CSF. Alternatively, intracerebroventricular (ICV) administration (a more invasive technique used to introduce anti-infective or anti-cancer drugs that do not penetrate the blood-brain barrier) can be used to instill the recombinant vector directly into the ventricles. Alternatively, intranasal administration can be used to deliver the recombinant vector to the CNS.

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

[0023] In certain embodiments, the recombinant nucleotide expression vector is administered at a dose that corresponds to the brain mass of the human subject, and 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 1.3 x 10 per gram of brain mass as determined by MRI. 10In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of 1.9 x 10 GC / g of brain mass as determined by MRI. 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of 9.6 x 10 GC / g of brain mass as determined by MRI. 10 In certain embodiments, the dose of the recombinant nucleotide expression vector is 1.3 x 10 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by polyA PCR assay). In certain embodiments, the dose of recombinant nucleotide expression vector is 1.9×10 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by transgene-specific PCR assay). In certain embodiments, the dose of recombinant nucleotide expression vector is 6.5×10 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by polyA PCR assay). In certain embodiments, the dose of recombinant nucleotide expression vector is 9.6 x 10 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by transgene-specific PCR assay). In certain embodiments, the dose of recombinant nucleotide expression vector is 2.0 x 10 11 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by polyA PCR assay). In certain embodiments, the dose of recombinant nucleotide expression vector is 2.9 x 10 11 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered to a subject at a genome count of 6.5 x 10 GC / g brain mass, as determined by MRI. 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of 2.0 x 10 GC / g brain mass as determined by MRI. 11In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x 10 GC / g brain mass. 11 The human subject's brain mass is administered at a dose of GC / g 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 recombinant nucleotide expression vector administered to the subject. In certain embodiments, the human subject's brain mass is calculated based on the human subject's brain volume cm 3 , 1.046g / cm 3 The brain volume of the human subject is converted by multiplying by a factor of , which is obtained from a brain MRI of the human subject.

[0024] By way of background, human IDS contains eight potential N-glycosylation sites (N) as listed in Figure 1. 31 , N 115 , N 144 , N 246 , N 280 , N 325 , N 513 , and N 537 ) and contains a 25 amino acid signal sequence that is cleaved during processing. An initial 76 kDa intracellular precursor is converted to a phosphorylated 90 kDa precursor after its oligosaccharide chains are modified in the Golgi apparatus. This precursor is processed through various intracellular intermediates by glycosylation modification and proteolytic cleavage to the major 55 kDa form. In summary, after removal of the 25 amino acid signal sequence, proteolytic processing continues through N 31 downstream of which an N-terminal proteolytic cleavage is performed to remove a propeptide of 8 amino acids (residues 26–33) and 513A C-terminal proteolytic cleavage occurs upstream of this enzyme, releasing an 18 kDa polypeptide and generating a 62 kDa intermediate, which is converted to the 55 kDa mature form. Further proteolytic cleavage generates the 45 kDa mature form, which is located in the lysosomal compartment. (See Figure 4 for a diagram excerpt from Millat et al., 1997, Exp Cell Res 230:362-367 ("Millat 1997"); see 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 herein by reference in its entirety.)

[0025] C required for enzyme activity 84 Formylglycine modification of N (shown in bold in Figure 1) most likely occurs in the endoplasmic reticulum, possibly as an early post- or co-translational event. (See Millat 1997a, citing Schmidt et al., 1995, Cell 82:271-278.) Post-translational processing continues in the Golgi apparatus, incorporating complex sialic acid-containing glycans and including the acquisition of mannose-6-phosphate residues that bind enzymes for delivery to the lysosomal compartment. (For a brief discussion, see Clarke, 2008, Expert Opin Pharmacother 9:311-317, the entire contents of which are incorporated herein by reference.) While there is no single glycosylation site essential for IDS stability, N 280 Glycosylation at this position is important for cellular internalization via the mannose-6-phosphate (M6P) receptor and for lysosomal targeting (Chung et al., 2014, Glycoconj J 31:309-315, first paragraph of p. 310). Under normal physiological conditions, IDS is produced at very low levels, and the enzyme is rarely, if ever, secreted from cells (Clarke, 2008, supra).

[0026] The present invention is based in part on the following principles. (i) Neurons and glial cells of 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, which are robust processes in the CNS. See, for example, Sleat et al., 2005, Proteomics 5:1520-1532, and Sleat 1996, J Biol Chem 271:19191-98, which describe the human brain mannose-6-phosphate glycoproteome and note that the brain contains many more proteins with many more individual isoforms and mannose-6-phosphorylated proteins than are 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, which report on the production of secreted tyrosine-sulfated glycoproteins by neuronal cells, the entire contents of each of which are incorporated by reference for purposes of post-translational modifications produced 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 from position 34 or 36 in the brain as follows: T 34 DALNVLLI and A 36 LNVLLIIV. (Sleat, 2005, Proteomics 5:1520-1532, Table S2). Two of the eight N-linked glycosylation sites, i.e., N 280 and N 116 was found to be mannose-6-phosphorylated in IDS from human brain (Sleat et al., 2006, Mol & Cell Proeomics 5.4:686-701, reported in Table V). (iii) During hIDS processing, neurons and glial cells secrete two polypeptides, 76 kDa and 90 kDa, but only the 90 kDa polypeptide is mannose-6-phosphorylated, indicating that the secreted form of the enzyme is required for cross-modification (Millat, 1997, Figure 1 for transduced lymphoblastoid cells; Froissart 1995, Figure 4 for transduced fibroblasts—only the 90 kDa form is phosphorylated in the culture medium). Interestingly, these results demonstrate that recombinant IDS produced by neurons and glial cells can be endocytosed more avidly by recipient CNS cells than recombinant IDS produced by other cells, such as the kidney. Daniele 2002 (Biochimica et Biophysica Acta 1588(3):203-9) demonstrated M6P receptor-mediated endocytosis of recombinant IDS from conditioned medium of transduced neuronal and glial cell cultures by recipient populations of untransduced neuronal and glial cells that properly processed the precursor to the 45 kDa mature active form. The uptake of recombinant IDS produced by neuronal and glial cell lines (74% endocytosis) far exceeded the uptake of the enzyme produced by kidney cell lines (5.6% endocytosis). In both cases, uptake was inhibited by M6P, indicating that uptake of recombinant IDS is mediated by the M6P receptor. (See Daniele 2002, Tables 2 and 4; discussion of the results on pp. 205-206 is summarized in Table 1 below.) [Table 1] (iv) The gene therapy approach described herein will result in the continuous secretion of an approximately 90 kDa hIDS glycoprotein precursor, as determined by polyacrylamide gel electrophoresis (depending on the assay used), that is enzymatically active. First, the C required for IDS activity will be identified. 84FGly synthase (FGE, also known as SUMF1), an enzyme involved in the formylglycine modification of rhIDS, is expressed in the cerebral cortex of the human brain (gene expression data for SUMF1 can be accessed, for example, at GeneCards, http: / / www.genecards.org). Glycosylated / phosphorylated rIDS secreted by transduced neurons and glial cells in situ may then be taken up by non-transduced neurons and glial cells in the CNS and processed appropriately. Without being bound by any theory, rhIDS precursors secreted in situ by gene therapy may be more avidly endocytosed by recipient cells in the CNS than conventional recombinant enzymes used in ERT. For example, Elaprase® (produced in the fibrosarcoma cell line HT1080) is a purified protein reported to have a molecular weight of approximately 76 kDa, rather than the 90 kDa species thought to be highly phosphorylated and secreted by neurons and glial cells. The eight N-linked glycosylation sites are fully occupied in Elaprase® and reported to contain two bismannose-6-phosphate terminal glycans as well as a highly sialylated glycan complex, but the C -required for enzyme activity is not present. 84 The post-translational modification of FGly from Elaprase to FGly is only about 50% (Clarke, 2008, Expert Opin Pharmacother 9:311-317; Elaprase® Full Prescribing Information and EMA filing). Another recombinant product, Hunterase®, is produced in CHO cells. It has been reported to have more FGly and higher activity than Elaprase®, but there were no differences in mannose-6-phosphorylation or incorporation (Chung, 2014, Glycoconj J 31:309-315). (v) The efficacy of IDS in vivo and outside the cells is determined by M6P and its active site formylglycine (FGly), i.e., post-translational modification by formylglycine-generating enzymes. 84The uptake (internalization in cells and lysosomes) of IDS is dependent on the FGly-mediated uptake (internalization in cells and lysosomes) of IDS, which is converted from IDS. As shown in Table 1 above, brain cells (neuronal and glial) exhibit higher enzyme activity when incubated with IDS precursor medium secreted by transduced neurons and glial cells than when incubated with IDS precursor medium secreted by genetically engineered kidney cells. The resulting five-fold increase in activity is largely due to efficient IDS uptake (see Daniele 2002, Tables 2 and 4). The FGly content of commercially available IDS produced by CHO cells or HT-1080 cells is approximately 50% to 70%, which determines the enzyme activity. However, this activity may be improved in neurons and glial cells due to improved IDS uptake. (vi) Cellular and intracellular transport / uptake of lysosomal proteins, such as IDS, is mediated by M6P. As reported in Daniele 2002 and Sleat, Proteomics, 2005, IDS derived from brain cells may contain higher amounts of M6P (indicating that the human brain contains more Man6-P glycoproteins (quantitatively and qualitatively) than other tissues). It is possible to measure the M6P content of IDS precursors, as performed in Daniele 2002. In the presence of inhibitory M6P (e.g., 5 mM), the uptake of IDS precursors produced by non-neuronal or non-glial cells, such as the genetically engineered kidney cells in Daniele 2002, is expected to be reduced to levels similar to those of control cells, as shown in Daniele 2002. In the presence of inhibitory M6P, uptake of IDS precursors produced by brain cells, including neurons and glial cells, is predicted to remain at high levels, as shown in Daniele 2002, with uptake fourfold greater than control cells and comparable to the level of IDS activity (or uptake) of IDS precursors produced by genetically engineered kidney cells in the absence of inhibitory M6P. This assay provides a method for predicting the M6P content of IDS precursors produced by brain cells and, in particular, for comparing the M6P content of IDS precursors produced by different cell types. The gene therapy approach described herein results in continuous secretion of hIDS precursors that can be taken up by neurons and glial cells at high levels in such assays in the presence of inhibitory M6P. (vii) The M6P content and incorporation of IDS precursors can be demonstrated by gel bands at 90 kDa and 76 kDa (e.g., SDS-PAGE gel bands). The 90 kDa band is reported to be highly glycosylated / phosphorylated and also contain M6P, whereas the 76 kDa band does not. Similar to the gel band of IDS precursors generated from genetically engineered kidney cells (Daniele 2002, Figure 1), very broad gel bands with average molecular weights ranging from 76 kDa to 95 kDa and 80 to 85 kDa can be contrasted with the gel band of IDS precursors generated from brain cells. In Daniele 2002, immunoprecipitation of the IDS precursor was unsuccessful, so no gel bands could be obtained. The gene therapy approach described herein will result in the continuous secretion of hIDS precursors, distinct from the IDS precursor gel band generated from genetically engineered kidney cells. (viii) The M6P content of commercially available IDS precursors is 2–2.5 mol / mol, with the majority present in the form of di-phosphorylated glycans. On average, all IDS precursors are phosphorylated, but the normal distribution of glycans, assuming multiple phosphorylation sites, includes some IDS precursors with two, one, and zero di-phosphorylated M6P glycans. The incorporation rate would be significantly higher with multiple phosphorylations. (ix) Glycosylation of hIDS by human cells of the CNS adds glycans that can improve stability, half-life, and reduce undesired aggregation of the transgene product. Importantly, the glycans added to the hIDS of the present invention contain 2,6-sialic acid and incorporate Neu5Ac ("NANA") but not its hydroxylated derivative, NeuGc (N-glycolylneuraminic acid, i.e., "NGNA" or "Neu5Gc"). Such glycans are absent in recombinant IDS products, such as Hunterase®, made in CHO cells. This is because CHO cells lack the 2,6-sialyltransferase required for this post-translational modification, nor do they produce bisecting GlcNAc, but instead add Neu5Gc(NGNA), an uncommon (and potentially immunogenic) sialic acid in 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, p. 5), and Hague et al., 1998 Electrophor 19:2612-2630 ("CHO cell lines are considered 'phenotypically restricted' with respect to glycosylation due to the lack of α2,6-sialyltransferase."). Furthermore, CHO cells can also produce α-Gal antigen, an immunogenic glycan that reacts with anti-α-Gal antibodies present in most individuals and can induce anaphylaxis at high concentrations. See, e.g., Bosques, 2010, Nat Biotech 28:1153-1156. The human glycosylation pattern of the hIDS of the present invention will reduce the immunogenicity and improve the efficacy of the transgene product. (x) The immunogenicity of transgene products can be induced by various factors, including the patient's immune status, the structure and characteristics of the injected protein drug, the route of administration, and the duration of treatment. Process-related impurities, such as host cell proteins (HCPs), host cell DNA, and chemical residues, as well as product-related impurities, such as protein degradation products and structural characteristics, such as glycosylation, oxidation, and aggregation (subvisible particles), can increase immunogenicity by acting as adjuvants to enhance the immune response. The amount of process-related and product-related impurities can be affected by the manufacturing process, i.e., cell culture, purification, formulation, storage, and handling, which can affect the commercially produced IDS product. In gene therapy, proteins are produced in vivo, and as a result, process-related impurities are absent, and protein products are unlikely to contain product-related impurities / degradants, such as protein aggregates and protein oxides, associated with recombinantly produced proteins. Aggregation can be associated with protein production and storage, resulting from high protein concentrations, surface interactions with manufacturing equipment and vessels, and the purification process using certain buffer systems. However, when transgenes are expressed in vivo, these conditions that promote aggregation are not present. Additionally, oxidation, e.g., of methionine, tryptophan, and histidine, is associated with protein production and storage and can be caused by, for example, stressful cell culture conditions, contact with metals and air, and impurities in buffers and excipients. Proteins expressed in vivo can oxidize under stressful conditions, but humans, like many organisms, possess antioxidant defense systems that not only reduce oxidative stress but also repair and / or reverse oxidation. Therefore, proteins produced in vivo are less likely to be in an oxidized form. Both aggregation and oxidation can affect potency, PK (clearance), and increase immunogenicity concerns. The gene therapy approach described herein will result in the continuous secretion of hIDS precursors with reduced immunogenicity compared to commercially produced products. (xi) In addition to N-linked glycosylation sites, hIDSs contain tyrosine ("Y") sulfation sites (PSSEKY 165ENTKTCRGPD). (See, e.g., Yang et al., 2015, Molecules 20:2138-2164, especially p. 2154, which is incorporated by reference in its entirety, for an analysis of amino acids surrounding tyrosine residues subject to protein tyrosine sulfation. The "rule" can be summarized as follows: Y residues with E or D within positions +5 to -5 of Y, and position -1 of Y is a neutral or acidic charged amino acid, but not a basic amino acid, e.g., R, K, or H, which abolishes sulfation.) Without being bound by any theory, sulfation of this site in hIDS may improve enzyme stability and binding affinity for substrates. Tyrosine sulfation of hIDS, a robust post-translational process in human CNS cells, may improve the processing and activity of transgene products. Although the importance of tyrosine sulfation of lysosomal proteins remains unclear, it has been shown to increase the affinity of protein-protein interactions (antibodies and receptors) and promote proteolytic processing (peptide hormones) in other proteins (see Moore, 2003, J. Biol. Chem. 278:24243-46; and Bundegaard et al., 1995, The EMBO J 14:3073-79). Tyrosylprotein sulfotransferase 1 (TPST1), involved in tyrosine sulfation (potentially the final step in IDS processing), is expressed at even higher levels (based on mRNA) in the brain (gene expression data for TPST1 can be found, for example, in the EMBL-EBI Expression Atlas, accessible at http: / / www.ebi.ac.uk / gxa / home). Such post-translational modifications are, at best, underrepresented in CHO cell products. Unlike human CNS cells, CHO cells are not secretory cells and also have a limited capacity for post-translational tyrosine sulfation (see, e.g., Mikkelsen & Ezban, 1991, Biochemistry 30:1533-1537, especially the discussion on p. 1537).

[0027] For the reasons discussed above, production of rhIDS by human neuronal and / or glial cells would provide a "bio-better" molecule for the treatment of MPS II, achieved by gene therapy, for example, by administering a viral vector or other DNA expression construct encoding rhIDS into the CSF of patients (human subjects) diagnosed with MPS II disease (including, but not limited to, Hunter) to create a permanent depot in the CNS that provides a continuous supply of fully human glycosylated, mannose-6-phosphorylated, sulfated transgene product secreted by transduced CNS cells. The rhIDS transgene product secreted from the depot into the CSF is endocytosed by cells in the CNS and "cross-corrects" the enzymatic defect in MPS II recipient cells.

[0028] It is not essential that all rhIDS molecules produced in a 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 the gene therapy treatment of the present invention is to slow or halt disease progression. Efficacy can be monitored by measuring cognitive function (e.g., prevention or reduction of neurocognitive decline); reduction in CSF and / or serum disease biomarkers (e.g., GAGs); and / or increased CSF and / or serum IDS enzyme activity. Signs of inflammation and other safety events can also be monitored.

[0029] As an alternative or adjunct to gene therapy, rhIDS glycoproteins can be produced in human neuronal or glial cells by recombinant DNA technology, and the glycoproteins can be administered systemically and / or into the CSF to patients diagnosed with MPS II 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-1A, HCN-2, NT2, SH-SY5y, hNSC11, or ReNcell VM (see, e.g., "Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives" in Dumont et al., 2016, Critical Rev in Biotech 36(6):1110-1122, for a review of human cell lines that can be used for recombinant production of rHuGlyIDS glycoproteins, the entire contents of which are incorporated herein by reference). To ensure complete glycosylation, particularly sialylation, and tyrosine sulfation, the cell lines used for production can be enhanced by genetically engineering the host cells to co-express the α-2,6-sialyltransferase (or both α-2,3- and α-2,6-sialyltransferases) responsible for tyrosine-O-sulfation, and / or the TPST-1 and TPST-2 enzymes.

[0030] While delivery of rhIDS will minimize immune responses, the most obvious potential source of toxicity for CNS-related gene therapy is the development of immunity to the expressed rhIDS protein in human subjects who are genetically deficient in IDS and therefore may not tolerate the vectors used to deliver the protein and / or transgene.

[0031] Therefore, in a preferred embodiment, co-treating the patient with immunosuppressive therapy is appropriate, especially when treating patients with severe disease whose IDS levels are close to zero. Immunosuppressive therapy involving a combination of tacrolimus or rapamycin (sirolimus) with mycophenolic acid, or other immunosuppressive regimens used in tissue transplantation procedures, can be utilized. Such immunosuppressive therapy can be administered during the course of gene therapy, and in certain embodiments, pretreatment with immunosuppressive therapy may be preferred. Immunosuppressive therapy can be continued after gene therapy treatment, at the discretion of the attending physician, and then discontinued, for example, after 180 days, if immune tolerance is induced.

[0032] The methods of the present invention include the combination of delivery of rhIDS to the CSF with the delivery of other available therapies. The additional therapies can be administered before, simultaneously with, or after the gene therapy treatment. Available therapies for MPS II that can be used in combination with the gene therapy of the present 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, a method of treating a human subject diagnosed with MPS II is provided, the method comprising delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or glial cells to the CSF of the human subject, wherein the glycosylated recombinant human IDS precursor is delivered by administering a recombinant nucleotide expression vector encoding the human IDS, the recombinant nucleotide expression vector being administered at a dose dependent on the brain mass of the human subject, the brain mass being determined by brain MRI of the human subject's brain.

[0034] In another aspect, provided herein is a method of treating a human subject diagnosed with MPS II, the method comprising, in order: (a) delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or glial cells to the CSF of the human subject; (b) measuring the 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 the level of heparan sulfate in a reference population; wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding the human IDS, the recombinant nucleotide expression vector is administered at a dose corresponding to the brain mass of the human subject, and 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 healthy individuals who do not have MPS II, preferably of a similar age, weight, and / or sex as the human subject.

[0035] In another aspect, provided herein are methods for treating a human subject diagnosed with MPS II, the method comprising, in order: (a) first measuring the level of heparan sulfate in the CSF of the human subject; (b) delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or glial cells to the CSF of the human subject; and (c) measuring the level of heparan sulfate after a period of time, wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding the human IDS, the recombinant nucleotide expression vector being administered at a dose dependent on the brain mass of the human subject, as 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 months, 4 months, 5 months, 6 months, 7 months, 8 months, 11 months, or 1 year.

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

[0037] In certain embodiments of the methods of treatment described herein, the brain mass of the human subject is 1.046 g / cm of brain volume of the human subject. 3 The brain volume of a human subject is calculated by multiplying the coefficient of 3 and the brain volume of a human subject is determined by brain MRI of the human subject.

[0038] In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered in an amount of about 1.3 x 10 per gram of brain mass as determined by MRI. 10 GC, or approximately 6.5 × 10 per gram of brain mass as determined by MRI. 10 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x 10 per gram of brain mass as determined by MRI. 11 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x 10 GC, as determined by MRI. 10 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x 10 GC / g brain mass, as determined by MRI. 10 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x 10 GC / g brain mass. 11 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by polyA-specific PCR assay). 11 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x 10 GC / g brain mass. 11In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x 10 GC / g brain mass (e.g., brain mass is determined by MRI and genome number is determined by transgene-specific PCR assay). 10 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x 10 GC / g brain mass (e.g., brain mass is determined by MRI and genome number is determined by polyA-specific PCR assay). 10 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x 10 GC / g brain mass (e.g., brain mass is determined by MRI and genome number is determined by transgene-specific PCR assay). 10 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x 10 GC / g brain mass (e.g., brain mass is determined by MRI and genome number is determined by polyA-specific PCR assay). 10 The dose is given in GC / g brain mass (eg, brain mass determined by MRI and genome number determined by transgene-specific PCR assay).

[0039] In various embodiments of the methods of treatment described herein, the human subject is 5 years of age or older and less than 18 years of age. In certain embodiments, the human subject is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years old. In certain embodiments, the human subject is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years old. In certain embodiments, the human subject is 5-6 years old, 6-7 years old, 7-8 years old, 8-9 years old, 9-10 years old, 10-11 years old, 11-12 years old, 12-13 years old, 13-14 years old, 14-15 years old, 15-16 years old, 16-17 years old, 17-18 years old, or 18-19 years old. In certain embodiments, the human subject is about 5-6 years old, 6-7 years old, 7-8 years old, 8-9 years old, 9-10 years old, 10-11 years old, 11-12 years old, 12-13 years old, 13-14 years old, 14-15 years old, 15-16 years old, 16-17 years old, 17-18 years old, or 18-19 years old. In certain embodiments, the recombinant nucleotide expression vector is administered at a concentration of about 6.5 x 10 per gram of brain mass as determined by MRI. 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose set forth in Table 7.

[0040] In various embodiments of the methods of treatment described herein, the human subject is at least 4 months old and less than 5 years old. In certain embodiments, the human subject is 4, 5, 6, 7, 8, 9, 10, or 11 months old. In certain embodiments, the human subject is about 4, 5, 6, 7, 8, 9, 10, or 11 months old. In certain embodiments, the human subject is 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months old. In certain 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 certain embodiments, the human subject is 1, 2, 3, 4, or 5 years old. In certain embodiments, the human subject is about 1, 2, 3, 4, or 5 years old. In certain embodiments, the human subject is 1-2 years old, 2-3 years old, 3-4 years old, 4-5 years old, or 5-6 years old. In certain embodiments, the human subject is about 1-2 years old, 2-3 years old, 3-4 years old, 4-5 years old, or 5-6 years old. In certain embodiments, the recombinant nucleotide expression vector is administered at a concentration of about 1.3 x 10 per gram of brain mass as determined by MRI. 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x 10 GC per gram of brain mass as determined by MRI. 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x 10 GC per gram of brain mass as determined by MRI. 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x 10 GC, as determined by MRI. 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x 10 GC / g brain mass, as determined by MRI. 11 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x 10 GC / g brain mass, as determined by MRI. 11 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x 10 GC / g brain mass, as determined by MRI. 11In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by polyA-specific PCR assay). 11 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by transgene-specific PCR assay). 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by polyA-specific PCR assay). 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by transgene-specific PCR assay). 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by polyA-specific PCR assay). 10 The recombinant nucleotide expression vector is administered at a dose of GC / g brain mass (e.g., brain mass is determined by MRI and genome number is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose selected from Dose 1 or Dose 2 according to Table 5. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose set forth in Table 6.

[0041] In some embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vector is administered via intracisternal (IC) administration. In other embodiments of the methods described herein, the recombinant nucleotide expression vector is administered via intracerebroventricular (ICV) administration.

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

[0043] In certain embodiments of the therapeutic methods described herein, the glycosylated recombinant human IDS precursor is secreted at detectable levels.

[0044] In certain embodiments of the methods of treatment described herein, the human neuronal or human glial cells have at least one mutation in an endogenous gene encoding a human IDS precursor.

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

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

[0047] In certain embodiments of the therapeutic methods described herein, the glycosylated recombinant human IDS precursor is expressed under the control of the CB7 promoter.

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

[0049] In certain embodiments of the methods of treatment described herein, the glycosylated recombinant human IDS precursor is about 90 kDa as determined by polyacrylamide gel electrophoresis.

[0050] In certain embodiments of the methods of treatment described herein, the glycosylated recombinant human IDS precursor contains formylglycine.

[0051] In certain embodiments of the therapeutic methods described herein, the glycosylated recombinant human IDS precursor (a) is α2,6-sialylated, (b) does not contain detectable NeuGc, (c) does not contain detectable α-Gal antigen, (d) contains tyrosine sulfation, and / or (e) is mannose-6-phosphorylated.

[0052] In certain embodiments of the methods of treatment 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 methods further include administering immunosuppressive therapy to the human subject prior to or concurrently with the human IDS precursor treatment, and optionally continuing the immunosuppressive therapy thereafter.

[0054] In some embodiments, the immunosuppressive therapy comprises administering one or more corticosteroids, sirolimus, and / or tacrolimus, hi certain embodiments, 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 prior to or concurrently with the immunosuppressive therapy, hi certain embodiments, the one or more antibiotics are trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atovaquone.

[0056] In some embodiments, the methods further comprise administering to the human subject one or more antifungal therapies prior to or concurrently with the immunosuppressive therapy.

[0057] In some embodiments, the method further comprises, after administering the recombinant nucleotide expression vector, measuring one or more of the following biomarkers: (a) glycosaminoglycan (GAG) levels in CSF, (b) iduronate-2-sulfatase (IS) levels in CSF, (c) GAG levels in plasma; (d) I2S levels in plasma, (e) leukocyte I2S enzyme activity levels, and (f) GAG levels in urine. In certain embodiments, the GAG ​​in CSF comprises heparin sulfate in CSF. In another specific embodiment, the GAG ​​in CSF is heparin sulfate in CSF. In another specific embodiment, the GAG ​​in plasma comprises heparin sulfate in plasma. In another specific embodiment, the GAG ​​in plasma is heparin sulfate in plasma. In another specific embodiment, the GAG ​​in urine comprises heparin sulfate in urine. In another specific embodiment, the GAG ​​in urine is heparin sulfate in urine. In certain embodiments, the measuring step comprises measuring the level of heparin sulfate in CSF. In another specific embodiment, the measuring step comprises measuring the level of leukocyte I2S enzyme activity.

[0058] In another aspect, provided herein are methods of treating a human subject diagnosed with MPS II, comprising: (a) administering to the human subject a therapeutically effective amount of an rAAV encoding a hIDS, wherein the human subject has been or is currently being treated with ERT; and (b) discontinuing ERT treatment in the human subject if a level of at least one biomarker in a biological sample from the human subject is lower than a baseline, wherein the biological sample is obtained from the human subject after administration, and 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%, or 95% lower than the baseline before discontinuing ERT. In some embodiments, the 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 a hIDS to the human subject, wherein the human subject has been 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 prior to administration, and the at least one biomarker is an anti-IDS antibody. In some embodiments, 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 baseline before discontinuing ERT. In some embodiments, the ERT is recombinant idursulfase. In some embodiments, the subject diagnosed with MPS II has hepatosplenomegaly.

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

[0061] In another aspect, provided herein are methods for treating a human subject diagnosed with MPS II, comprising: (a) discontinuing ERT treatment in the human subject, where the human subject has been or is being treated with ERT; and (b) administering a therapeutically effective amount of an rAAV encoding a hIDS to the human subject, wherein the administration occurs after discontinuing ERT treatment in the human subject. In some embodiments, 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 1 day before administering the rAAV encoding the hIDS to the human subject. In some embodiments, ERT treatment is discontinued if the level of at least one biomarker is not detectable 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 or monitoring the effectiveness of an MPS II treatment in a human subject, comprising administering to the human subject a therapeutically effective amount of an rAAV encoding a hIDS, wherein a decrease in the level of D2S6 in a biological sample from the human subject compared to a reference indicates the effectiveness of the MPS II treatment in the human subject, and wherein the biological sample is at least 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, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, , 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 days later, or 1, 2, 3, 4, 5, 6, 7, 8, 10, 16, 20, 24, 30, 35, 40, 45, 48, 50, 52, 56, 104 weeks later, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months later, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years later. In some embodiments, the human subject received treatment with ERT before administering the rAAV encoding the hIDS to the human subject. In some embodiments, the human subject received ERT treatment after administering the rAAV encoding the hIDS to the human subject. In some embodiments, the ERT is enzyme replacement therapy using recombinant idursulfase. In some embodiments, the biological sample is CSF. In some embodiments, the patient is a pediatric patient.

[0063] In some embodiments, the efficacy of treatment is demonstrated by a decrease in D2S6 levels 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% compared to a baseline (e.g., the D2S6 level in the same patient prior to the administration 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) compared to a baseline. In some embodiments, the at least one subtest is an age-equivalent score, a cognitive development quotient (DQ), an expressive language DQ, a receptive language DQ, a gross motor DQ, and / or a 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-equivalent score increases by about or at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, or more than 24 months. In some embodiments, the baseline is a score on at least one subtest of the BSID-III obtained before administration. In some embodiments, the baseline is the average score on 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 administration.

[0064] In some embodiments, the rAAV is administered intrathecally to a human subject using 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% (vol / vol), (g) sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and (h) disodium phosphate anhydrous at a concentration of about 0.114 g / L.

[0065] One aspect provided herein is a method for identifying or diagnosing the presence or absence of neuropathic MPS II in a subject, the method comprising: (a) determining the level of one or more heparan sulfate disaccharides in a biological sample from the subject; (b) identifying or diagnosing the subject as having neuropathic MPS II if the level of the one or more heparan sulfate disaccharides is elevated compared to a reference level; and (c) administering a therapeutically effective amount of an rAAV encoding a hIDS to the subject identified or diagnosed as having neuropathic MPS II. In some embodiments, the one or more heparan sulfate disaccharides comprise one or more of D0A0, D0S0, D0A6, D2S6, or a combination thereof. In some embodiments, the one or more heparan sulfate disaccharides is D2S6.

[0066] One embodiment provided herein is a method of identifying or diagnosing the presence or absence of neuropathic MPS II in a subject, wherein the subject is identified or diagnosed as having neuropathic MPS II if the level of D2S6 in a biological sample from the subject is elevated compared to a reference level, and a therapeutically effective amount of an rAAV encoding a hIDS is administered to the subject identified or diagnosed as having neuropathic MPS II.

[0067] In some embodiments, the biological sample is cerebrospinal fluid. In some embodiments, the subject is pre-symptomatic or has no visible or detectable MPS II symptoms. In some embodiments, the subject has MPS II. In some embodiments, the reference level is the level of at least one or more heparan sulfate disaccharides in a biological sample from one or more healthy individuals and / or one or more non-neurodisordered subjects. In some embodiments, the reference level is the level of D2S6 in a biological sample from one or more healthy individuals and / or one or more non-neurodisordered subjects. In some embodiments, the biological sample from one or more healthy individuals and / or one or more non-neurodisordered subjects is a CSF sample. In some embodiments, the reference level is a predetermined 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 more than 40% total heparan sulfate disaccharides (HS) in a biological sample from the subject. In some embodiments, the level of D2S6 of total heparan sulfate disaccharides (HS) in a biological sample from a subject is about or at least about 20%.In some embodiments, the level of one or more heparan sulfate disaccharides or the level of D2S6 in a biological sample from a subject is about or at least about 50 ng / mL, about 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, 176 ng / mL, 177 ng / mL, 178 ng / mL, 179 ng / mL, 180 ng / mL, 181 ng / mL, 182 ng / mL, 183 ng / mL, 184 ng / mL, 185 ng / mL, 186 ng / mL, 187 ng / mL, 188 ng / mL, 189 ng / mL, 190 ng / mL, 191 ng / mL, 192 ng / mL, 193 ng / mL, 194 ng / mL, 195 ng / mL, 196 ng / mL, 197 ng / mL, 198 ng / mL, 199 ng / mL, 200 ng / mL, 201 ng / mL, 202 ng / mL, 203 ng / mL, 204 ng / mL, 205 ng / mL, 206 ng / mL, 75ng / mL, 180ng / mL, 185ng / mL, 190ng / mL, 195ng / mL, 200ng / mL, 210ng / mL, 220ng / mL, 230ng / mL, 240ng / mL, 250ng / mL, 260ng / mL, 270ng / mL, 280ng / mL , 290ng / mL, 300ng / mL, 310ng / mL, 320ng / mL, 330ng / mL, 340ng / mL, 350ng / mL, 360ng / mL, 370ng / mL, 380ng / mL, 390ng / mL, 400ng / mL, or greater than 400ng / mL. In some embodiments, the level of one or more heparan sulfate disaccharides or D2S6 in a biological sample from a subject is about or at least about 100 ng / mL, about 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 greater than 200 ng / mL.In some embodiments, the level of one or more heparan sulfate disaccharides or D2S6 levels in a biological sample from a subject is 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 / g / mL, 260ng / mL, 270ng / mL, 280ng / mL, 290ng / mL, 300ng / mL, 310ng / mL, 320ng / mL, 330ng / mL, 340ng / mL, 350ng / mL, 360ng / mL, 370ng / mL, 380ng / mL, 39 0ng / mL, 400ng / mL, 410ng / mL, 420ng / mL, 430ng / mL, 440ng / mL, 450ng / mL, 460ng / mL, 470ng / mL, 480ng / mL, 490ng / mL, 500ng / mL, or elevated above 500ng / mL.

[0068] One aspect provided herein is a method for determining the efficacy or monitoring efficacy of a treatment for MPS I in a human subject, comprising administering to the human subject a therapeutically effective amount of an rAAV encoding a human IDUA, wherein a decreased level of I0S6 in a biological sample from the human subject compared to a reference indicates the efficacy of the treatment for MPS I in the human subject, and wherein the biological sample is obtained from the human subject following administration.

[0069] In some embodiments, the biological sample is plasma. In some embodiments, the human subject is treated with ERT before administration and / or receives ERT treatment after administration. In some embodiments, the ERT is enzyme replacement therapy with recombinant idursulfase. In some embodiments, the reduction in the level of I0S6 is about or at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction compared to the reference level. In some embodiments, the reference level is the level of I0S6 in a biological sample obtained from the human subject before administration. In some embodiments, the reference level is a predetermined value. In some embodiments, the reference level is the level of I0S6 in a biological sample obtained from another human subject diagnosed with MPS I or from a population of human subjects diagnosed with MPS I. In some embodiments, the efficacy of treating MPS I is an improvement in at least one subtest of the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III) compared to a baseline. In some embodiments, the at least one subtest is an age-equivalent score, a Cognitive Development Quotient (DQ), an Expressive Language DQ, a Receptive Language DQ, a Gross Motor DQ, and / or a Fine Motor DQ. In some embodiments, the baseline is a score on at least one subtest of the BSID-III obtained from the human subject prior to administration. In some embodiments, the baseline is the average score on at least one subtest of the BSID-III obtained from human subjects with MPS I of the same age as the human subject.

[0070] In some embodiments, the rAAV is administered to a human subject using 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% (vol / vol); (g) sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L; and (h) disodium phosphate anhydrous at a concentration of about 0.114 g / L. 3.1 Illustrative Embodiments 3.1.1. Set 1 1. A method for treating a human subject diagnosed with Mucopolysaccharidosis Type II (MPS II), comprising delivering a therapeutically effective amount of a glycosylated recombinant human iduronate-2-sulfatase (IDS) precursor produced by human neuronal or glial cells to the cerebrospinal fluid (CSF) of the human subject, wherein the glycosylated recombinant human IDS precursor is delivered by administering a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose dependent on the brain mass of the human subject, 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 detectable levels. 3. The method of paragraph 1 or 2, wherein the human neuronal or human glial cells have at least one mutation in an endogenous gene encoding a human IDS precursor. 4. The method of any one of paragraphs 1 to 3, wherein the human neuronal or glial cells are transduced with a recombinant adeno-associated viral vector (rAAV). 5. The method of any one of paragraphs 1 to 4, wherein the glycosylated recombinant human IDS precursor is expressed under the control of the CB7 promoter. 6. The method of any one of paragraphs 1 to 5, wherein the glycosylated recombinant human IDS precursor is expressed from a cDNA encoding the human IDS precursor. 7. The method of any one of paragraphs 1 to 6, wherein the glycosylated recombinant human IDS precursor is about 90 kDa as determined by polyacrylamide gel electrophoresis. 8. The method of any one of paragraphs 1 to 7, wherein the glycosylated recombinant human IDS precursor contains formylglycine. 9. The method of any one of paragraphs 1 to 8, wherein the glycosylated recombinant human IDS precursor (a) is α2,6-sialylated, (b) contains no detectable NeuGc, (c) contains no detectable α-Gal antigen, (d) contains tyrosine-sulfation, and / or (e) is mannose-6-phosphorylated. 10. The method of any one of paragraphs 1 to 9, wherein 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 to 10, wherein the recombinant nucleotide expression vector is an AAV9 or AAVrh10 vector. 12. Calculate the brain mass of the human subject by dividing the brain volume cm of the human subject by the 3 , 1.046g / cm 3 12. The method of any one of paragraphs 1 to 11, wherein the brain volume of the human subject is obtained from a brain MRI of the human subject by multiplying the brain volume of the human subject by a coefficient of: 13. The recombinant nucleotide expression vector is administered to approximately 1.3 x 10 cells per gram of brain mass as determined by MRI. 10 GC, or approximately 6.5 × 10 per gram of brain mass as determined by MRI. 10 13. The method of any one of paragraphs 1 to 12, wherein the GC is administered in a dose of 0.1 mg / kg. 14. The method of any one of paragraphs 1 to 13, wherein the recombinant nucleotide expression vector is administered via intracisternal (IC) administration. 15. The method of any one of paragraphs 1 to 13, wherein the recombinant nucleotide expression vector is administered via intracerebroventricular (ICV) administration. 16. The method of any one of paragraphs 1 to 15, wherein the recombinant nucleotide expression vector is administered in a volume not exceeding 10% of the total volume of cerebrospinal fluid of the human subject. 17. The method of any one of paragraphs 1 to 16, wherein the glycosylated recombinant human IDS precursor is delivered to a lysosome within a cell of the CNS of the human subject. 18. The method of any one of paragraphs 1 to 17, further comprising administering immunosuppressive therapy to the human subject prior to or concurrently with administering the human IDS precursor treatment, and optionally continuing the immunosuppressive therapy thereafter. 19. The method of paragraph 18, wherein the immunosuppressive therapy comprises administering one or more corticosteroids, sirolimus, and / or tacrolimus. 20. The method of paragraph 19, wherein the one or more corticosteroids is methylprednisolone and / or prednisone. 21. The method of any one of paragraphs 18 to 20, further comprising administering one or more antibiotics to the human subject prior to or concurrently with the immunosuppressive 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 to 22, further comprising administering one or more antifungal therapies to the human subject prior to or concurrently with the immunosuppressive therapy. 24. The method of any one of paragraphs 1 to 23, further comprising measuring one or more of the following biomarkers after administering the recombinant nucleotide expression vector: (a) levels of glycosaminoglycans (GAGs) in the CSF, (b) levels of iduronate-2-sulfatase (I2S) in the CSF, (c) levels of GAGs in plasma, (d) levels of I2S in plasma, (e) levels of leukocyte I2S enzyme activity, and (f) levels of GAGs in urine. 25. The method of paragraph 24, wherein the GAG ​​in the CSF comprises heparin sulfate in the CSF. 26. The method of paragraph 24, wherein the GAG ​​in the CSF is heparin sulfate in the CSF. 27. The method of any one of paragraphs 24 to 26, wherein the GAGs in the plasma comprise heparin sulfate in the plasma. 28. The method of any one of paragraphs 24 to 26, wherein the GAG ​​in plasma is heparin sulfate in plasma. 29. The method of any one of paragraphs 24-28, wherein the urinary GAG comprises urinary heparin sulfate. 30. The method of any one of paragraphs 24 to 28, wherein the urinary GAG is urinary heparin sulfate. 31. The method of any one of paragraphs 24 to 30, wherein the measuring step includes measuring the level of heparin sulfate in the CSF. 32. The method of any one of paragraphs 24 to 31, wherein the measuring step comprises measuring the 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 a therapeutically effective amount of a glycosylated recombinant human iduronate-2-sulfatase (IDS) precursor produced by human neuronal or glial cells to the cerebrospinal fluid (CSF) of the human subject, wherein the glycosylated recombinant human IDS precursor is delivered by administering a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose dependent on the brain mass of the human subject, and wherein the brain mass is determined by magnetic resonance imaging (MRI) of the brain of the human subject. 2. The method of paragraph 1, wherein the glycosylated recombinant human IDS precursor is secreted at detectable levels. 3. The method of paragraph 1 or 2, wherein the human neuronal or human glial cells have at least one mutation in an endogenous gene encoding a human IDS precursor. 4. The method of any one of paragraphs 1 to 3, wherein the human neuronal or glial cells are transduced with a recombinant adeno-associated viral vector (rAAV). 5. The method of any one of paragraphs 1 to 4, wherein the glycosylated recombinant human IDS precursor is expressed under the control of the CB7 promoter. 6. The method of any one of paragraphs 1 to 5, wherein the glycosylated recombinant human IDS precursor is expressed from a cDNA encoding the human IDS precursor. 7. The method of any one of paragraphs 1 to 6, wherein the glycosylated recombinant human IDS precursor is about 90 kDa as determined by polyacrylamide gel electrophoresis. 8. The method of any one of paragraphs 1 to 7, wherein the glycosylated recombinant human IDS precursor contains formylglycine. 9. The method of any one of paragraphs 1 to 8, wherein the glycosylated recombinant human IDS precursor (a) is α2,6-sialylated, (b) contains no detectable NeuGc, (c) contains no detectable α-Gal antigen, (d) contains tyrosine-sulfation, and / or (e) is mannose-6-phosphorylated. 10. The method of any one of paragraphs 1 to 9, wherein 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 to 10, wherein the recombinant nucleotide expression vector is an AAV9 or AAVrh10 vector. 12. Calculate the brain mass of the human subject by dividing the brain volume cm of the human subject by the 3 , 1.046g / cm 3 12. The method of any one of paragraphs 1 to 11, wherein the brain volume of the human subject is obtained from a brain MRI of the human subject by multiplying the brain volume of the human subject by a coefficient of: 13. The recombinant nucleotide expression vector is administered to approximately 1.3 x 10 cells per gram of brain mass as determined by MRI. 10 GC, or approximately 6.5 × 10 per gram of brain mass as determined by MRI. 10 13. The method of any one of paragraphs 1 to 12, wherein the GC is administered in a dose of 0.1 mg / kg. 14. The recombinant nucleotide expression vector is administered to approximately 2.0 x 10 cells per gram of brain mass as determined by MRI. 11 GC, or approximately 2.9 × 10 per gram of brain mass. 11 13. The method of any one of paragraphs 1 to 12, wherein the GC is administered in a dose of 0.1 mg / kg. 15. The method of any one of paragraphs 1-12, wherein the human subject is at least 5 years old and less than 18 years old. 16. The recombinant nucleotide expression vector is administered to approximately 6.5 x 10 cells per gram of brain mass as determined by MRI. 10 16. The method of paragraph 15, wherein the GC is administered in a dose of 100 mg / kg. 17. The method of paragraph 15, wherein the recombinant nucleotide expression vector is administered in the dosages set forth in the table below. [Table 2] 18. The method of any one of paragraphs 1-12, wherein the human subject is at least 4 months old and less than 5 years old. 19. The method of paragraph 18, wherein the recombinant nucleotide expression vector is administered at a dose selected from dose 1 or dose 2 according to the table below. [Table 3] 20. The method of paragraph 18, wherein the recombinant nucleotide expression vector is administered in the dosages set forth in the table below. [Table 4] TIFF2024505739000005.tif13416521.The method of any one of paragraphs 1 to 20, wherein the recombinant nucleotide expression vector is administered via intracisternal (IC) administration. 22. The method of any one of paragraphs 1 to 20, wherein the recombinant nucleotide expression vector is administered via intracerebroventricular (ICV) administration. 23. The method of any one of paragraphs 1 to 22, wherein the recombinant nucleotide expression vector is administered in a volume not exceeding 10% of the total volume of cerebrospinal fluid of the human subject. 24. The method of any one of paragraphs 1 to 23, wherein the glycosylated recombinant human IDS precursor is delivered to lysosomes within cells of the CNS of the human subject. 25. The method of any one of paragraphs 1 to 24, further comprising administering immunosuppressive therapy to the human subject prior to or concurrently with administering the human IDS precursor treatment, and optionally continuing the immunosuppressive therapy thereafter. 26. The method of paragraph 25, wherein the immunosuppressive therapy comprises administering one or more corticosteroids, sirolimus, and / or tacrolimus. 27. The method of paragraph 26, wherein the one or more corticosteroids is methylprednisolone and / or prednisone. 28. The method of any one of paragraphs 25 to 27, further comprising administering one or more antibiotics to the human subject prior to or concurrently with the immunosuppressive therapy. 29. The method of paragraph 28, wherein the one or more antibiotics is trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atovaquone. 30. The method of any one of paragraphs 25 to 29, further comprising administering one or more antifungal therapies to the human subject prior to or concurrently with the immunosuppressive therapy. 31. The method of any one of paragraphs 1 to 30, further comprising measuring one or more of the following biomarkers after administering the recombinant nucleotide expression vector: (a) levels of glycosaminoglycans (GAGs) in the CSF, (b) levels of iduronate-2-sulfatase (I2S) in the CSF, (c) levels of GAGs in plasma, (d) levels of I2S in plasma, (e) levels of leukocyte I2S enzyme activity, and (f) levels of GAGs in urine. 32. The method of paragraph 31, wherein the GAG ​​in the CSF comprises heparin sulfate in the CSF. 33. The method of paragraph 31, wherein the GAG ​​in the CSF is heparin sulfate in the CSF. 34. The method of any one of paragraphs 31 to 33, wherein the GAGs in the plasma comprise heparin sulfate in the plasma. 35. The method of any one of paragraphs 31 to 33, wherein the GAG ​​in plasma is heparin sulfate in plasma. 36. The method of any one of paragraphs 31-35, wherein the urinary GAG comprises urinary heparin sulfate. 37. The method of any one of paragraphs 31 to 35, wherein the urinary GAG is urinary heparin sulfate. 38. The method of any one of paragraphs 31 to 37, wherein the measuring step includes measuring the level of heparin sulfate in the CSF. 39. The method of any one of paragraphs 31 to 38, wherein the measuring step comprises measuring the 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), comprising administering a recombinant adeno-associated viral vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) to the cerebrospinal fluid (CSF) of said human subject in need of treatment. 2. A method for treating hepatosplenomegaly and central nervous system (CNS) symptoms in a human subject diagnosed with MPS II, comprising administering a single dose of rAAV encoding hIDS to the CSF of said human subject in need of treatment, without further treatment for MPS II outside of 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 for delivering an rAAV encoding a hIDS to the liver and / or spleen of a human subject diagnosed with MPS II, comprising administering a single dose of said rAAV into the CSF of said human subject. 7. The method of any one of paragraphs 1-6, wherein the method reduces the size of the liver by about 10%, about 20%, or about 30% compared to the size of the liver before administration of the rAAV. 8. The method of any one of paragraphs 1-7, wherein the method reduces the size of the spleen by about 10%, about 20%, or about 30% compared to the size of the spleen before administration of the rAAV. 9. A method for treating a human subject diagnosed with MPS II, comprising: (i) administering a therapeutically effective amount of an rAAV encoding a hIDS to the CSF of the human subject; and (ii) measuring the level of heparin sulfate (HS) D2S6 in the CSF of the subject. 10. The method of paragraph 9, wherein a decrease in HS D2S6 correlates with an improvement in neurocognitive parameters in a human subject. 11. The method of paragraph 9 or 10, further comprising the step of determining whether further treatment is required. 12. The method of paragraph 11, further comprising administering a further treatment. 13. The method of paragraph 12, wherein the further treatment is a second administration of the same rAAV as in step (i). 14. The method of paragraph 12, wherein the further treatment is a second administration of the same rAAV as in step (i) at a higher dose. 15. The method according to paragraph 12, wherein said further treatment is enzyme replacement therapy utilizing recombinant idursulfase. 16. The method of any one of paragraphs 1-15, wherein the method results in a decrease in the level of HS D2S6 in the CSF of the subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than about 95% compared to the level of HS D2S6 in the CSF of the subject before administering the rAAV. 17. The method of paragraph 16, wherein the level of HS D2S6 is 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 reduction in HS D2S6 levels persists for at least 6 months, at least 9 months, at least 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 rAAV has a virulence of approximately 1.3 x 10, as determined by MRI. 10 GC / g brain mass, approximately 6.5×10 10 GC / g brain mass, or 2 × 10 11 19. The method of any one of paragraphs 1 to 18, wherein the dose is administered at a dose of GC / g brain mass. 20. The method of any one of paragraphs 1-19, wherein the human subject is 5 years of age or older and less than 18 years of age. 21. The method of any one of paragraphs 1-19, wherein the human subject is at least 4 months old and less than 5 years old. 22. The method of any one of paragraphs 1 to 21, wherein the human subject is undergoing enzyme replacement therapy (ERT) at the time of administration of the rAAV. 23. The method of paragraph 22, wherein the human subject is non-responsive to ERT. 24. The method of paragraph 22 or 23, wherein the ERT is recombinant idursulfase. 25. The method of any one of paragraphs 22 to 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 administration of the rAAV. 26. The method of any one of paragraphs 1 to 25, wherein the rAAV is administered via intracisternal (IC) administration. 27. The method of any one of paragraphs 1 to 25, wherein the rAAV is administered via intracerebroventricular (ICV) administration. 28. The method of any one of paragraphs 1 to 27, wherein the rAAV is administered in a volume not exceeding 10% of the total volume of cerebrospinal fluid of the human subject. 29. The method of any one of paragraphs 1 to 28, wherein the rAAV is recombinant adeno-associated virus serotype 9. 30. The method of paragraph 29, wherein the rAAV contains a human IDS expression cassette, and 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) a chicken beta-actin intron, and (iii) a rabbit beta-globin polyadenylation (polyA) signal. 32. The method of paragraph 31, wherein the ITR is an AAV2 ITR. 33. The method of any one of paragraphs 30 to 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 to 33, further comprising administering immunosuppressive therapy to the human subject prior to or at the time of administration of the rAAV, and optionally continuing the immunosuppressive therapy thereafter. 35. The method of paragraph 34, wherein the immunosuppressive therapy comprises administering one or more of a corticosteroid, sirolimus, and / or tacrolimus. 36. The method of paragraph 35, wherein the one or more corticosteroids is methylprednisolone and / or prednisone. 37. The method of any one of paragraphs 1 to 36, further comprising administering one or more antibiotics to the human subject prior to or simultaneously 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 to 38, further comprising administering one or more antifungal therapies to the human subject prior to or concurrently with the rAAV. 40. A method of treating a human subject diagnosed with MPS II, comprising: (a) administering to the human subject a therapeutically effective amount of an rAAV encoding a hIDS, wherein the human subject has been treated with or is undergoing treatment 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 is obtained from the human subject after administration, and 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 to the human subject a therapeutically effective amount of an rAAV encoding a hIDS, wherein the human subject has been treated with or is undergoing treatment 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 prior to administration and 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 said 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 baseline before discontinuing ERT. 44. The method of paragraph 41, wherein said 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 baseline before discontinuing ERT. 45. The method of any one of paragraphs 40 to 44, wherein the criterion is a predetermined value. 46. ​​A method according to any one of paragraphs 40 to 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 to 44, wherein the criterion is the level of 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 to 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 rAAV is administered to the human subject and the ERT treatment is discontinued 52 weeks later. 50. The method of any one of paragraphs 40 to 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 said human subject, wherein said human subject has been treated with or is undergoing treatment with ERT; and (b) administering a therapeutically effective amount of rAAV encoding a hIDS to a human subject, said administration occurring after ERT treatment in said human subject has ceased. 52. The method of paragraph 51, wherein said 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 1 day before said administration. 53. The method of any one of paragraphs 51-52, wherein the level of at least one biomarker is undetectable in a biological sample from the human subject prior to discontinuation. 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 to 55, wherein the biological sample is serum. 57. A method for determining or monitoring the effectiveness of a treatment for MPS II in a human subject, comprising administering to the human subject a therapeutically effective amount of an rAAV encoding a hIDS, wherein a decrease in the level of D2S6 in a biological sample from the human subject compared to a reference indicates the effectiveness of the treatment for MPS II in the human subject, and wherein the biological sample is obtained from the human subject after administration. 58. The method of any one of paragraphs 40 to 57, wherein the human subject has hepatosplenomegaly. 59. The method of paragraph 58, wherein the human subject has been treated with ERT prior to administration and / or has been treated with ERT after administration. 60. The method of any one of paragraphs 40 to 59, wherein the ERT is enzyme replacement therapy using recombinant idursulfase. 61. The method of any one of paragraphs 57 to 60, wherein the biological sample is CSF. 62. The method of any one of paragraphs 57-61, wherein the reduction in the level of D2S6 is about or at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% reduction compared to said reference standard. 63. The method of any one of paragraphs 57-62, wherein said efficacy of treating MPS II is improvement in at least one subtest of the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III) compared to baseline. 64. The method of paragraph 63, wherein the at least one subtest is an age-equivalent score, a cognitive development quotient (DQ), an expressive language DQ, a receptive language DQ, a gross motor DQ, and / or a 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-equivalent score increases over about or at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 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 to 66, wherein the criterion is a score on at least one subtest of the BSID-III obtained from the human subject prior to administration. 68. The method of any one of paragraphs 63 to 66, wherein the reference standard is the mean score of at least one subtest of the BSID-III obtained from human subjects with MPS II who are age-matched to the human subject. 69. The method of any one of paragraphs 57 to 68, wherein the reference is the level of D2S6 in a biological sample obtained from the human subject prior to administration. 70. The biological sample is administered to a patient in a range of 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, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 810, 820, 830, 840, 850, 860, 870, 880, 890 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 later from the human subject. 71. The method of any one of paragraphs 51 to 70, wherein the human subject is a pediatric subject. 72. The method of any one of paragraphs 57 to 71, wherein said efficacy of treating MPS II 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%, compared to the level of D2S6 in said human subject prior to administration. 73. The method of any one of paragraphs 1 to 72, wherein the rAAV is administered intrathecally to the human subject. 74. The rAAV: (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% (vol / vol); (g) monosodium phosphate monohydrate at a concentration of about 0.0278 g / L, and (h) disodium phosphate anhydrous at a concentration of about 0.114 g / L. 75. A method for identifying or diagnosing a subject as having neuropathic MPS II, comprising: (a) determining the level of one or more heparan sulfate disaccharides in a biological sample from said subject; (b) identifying or diagnosing the subject as having neuropathic MPS II if the level of one or more heparan sulfate disaccharides is elevated compared to a reference level; and (c) administering a therapeutically effective amount of an rAAV encoding a hIDS to the subject identified or diagnosed as having neuropathic MPS II. 76. The method of paragraph 75, wherein the one or more heparan sulfate disaccharides include 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 disaccharides is D2S6. 78. A method for identifying or diagnosing a subject as having neuropathic MPS II, comprising: If the level of D2S6 in a biological sample from a subject is higher compared to a reference level, the subject is identified or diagnosed as having neuropathic MPS II, and a therapeutically effective amount of an rAAV encoding a hIDS is administered to the subject identified or diagnosed as having neuropathic MPS II. 79. The method of any one of paragraphs 75 to 78, wherein the biological sample is cerebrospinal fluid. 80. The method of any one of paragraphs 75-79, wherein the subject is pre-symptomatic or has no visible or detectable MPS II symptoms. 81. The method of any one of paragraphs 75 to 80, wherein the subject has MPS II. 82. The method of any one of paragraphs 75 to 77 and 79 to 81, wherein the reference level is the level of the at least one or more heparan sulfate disaccharides in a biological sample from one or more healthy individuals and / or one or more non-neuropathic subjects. 83. The method of any one of paragraphs 77 to 81, wherein the reference level is the level of D2S6 in a biological sample from one or more healthy individuals and / or one or more non-neuropathic subjects. 84. The method of paragraph 82 or 83, wherein the biological sample from one or more healthy and / or one or more non-neuropathic subjects is a CSF sample. 85. The method of any one of paragraphs 75 to 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 more than 40% of the total heparan sulfate disaccharides (HS) in said biological sample from said 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 said biological sample from said subject. 88. The level of one or more heparan sulfate disaccharides or the level of D2S6 in the biological sample from the subject is about or at least about 50ng / mL, 75ng / mL, 80ng / mL, 85ng / mL, 90ng / mL, 95ng / mL, 100ng / mL, 105ng / mL, 110ng / mL, 115ng / mL, 120ng / mL, 125ng / mL, 130ng / mL, 135ng / mL, 140ng / mL, 145ng / mL, 150ng / mL, 155ng / mL, 160ng / mL, 165ng / mL, 170ng / mL, 175ng / mL, 180ng / mL, 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 greater than 400 ng / mL. 89. The method of any one of paragraphs 75 to 88, wherein the level of the one or more heparan sulfate disaccharides 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, 200 ng / mL, or greater than 200 ng / mL. 90. The level of one or more heparan sulfate disaccharides or the level of D2S6 in the biological sample from the subject is 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, or 89. The method of any one of paragraphs 75-89, wherein the serum albumin exhibits an increase of 270ng / mL, 280ng / mL, 290ng / mL, 300ng / mL, 310ng / mL, 320ng / mL, 330ng / mL, 340ng / mL, 350ng / mL, 360ng / mL, 370ng / mL, 380ng / mL, 390ng / mL, 400ng / mL, 410ng / mL, 420ng / mL, 430ng / mL, 440ng / mL, 450ng / mL, 460ng / mL, 470ng / mL, 480ng / mL, 490ng / mL, 500ng / mL, or greater than 500ng / mL. 91. A method for determining or monitoring the effectiveness of a treatment for MPS I in a human subject, comprising administering to the human subject a therapeutically effective amount of an rAAV encoding human IDUA, wherein a decrease in the level of I0S6 in a biological sample from the human subject compared to a reference indicates the effectiveness of the treatment for MPS I in the human subject, and wherein the biological sample is obtained from the human subject after administration. 92. The method of paragraph 91, wherein the biological sample is plasma. 93. The method of paragraph 90 or 91, wherein the human subject has been treated with ERT prior to administration and / or has been treated with ERT after administration. 94. The method of any one of paragraphs 91 to 93, wherein the ERT is enzyme replacement therapy using recombinant idursulfase. 95. The method of any one of paragraphs 91 to 94, wherein the reduction in the level of I0S6 is about or at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction compared to the reference standard. 96. The method of any one of paragraphs 91 to 95, wherein the reference is the level of I0S6 in a biological sample obtained from the human subject prior to administration. 97. The method of any one of paragraphs 91 to 95, wherein the criterion is a predetermined value. 98. The method of any one of paragraphs 91 to 95, wherein the reference is the level of I0S6 in a biological sample obtained from another human subject diagnosed with MPS I or from a population of human subjects diagnosed with MPS I. 99. The method of any one of paragraphs 91-98, wherein said efficacy of MPS I treatment is improvement in at least one subtest of the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III) compared to baseline. 100. The method of paragraph 99, wherein the at least one subtest is an age-equivalent score, a cognitive development quotient (DQ), an expressive language DQ, a receptive language DQ, a gross motor DQ, and / or a fine motor DQ. 101. The method of paragraph 99 or 100, wherein the criterion is the score of at least one bouton of the BSID-III obtained from the human subject prior to administration. 102. The method of any one of paragraphs 99-101, wherein the reference standard is the mean score of 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 rAAV: (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% (vol / vol); (g) monosodium phosphate monohydrate at a concentration of about 0.0278 g / L, and 103. The method of any one of paragraphs 91 to 102, wherein the human subject is administered a solution comprising (h) disodium phosphate anhydrous at a concentration of about 0.114 g / L. [Brief explanation of the drawings]

[0071] 4. Brief description of the drawings [Figure 1] Figure 1 shows the amino acid sequence of human IDS. The post-translational formylglycine modification at C84 (shown in bold in Figure 1) is required for enzymatic activity. Eight N-linked glycosylation sites (N31, N115, N144, N246, N280, N325, N513, and N537) are bolded and boxed. One tyrosine-O-sulfation site (Y) is bolded, and the entire sulfation site sequence (PSSEKY165ENTKTCRGPD) is boxed. The N-terminal mature 42 kDa and mature 14 kDa polypeptides are indicated by horizontal arrows. In the brain, the N-terminal mature 42 kDa form begins at position 34 or 36, as shown in Figure 1: T34DALNVLLI and A36LNVLLIIV. (See Sleat, 2005, Proteomics 5:1520-1532, Table S2.) Two of the eight N-linked glycosylation sites, N280 and N116, undergo mannose-6-phosphorylation in IDS from human brain (reported in Sleat et al., 2006, Mol & Cell Proteomics 5.4:686-701, Table V).

[0072] [Figure 2]Multiple sequence alignment of hIDS with known orthologues. The species names and protein IDs are as follows: SP|P22304|IDS_HUMAN [human]; 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 marmoset)], TR|U3DTL8_CALJA [Callithrix jacchus (white-tufted marmoset)], TR|G7NRX7_MACMU [Macaca mulatta (rhesus macaque)], TR|G7Q1V9_MACFA [Macaca fascicularis (cynomolgus monkey)], TR|H2PX10_PONAB [Pongo abelii (Sumatran orangutan)], TR|A0A0D9R4D1_CHLSB [Chlorocebus sabaeus (Green monkey)], TR|G1RST8|G1RST8_N0MLE [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 (Craniomolgus macaque, Cynomolgus monkey)], UPI000533297F [Rhinopithecus roxellana (Golden snub-nosed monkey, Pygathrix roxellana)), UPI0005F40BD2 [Colobus angolensis palliates (Peters' Angola colobus)] (SEQ ID NOs: 27 to 44).

[0073] [Figure 3]hIDS and MPS II mutations with corresponding mild, moderate or severe disease phenotypes (obtained from Uniprot).

[0074] [Figure 4] Human IDS processing reported in Figure 7 of Millat et al., 1997, Exp. Cell. Res. 230:362-367.

[0075] [Figure 5] FIG. 1 is a schematic diagram of Construct 1.

[0076] [Figure 6] Clustal multiple sequence alignment of AAV capsids 1-9 (SEQ ID NOS: 16-26). Amino acid substitutions (shown in bold in the bottom row) can be made into AAV9 and AAV8 capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequence regions labeled "HVR" = hypervariable regions.

[0077] [Figure 7] Heparan sulfate (HS) digestion using heparinase.

[0078] [Figure 8] Graph showing cerebrospinal fluid (CSF) heparan sulfate biomarkers (ng / mL). The graph demonstrated consistent HS reduction in CSF after administration of Construct 1. 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 time point (N=6) was -35.8% (p-value = 0.03 as measured by Wilcoxon signed-rank test). The graph showed measurable CSF I2S enzyme concentrations in Cohort 2 after administration of Construct 1, ranging from 1170 to 1940 pg / mL.

[0079] [Figure 9]Graph showing cerebrospinal 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 time point (N=6) was -39.2% (p-value=0.03 as measured by Wilcoxon signed-rank test).

[0080] [Figure 10A] Graphs showing neurodevelopmental functioning compared with age peers (months) and age (months). Graph A shows continued cognitive development in four of the five patients at >6 months follow-up. Patients 1, 3, and 5 showed continued cognitive development within normal limits. Patients 2 and 4 showed significant cognitive delays at baseline. Patient 2 continued to develop cognitively. Patient 4 acquired expressive and receptive language skills. [Figure 10B] Graphs showing neurodevelopmental functioning compared with age equivalence (months) and age (months). A and C show the cognitive development index and age equivalence, respectively, for patients in the Construct 1 gene therapy clinical trial. Graphs B and C show that three of four patients who entered the trial with cognitive skills above -2 SD of normal remained above -2 SD at greater than 6 months of follow-up. One patient entered the trial with significant delays in neurocognitive development at baseline, but showed relative stability after receiving Construct 1 at an older age (59 months of age), continuing to acquire expressive and receptive language skills. [Figure 10C]Graphs showing neurodevelopmental function comparing age equivalence (months) with age (months). A and C show the cognitive development index and age equivalence, respectively, for patients in the Construct 1 gene therapy clinical trial. Graphs B and C show that three of four patients who entered the trial with cognitive skills above -2 SD of normal remained above -2 SD at greater than 6 months of follow-up. One patient entered the trial with significant delays in neurocognitive development at baseline, but showed relative stability after receiving Construct 1 at an older age (59 months of age), continuing to acquire expressive and receptive language skills.

[0081] [Figure 11-1] Graphs A-B show neurodevelopmental function in terms of language and motor skills. A shows expressive communication. B shows receptive communication. The graphs show the continued acquisition of language and / or motor skills for patients over a 6-month follow-up period. [Figure 11-2] C-D are graphs showing neurodevelopmental function in terms of language and motor skills. C shows gross motor skills. D shows fine motor skills. The graphs show the continued acquisition of language and / or motor skills for patients over a 6-month follow-up period.

[0082] [Figure 12] Graph showing systemic efficacy measured by plasma I2S protein concentration (pg / mL). This graph shows a general increase in plasma I2S enzyme levels in five of six patients after administration of Construct 1 (normal range (14,706 pg / mL to <100,000 pg / mL)).

[0083] [Figure 13] Graph showing systemic efficacy measured by measuring urinary total GAG levels (g / mol CK) in ERT-treated patients. The graph showed a sustained decrease in urinary GAG levels in all patients treated with ERT.

[0084] [Figure 14] Graph showing systemic efficacy measured by measuring urinary total GAG levels (g / mol CK) in ERT-naive and ERT-discontinued patients. The graph shows a rapid decrease in urinary GAGs in ERT-naive patients after administration of Construct 1 (lack of urinary GAG rebound after ERT discontinuation).

[0085] [Figure 15] Ultrasound of the liver or spleen in ERT-naive patients. Liver and spleen dimensions in ERT-naive patients decreased over 24 weeks after administration of Construct 1.

[0086] [Figure 16] Proof-of-concept research activities.

[0087] [Figure 17] Dosage rationale for first-in-human clinical trials.

[0088] [Figure 18] Total heparan sulfate in CSF samples from healthy individuals, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects.

[0089] [Figure 19A] Graph showing levels of D0S0 disaccharide in healthy individuals, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects. [Figure 19B] Graph showing levels of D0A6 disaccharide in healthy individuals, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects. [Figure 19C] Graph showing levels of D0A0 in healthy individuals, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects.

[0090] [Figure 20]Graph showing levels of D2S6 disaccharide in healthy individuals, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects.

[0091] [Figure 21] Graph showing percent composition of heparan sulfate disaccharides in healthy individuals, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects.

[0092] [Figure 22] Schematic diagram of the MPS II Phase 1 / 2 clinical trial.

[0093] [Figure 23] Graph showing widespread CNS and systemic biodistribution in non-humans following IC administration of Construct 1. The terms "LD" is low dose, "IS" is immunosuppressive, and "HD" is high dose.

[0094] [Figure 24] Graph showing the cerebrospinal fluid (CSF) biomarker heparan sulfate (HS) in subjects from the Phase 1 / 2 study. CSF HS measurements showed a dose-dependent decrease in cohorts 1-3 at weeks 8 and 24.

[0095] [Figure 25] Graph showing D2S6 concentrations in patients in the Phase 1 / 2 study. CSF D2S6 measurements showed a dose-dependent decrease in D2S6 levels in cohorts 1-3 at weeks 8 and 24, with levels approaching normal in cohort 3 participants.

[0096] [Figure 26] Graph showing cognitive, expressive language, and fine neurodevelopmental functioning of Cohort 1 patients in the Phase 1 / 2 study.

[0097] [Figure 27] Graph showing cognitive, expressive language, and fine neurodevelopmental functioning of Cohort 2 patients in the Phase 1 / 2 study.

[0098] [Figure 28] Graph showing maladaptive behavior index and toileting skills of patients in a Phase 1 / 2 study.

[0099] [Figure 29] Graph showing plasma I2S protein levels and urinary GAG levels in patients in a Phase 1 / 2 study.

[0100] [Figure 30] Schematic diagram of the MPS I Phase 1 / 2 clinical trial.

[0101] [Figure 31] Graphs showing cerebrospinal fluid (CSF) biomarker and heparin sulfate concentrations in participants (A) and single participants (B) in the MPS I Phase 1 / 2 study. The graphs show the reduction in CSF heparin sulfate for all participants at the last available time point (e.g., Week 24 for Phase 1 / 2 (A) and Week 59 for single participants (B)). The study showed measurable CSF IDUA enzyme activity in the majority of Phase 1 / 2 participants and single participants.

[0102] [Figure 32-1] Graphs A-C show neurodevelopmental function BSID-III as cognition, expressive language, and fine motor in the MPS I Phase 1 / 2 study and single participant study. [Figure 32-2] D-F are graphs showing neurodevelopmental function BSID-III as cognition, expressive language, and fine motor in the MPS I Phase 1 / 2 study and single participant study.

[0103] [Figure 33] 13 is a graph showing neurodevelopmental function BSID-III cognition in the MPS I single participant study.

[0104] [Figure 34]1 is a table showing neurodevelopmental functioning (WASI-II and VABS-III) of 13-year-old MPS I Phase 1 / 2 study participants.

[0105] [Figure 35] Graph showing levels of I0S6 in MPS I Phase 1 / 2 participants (A) and single participant study (B).

[0106] [Figure 36] Graph showing urinary total GAG levels in MPS I Phase 1 / 2 participants (A) and single participant study (B). DETAILED DESCRIPTION OF THE INVENTION

[0107] 5. Detailed Description of the Invention The present 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 human subjects diagnosed with Mucopolysaccharidosis Type 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 October 19, 2017 as WO / 2017 / 181113), the entire contents of which are incorporated herein by reference, for compositions and methods that can be used in accordance with the invention described herein.

[0108] In a preferred embodiment, treatment is via gene therapy, e.g., administering a viral vector or other DNA expression construct encoding human IDS (hIDS) or a derivative of hIDS into the CSF of a patient (human subject) diagnosed with MPS II to create a permanent depot of transduced neuronal and / or glial cells that continuously delivers the transgene product to the CNS. The rhIDS secreted into the CSF from the neuronal / glial depot is taken up by cells in the CNS, "cross-correcting" the enzyme deficiency in the recipient cells. Furthermore, it has been unexpectedly discovered that the depot of transduced neuronal and glial cells in the CNS allows for the delivery of recombinant enzyme both to the CNS and throughout the body, potentially reducing or eliminating the need for systemic therapy, e.g., weekly intravenous injections of the enzyme. Also provided herein is treating MPS I by administering to a subject a viral vector or other DNA expression construct encoding human IDUA (e.g., a non-replicating recombinant AAV of serotype 9 capsid containing a hIDUA expression cassette; Construct 2; see PCT / US2021 / 014129; PCT / US2018 / 015910; and PCT / US2019 / 042205, each of which is incorporated by reference in its entirety).

[0109] In alternative embodiments, hIDS can be produced in cell cultures (e.g., bioreactors) of human neuronal or glial cells and administered as enzyme replacement therapy ("ERT"), e.g., by injection of the enzyme, into the CSF, directly into the CNS, and / or systemically. However, gene therapy approaches offer several advantages over ERT, as enzymes cannot cross the blood-brain barrier, systemic delivery of enzymes does not result in treatment of the CNS, and unlike the gene therapy approaches of the present invention, direct delivery of enzymes to the CNS requires repeated injections, which are not only burdensome but also introduce the risk of infection.

[0110] 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 (shown in FIG. 1), and derivatives of hIDS having amino acid substitutions, deletions, or additions, such as, but not limited to, amino acid substitutions selected from non-conserved residues corresponding to orthologs of IDS shown in FIG. 2, provided that such mutations do not include substitutions of the cysteine ​​residue at position 84 (C84) required for enzymatic activity (Millat et al., 1997, Biochem J 326:243-247), or as shown in FIG. 3, or as described in Sukegawa-Hayasaka et al., 2006, J Inherit Metab Dis, each of which is incorporated herein by reference in its entirety. 29:755-761 (reporting the "weak" variants R48P, A85T, W337R, and the truncating variant Q531X, and the "severe" variants P86L, S333L, S349I, R468Q, R468L), Millat et al., 1998, BBA 1406:214-218 (reporting the "weak" variants P480L and P480Q; and the "severe" variant P86L), and Bonucelli et al., 2001, BBA 1537:233-238.

[0111] For example, amino acid substitutions at specific positions in hIDS can be selected from the corresponding nonconservative amino acid residues found at those positions in the IDS orthologs set out in Figure 2, provided that they do not contain any deleterious mutations, such as those shown in Figure 3 or those reported in Sukegawa-Hayasaka et al., 2006, supra; Millat et al., 1998 (supra); and Bonucelli et al., 2001, supra, each of which is incorporated by reference in its entirety. The resulting transgene product can be tested in vitro in cell culture or in test animals using conventional assays to ensure that the mutations do not impair IDS function. Preferred amino acid substitutions, deletions, or additions selected should maintain or increase the enzymatic activity, stability, or half-life of the IDS when tested in conventional in vitro assays for MPS II in cell culture or animal models. For example, the enzymatic activity of the transgene product can be assessed using a conventional enzyme assay using, for example, 4-methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate or 4-methylumbelliferyl sulfate as a substrate (for exemplary IDS enzyme assays that can be used, see, for example, Lee et al., 2015, Clin. Biochem. 48(18):1350-1353; Dean et al., 2006, Clin. Chem. 52(4):643-649, the entire contents of each of which are incorporated herein by reference). The ability of the transgene product to correct the MPS II phenotype can be assessed in cell culture. For example, cultured MPS II cells can be transduced with a viral vector or other DNA expression construct encoding rhIDS or a derivative, the transgene product or derivative can be added to cultured MPS II cells, or MPS II cells can be co-cultured with human neuronal / glial host cells genetically engineered to express and secrete rhIDS or a derivative, and correction of the defect in the cultured MPS II cells can be determined, e.g., by detecting a decrease in IDS enzyme activity and / or GAG storage in the cultured MPS II cells.(See, eg, Stroncek et al., 1999, Transfusion 39(4):343-350, the entire contents of which are incorporated herein by reference).

[0112] Animal models for MPS II that can be used to evaluate the therapeutic agents described herein have been described. For example, a knockout mouse model of MPS II (IDS knockout) was engineered by replacing exons 4 and 5 of the IDS gene with a neomycin resistance gene (Garcia et al., 2007, J Inherit Metab Dis 30:924-34). This IDS knockout mouse exhibits many of the hallmarks of MPS II, including skeletal abnormalities, hepatosplenomegaly, elevated urinary and tissue GAGs, and brain storage lesions (Muenzer et al., 2001, Acta Paediatr Suppl 91:98-99), and has been used to evaluate the effects of enzyme replacement therapy in MPS II to support clinical trials of ERT. Therefore, this mouse model is suitable 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, the entire contents of which are incorporated herein by reference).

[0113] Preferably, the hIDS transgene produced by human neuronal / glial cells should be controlled by expression control elements functional in neuronal and / or glial cells, such as the CB7 promoter (chicken β-actin promoter and CMV enhancer), and may include other expression control elements (e.g., chicken β-actin intron and rabbit β-globin polyA signal) that enhance vector-driven transgene expression. 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 for use in CNS cells include, but are not limited to, the following: Oligodendrocyte myelin glycoprotein (hOMG) signal peptide: MEYQILKMSLCLFILLFLTPGILC (SEQ ID NO: 2) Cellular repressor of E1A-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 (hPCADHA1) 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). A signal peptide may also be referred to herein as a leader sequence or leader peptide.

[0114] The recombinant vector used to deliver the transgene should have tropism for cells in the CNS, such as, but not limited to, neurons and / or glial cells. Such vectors can include non-replicating recombinant adeno-associated viral vectors ("rAAV"), with viral vectors having AAV9 or AAVrh10 capsids being particularly preferred. AAV variant capsids, particularly AAV / hu.31 and AAV / hu.32, described by Wilson in U.S. Pat. No. 7,906,111, the entire contents of which are incorporated herein by reference, as well as AAV variant capsids described by Chatterjee in U.S. Pat. Nos. 8,628,966, 8,927,514, and Smith et al., 2014, Mol Ther 22:1625-1634, the entire contents of which are incorporated herein by reference, can be used. However, other viral vectors can be used, including but not limited to lentiviral vectors, vaccinia viral vectors, or non-viral expression vectors, referred to as "naked DNA" constructs.

[0115] Pharmaceutical compositions suitable for administration to the CSF include suspensions of rhIDS vectors in a formulation buffer containing a physiologically compatible aqueous buffer, a surfactant, and optional excipients. In certain embodiments, the pharmaceutical composition is suitable for intrathecal administration. In certain embodiments, the pharmaceutical composition is suitable for intracisternal administration (injection into the cisterna magna). In certain embodiments, the pharmaceutical composition is suitable for injection into the subarachnoid space via C1-2 puncture. In certain embodiments, the pharmaceutical composition is suitable for intraventricular administration. In certain embodiments, the pharmaceutical composition is suitable for administration via lumbar puncture. In some embodiments, a pharmaceutical composition comprising an rAAV of the disclosure comprises sodium chloride at a concentration of about 8.77 g / L, magnesium chloride hexahydrate 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 dehydrate at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.001% (vol / vol), sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and disodium phosphate anhydrous at a concentration of about 0.114 g / L.

[0116] A therapeutically effective dose of the recombinant vector should be administered into the CSF via intrathecal administration (i.e., injection into the subarachnoid space, allowing the recombinant vector to diffuse through the CSF and transduce cells of the CNS). In some embodiments, the recombinant vector is administered in a solution containing sodium chloride at a concentration of about 8.77 g / L, magnesium chloride hexahydrate 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 disodium phosphate anhydrous at a concentration of about 0.114 g / L. This can be accomplished in several ways, for example, by intracranial (cisternal or intraventricular) injection or injection into the lumbar cisterna. For example, intracisternal (IC) injection (into the cisterna magna) can be performed via CT-guided suboccipital puncture, or if feasible for the patient, intrathecal injection can be performed via C1-2 puncture, or lumbar puncture (a diagnostic procedure commonly performed to collect a CSF sample) can be used to access the CSF. Alternatively, intracerebroventricular (ICV) administration (a more invasive technique used to introduce anti-infective or anti-cancer drugs that do not penetrate the blood-brain barrier) can be used to instill the recombinant vector directly into the ventricles. Alternatively, intranasal administration can be used to deliver the recombinant vector to the CNS.

[0117] Due to the relatively rapid brain growth that occurs early in childhood development, the total dose of AAV9.hIDS administered IC varies depending on the estimated brain mass in different age groups, see, e.g., Table 2 below. For brain mass at the age of study subjects, see, e.g., AS Dekaban, Ann Neurol, 1978 Oct;4(4):345-56. [Table 5]

[0118] CSF concentrations can be monitored by directly measuring the concentration of rhIDS in CSF fluid obtained from an occipital or lumbar puncture, or can be estimated by extrapolation from the concentration of rhIDS detected in the patient's serum.

[0119] By way of background, human IDS contains eight potential N-glycosylation sites (N) as listed in Figure 1. 31 , N 115 , N 144 , N 246 , N 280 , N 325 , N 513 , and N 537 ) and contains a 25 amino acid signal sequence that is cleaved during processing. The initial 76 kDa intracellular precursor is converted to a phosphorylated 90 kDa precursor after its oligosaccharide chains are modified in the Golgi apparatus. This precursor is processed through various intracellular intermediates by glycosylation modification and proteolytic cleavage to the major 55 kDa form. In summary, after removal of the 25 amino acid signal sequence, proteolytic processing continues through N 31 downstream of which an N-terminal proteolytic cleavage is performed to remove a propeptide of 8 amino acids (residues 26–33) and 513 A C-terminal proteolytic cleavage occurs upstream of this enzyme, releasing an 18 kDa polypeptide and generating a 62 kDa intermediate, which is converted to the 55 kDa mature form. Further proteolytic cleavage generates the 45 kDa mature form, which is located in the lysosomal compartment. (See Figure 4 for a drawing taken from Millat et al., 1997, Exp Cell Res 230:362-367 ("Millat 1997"); see 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 herein by reference in its entirety.)

[0120] C required for enzyme activity84 Formylglycine modification of N (shown in bold in Figure 1) most likely occurs in the endoplasmic reticulum, possibly as an early post-translational or co-translational event. (See Millat 1997a, citing Schmidt et al., 1995, Cell 82:271-278.) Post-translational processing continues in the Golgi apparatus, yielding mannose-6-phosphate residues that are attached to enzymes incorporating complex sialic acid-containing glycans for delivery to the lysosomal compartment. (For a brief discussion, see Clarke, 2008, Expert Opin Pharmacother 9:311-317, the entire contents of which are incorporated herein by reference.) While there is no single glycosylation site essential for IDS stability, N 280 Glycosylation at this position is important for cellular internalization via the mannose-6-phosphate (M6P) receptor and for lysosomal targeting (Chung et al., 2014, Glycoconj J 31:309-315, first paragraph of p. 310). Under normal physiological conditions, IDS is produced at very low levels, and the enzyme is rarely, if ever, secreted from cells (Clarke, 2008, supra).

[0121] The present invention is based in part on the following principles. (i) Neurons and glial cells of 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, which are robust processes in the CNS. See, for example, Sleat et al., 2005, Proteomics 5:1520-1532, and Sleat 1996, J Biol Chem 271:19191-98, which describe the human brain mannose-6-phosphate glycoproteome and note that the brain contains many more proteins with many more individual isoforms and mannose-6-phosphorylated proteins than are 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, which report the production of secreted tyrosine-sulfated glycoproteins by neuronal cells. The entire contents of each are incorporated by reference for information regarding post-translational modifications produced by human CNS cells. (ii) The human brain produces multiple isoforms of 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 from position 34 or 36 in the brain as follows: 34 DALNVLLI; and A 36 LNVLLIIV. (Sleat, 2005, Proteomics 5:1520-1532, Table S2). Two of the eight N-linked glycosylation sites, i.e., N 280 and N 116 was found to be mannose-6-phosphorylated in IDS from human brain (Sleat et al., 2006, Mol & Cell Proeomics 5.4:686-701, reported in Table V). (iii) During hIDS processing, neurons and glial cells secrete two polypeptides, 76 kDa and 90 kDa, but only the 90 kDa polypeptide is mannose-6-phosphorylated, which is the secreted form of the enzyme required for cross-correction (Millat, 1997, Figure 1 for transduced lymphoblastoid cells; Froissart 1995, Figure 4 for transduced fibroblasts—only the 90 kDa form is phosphorylated in the culture medium). Interestingly, these results demonstrate that recombinant IDS produced by neurons and glial cells can be endocytosed more avidly by recipient CNS cells than recombinant IDS produced by other cells, such as the kidney. Daniele 2002 (Biochimica et Biophysica Acta 1588(3):203-9) demonstrated M6P receptor-mediated endocytosis of recombinant IDS from conditioned medium of transduced neuronal and glial cell cultures by recipient populations of untransduced neuronal and glial cells appropriately treated with the precursor to the 45 kDa mature active form. The uptake of recombinant IDS produced by neuronal and glial cell lines (74% endocytosis) far exceeded the uptake of the enzyme produced by a kidney cell line (5.6% endocytosis). In both cases, uptake was inhibited by M6P, indicating that uptake of recombinant IDS is mediated by the M6P receptor. (See Daniele 2002, Tables 2 and 4; discussion of the results on pp. 205-206 is summarized in Table 3 below.) [Table 6] (iv) The gene therapy approach described herein will result in the continuous secretion of an approximately 90 kDa hIDS glycoprotein precursor, as determined by polyacrylamide gel electrophoresis (depending on the assay used), that is enzymatically active. First, the C required for IDS activity will be identified. 84FGly synthase (FGE, also known as SUMF1), an enzyme involved in the formylglycine modification of rhIDS, is expressed in the cerebral cortex of the human brain (gene expression data for SUMF1 can be accessed, for example, at GeneCards, http: / / www.genecards.org). Glycosylated / phosphorylated rIDS secreted by transduced neurons and glial cells in situ may then be taken up and appropriately processed by non-transduced neurons and glial cells in the CNS. Without being bound by any theory, rhIDS precursors secreted in situ by gene therapy may be more avidly endocytosed by recipient cells in the CNS than conventional recombinant enzymes used in ERT. For example, Elaprase® (produced in the fibrosarcoma cell line HT1080) is a purified protein reported to have a molecular weight of approximately 76 kDa, rather than the 90 kDa species thought to be highly phosphorylated and secreted by neurons and glial cells. The eight N-linked glycosylation sites are fully occupied in Elaprase® and reported to contain two bismannose-6-phosphate terminal glycans as well as a highly sialylated glycan complex, but the C -terminated glycan, which is an absolute requirement for enzymatic activity, is not present. 84 Only about 50% of the protein is post-translationally modified to FGly (Clarke, 2008, Expert Opin Pharmacother 9:311-317; Elaprase® Full Prescribing Information and EMA filing). Another recombinant product, Hunterase®, is produced in CHO cells. It has been reported to have more FGly and higher activity than Elaprase®, but no differences were found in mannose-6-phosphorylation or incorporation (Chung, 2014, Glycoconj J 31:309-315). (v) The efficacy of IDS in vivo and outside the cells is determined by M6P and its active site formylglycine (FGly), i.e., post-translational modification by formylglycine-generating enzymes. 84The uptake (cellular and lysosomal internalization) of IDS is dependent on the FGly-mediated uptake of IDS, which is converted from IDS. As shown in Table 3 above, brain cells (neuronal and glial) exhibit higher enzyme activity when incubated with IDS precursor medium secreted by transduced neurons and glial cells than when incubated with IDS precursor medium secreted by genetically engineered kidney cells. The resulting five-fold increase in activity is largely due to efficient IDS uptake (see Daniele 2002, Tables 2 and 4). The FGly content of commercially available IDS produced by CHO cells or HT-1080 cells is approximately 50% to 70%, which determines the enzyme activity. However, neurons and glial cells may enhance this activity due to improved IDS uptake. (vi) Cellular and intracellular transport / uptake of lysosomal proteins, such as IDS, is mediated by M6P. As reported in Daniele 2002 and Sleat, Proteomics, 2005, IDS derived from brain cells can contain high levels of M6P (indicating that the human brain contains more Man6-P glycoproteins (quantitatively and qualitatively) than other tissues). It is possible to measure the M6P content of IDS precursors, as performed in Daniele 2002. In the presence of inhibitory M6P (e.g., 5 mM), the uptake of IDS precursors produced by non-neuronal or non-glial cells, such as the genetically engineered kidney cells in Daniele 2002, is predicted to be reduced to levels similar to those of control cells, as shown in Daniele 2002. In the presence of inhibitory M6P, uptake of IDS precursors produced by brain cells, including neurons and glial cells, is predicted to remain at high levels, as shown in Daniele 2002, with uptake fourfold greater than control cells and comparable to the level of IDS activity (or uptake) of IDS precursors produced by genetically engineered kidney cells in the absence of inhibitory M6P. This assay provides a method for predicting the M6P content of IDS precursors produced by brain cells and, in particular, for comparing the M6P content of IDS precursors produced by different cell types. The gene therapy approach described herein results in continuous secretion of hIDS precursors that can be taken up by neurons and glial cells at high levels in such assays in the presence of inhibitory M6P. (vii) The M6P content and incorporation of IDS precursors can be demonstrated by gel bands at 90 kDa and 76 kDa (e.g., SDS-PAGE gel bands). The 90 kDa band is reported to be highly glycosylated / phosphorylated and also contain M6P, whereas the 76 kDa band does not. Similar to the gel bands of IDS precursors generated from genetically engineered kidney cells (Daniele 2002, Figure 1), very broad gel bands with average molecular weights ranging from 76 kDa to 95 kDa and 80 to 85 kDa can be contrasted with the gel bands of IDS precursors generated from brain cells. In Daniele 2002, immunoprecipitation of the IDS precursor was unsuccessful, so no gel bands could be obtained. The gene therapy approach described herein will result in the continuous secretion of hIDS precursors, distinct from the IDS precursor gel bands generated from genetically engineered kidney cells. (viii) The M6P content of commercially available IDS precursors is 2–2.5 mol / mol, with the majority present in the form of di-phosphorylated glycans. On average, all IDS precursors are phosphorylated, but the normal distribution of glycans, assuming multiple phosphorylation sites, includes some IDS precursors with two, one, and zero di-phosphorylated M6P glycans. The incorporation rate would be significantly higher with multiple phosphorylations. (ix) Glycosylation of hIDS by human cells of the CNS adds glycans that can enhance stability, half-life, and reduce undesired aggregation of the transgene product. Importantly, the glycans added to the hIDS of the present invention contain 2,6-sialic acid and incorporate Neu5Ac ("NANA"), but not its hydroxylated derivative, NeuGc (N-glycolylneuraminic acid, or "NGNA" or "Neu5Gc"). Such glycans are absent in recombinant IDS products, such as Hunterase®, made in CHO cells. This is because CHO cells lack the 2,6-sialyltransferase required for this post-translational modification; they also do not produce the bisected GlcNAc, adding Neu5Gc(NGNA) instead of Neu5Ac(NANA), an uncommon (and potentially immunogenic) sialic acid in humans. See, e.g., Dumont et al., 2016, Critical Rev in Biotech 36(6):1110-1122 (Early Online pp. 1-13, p. 5), and Hague et al., 1998 Electrophor 19:2612-2630 ("CHO cell lines are considered 'phenotypically restricted' with respect to glycosylation due to the lack of α2,6-sialyltransferase."). Furthermore, CHO cells can also produce α-Gal antigen, an immunogenic glycan that reacts with anti-α-Gal antibodies present in most individuals and can induce anaphylaxis at high concentrations. See, e.g., Bosques, 2010, Nat Biotech 28:1153-1156. The human glycosylation pattern of the rhIDS of the present invention will reduce the immunogenicity and improve efficacy of the transgene product. (x) The immunogenicity of transgene products can be induced by various factors, including the patient's immune status, the structure and characteristics of the injected protein drug, the route of administration, and the duration of treatment. Process-related impurities, such as host cell proteins (HCPs), host cell DNA, and chemical residues, as well as product-related impurities, such as protein degradation products and structural features, such as glycosylation, oxidation, and aggregation (subvisible particles), can also enhance immunogenicity by functioning as adjuvants to enhance the immune response. The amounts of process-related and product-related impurities are affected by the manufacturing process, i.e., cell culture, purification, formulation, storage, and handling, and they affect the commercially produced IDS product. In gene therapy, proteins are produced in vivo, so there are no process-related impurities, and the protein product is unlikely to contain product-related impurities / degradants, such as protein aggregates and protein oxides, associated with recombinantly produced proteins. Aggregation is associated with protein production and storage, resulting from high protein concentrations, surface interactions with manufacturing equipment and vessels, and purification processes using certain buffer systems. However, these conditions that promote aggregation are not present when transgenes are expressed in vivo. Oxidation, such as oxidation of methionine, tryptophan, and histidine, is also associated with protein production and storage and occurs due to, for example, stressed cell culture conditions, contact with metals and air, and impurities in buffers and excipients. Proteins expressed in vivo can also oxidize under stressed conditions, but humans, like many organisms, possess antioxidant defense systems that not only reduce oxidative stress but also repair and / or reverse oxidation. Therefore, proteins produced in vivo are less likely to be in an oxidized form. Both aggregation and oxidation can affect efficacy, pharmacokinetics (clearance), and increase concerns about immunogenicity. The gene therapy approach described herein will result in the continuous secretion of hIDS precursors with reduced immunogenicity compared to commercially produced products. (xi) In addition to N-linked glycosylation sites, hIDSs contain tyrosine ("Y") sulfation sites (PSSEKY165 ENTKTCRGPD). (See, e.g., Yang et al., 2015, Molecules 20:2138-2164, especially p. 2154, which is incorporated by reference in its entirety for its analysis of amino acids surrounding tyrosine residues subject to protein tyrosine sulfation. The "rule" can be summarized as follows: Y residues with E or D within positions +5 to -5 of Y, and position -1 of Y is a neutral or acidic charged amino acid, but not a basic amino acid, such as R, K, or H, which abolishes sulfation.) Without being bound by any theory, sulfation of this site in hIDS may improve the enzyme's stability and binding affinity for substrates. Tyrosine sulfation of hIDS, a robust post-translational process in human CNS cells, may improve the processing and activity of transgene products. Although the importance of tyrosine sulfation of lysosomal proteins remains unclear, it has been shown to increase the affinity of protein-protein interactions (antibodies and receptors) and facilitate proteolytic processing (peptide hormones) in other proteins (see Moore, 2003, J. Biol. Chem. 278:24243-46; and Bundegaard et al., 1995, The EMBO J 14:3073-79). Tyrosylprotein sulfotransferase 1 (TPST1), involved in tyrosine sulfation (potentially the final step in IDS processing), is expressed at even higher levels (based on mRNA) in the brain (gene expression data for TPST1 can be found, for example, in the EMBL-EBI Expression Atlas, accessible at http: / / www.ebi.ac.uk / gxa / home). Such post-translational modifications are, at best, underrepresented in CHO cell products. Unlike human CNS cells, CHO cells are not secretory cells and also have a limited capacity for post-translational tyrosine sulfation (see, e.g., Mikkelsen & Ezban, 1991, Biochemistry 30:1533-1537, especially the discussion on p. 1537).

[0122] For the reasons discussed above, production of rhIDS by human neuronal and / or glial cells would provide a "bio-better" molecule for the treatment of MPS II, achieving gene therapy by, for example, administering a viral vector or other DNA expression construct encoding rhIDS into the CSF of a patient (human subject) diagnosed with MPS II disease (including, but not limited to, Hunter) to create a permanent depot in the CNS that provides a continuous supply of fully human glycosylated, mannose-6-phosphorylated, sulfated transgene product secreted by transduced CNS cells. The rhIDS transgene product secreted from the depot into the CSF is endocytosed by cells in the CNS and "cross-corrects" the enzymatic defect in MPS II recipient cells.

[0123] It is not essential that all rhIDS molecules produced in a 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 the gene therapy treatment of the present invention is to slow or halt disease progression. Efficacy can be monitored by measuring cognitive function (e.g., prevention or suppression of neurocognitive decline); reduction in disease biomarkers (e.g., GAGs) in CSF and / or serum; and / or increased IDS enzyme activity in CSF and / or serum. Signs of inflammation and other safety events can also be monitored.

[0124] As an alternative or adjunct to gene therapy, rhIDS glycoproteins can be produced in human neuronal or glial cells by recombinant DNA technology, and the glycoproteins can be administered systemically and / or into the CSF to patients diagnosed with MPS II 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-1A, HCN-2, NT2, SH-SY5y, hNSC11, or ReNcell VM (see, e.g., "Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives" in Dumont et al., 2016, Critical Rev in Biotech 36(6):1110-1122, for a review of human cell lines that can be used for recombinant production of rHuGlyIDS glycoproteins, the entire contents of which are incorporated herein by reference). To ensure complete glycosylation, particularly sialylation, and tyrosine sulfation, the cell lines used for production can be enhanced by genetically engineering the host cells to co-express the α-2,6-sialyltransferase (or both α-2,3- and α-2,6-sialyltransferases) responsible for tyrosine-O-sulfation, and / or the TPST-1 and TPST-2 enzymes.

[0125] While delivery of rhIDS will minimize immune responses, the most obvious potential source of toxicity for CNS-related gene therapy is the development of immunity to the expressed rhIDS protein in human subjects who are genetically deficient in IDS and therefore may not tolerate the vectors used to deliver the protein and / or transgene.

[0126] Therefore, in a preferred embodiment, co-treating the patient with immunosuppressive therapy is appropriate, especially when treating patients with severe disease whose IDS levels are close to zero. Immunosuppressive therapy involving a combination of tacrolimus or rapamycin (sirolimus) with mycophenolic acid, or other immunosuppressive regimens used in tissue transplantation procedures, can be utilized. Such immunosuppressive therapy can be administered during the course of gene therapy, and in certain embodiments, pretreatment with immunosuppressive therapy may be preferred. Immunosuppressive therapy can be continued after gene therapy treatment, at the discretion of the attending physician, and then discontinued, for example, after 180 days, if immune tolerance is induced.

[0127] The methods of the present invention include the combination of delivery of rhIDS to the CSF with the delivery of other available therapies. The additional therapies can be administered before, simultaneously with, or after the gene therapy treatment. Available therapies for MPS II that can be used in combination with the gene therapy of the present invention include, but are not limited to, enzyme replacement therapy using Elaprase® administered systemically or to the CSF, and / or HSCT therapy.

[0128] In certain aspects, the methods described herein are methods 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 recombinant human iduronate-2-sulfatase (IDS) precursor produced by human neuronal or glial cells.

[0129] In certain embodiments, the methods described herein are methods 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 recombinant human iduronate-2-sulfatase (IDS) precursor, the precursor being approximately 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, and 84 It contains a formylglycine residue at 1 (Fig. 1), is α2,6-sialylated, does not contain detectable NeuGc, and is mannose-6-phosphorylated.

[0130] In certain embodiments, the methods described herein are methods 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 recombinant human iduronate-2-sulfatase (IDS) glycoprotein precursor, the precursor being approximately 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, and 84 It contains a formylglycine residue at the α-terminus (Figure 1), is α2,6-sialylated, does not contain detectable NeuGc and / or α-Gal, and is mannose-6-phosphorylated.

[0131] In certain embodiments, the human IDS precursor is delivered to the CSF from a depot of cells in the central nervous system that are genetically engineered to secrete the IDS precursor into the CSF. In certain embodiments, the depot is formed in the brain of the subject. In certain embodiments, the human subject lacks IDS activity. In certain embodiments, the human IDS comprises the amino acid sequence of SEQ ID NO: 1.

[0132] In certain embodiments, the methods described herein are methods for treating a human subject diagnosed with Mucopolysaccharidosis Type II (MPS II), comprising administering to the cerebrospinal fluid (CSF) of the human subject a recombinant nucleotide expression vector encoding human iduronate-2-sulfatase (IDS), wherein the expression vector, when used to transduce primary human neuronal cells in culture, 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; 84 It contains a formylglycine residue at the α2,6-sialylated position (Figure 1), and is mannose-6-phosphorylated.

[0133] In certain embodiments, the methods described herein are methods for treating a human subject diagnosed with Mucopolysaccharidosis Type II (MPS II), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS to the cerebrospinal fluid of the human subject, thereby forming depots in the central nervous system of the subject that secrete α2,6-sialylated and mannose-6-phosphorylated recombinant human IDS glycoprotein precursor.

[0134] In certain embodiments, secretion of the α2,6-sialylated recombinant human IDS glycoprotein precursor is confirmed in cell culture by transducing a human neuronal cell line with the recombinant nucleotide expression vector. In certain embodiments, secretion of the mannose-6-phosphorylated recombinant human IDS glycoprotein precursor is confirmed in cell culture by transducing a human neuronal cell line with the recombinant nucleotide expression vector. In certain embodiments, secretion is confirmed in the presence and absence of mannose-6-phosphate.

[0135] In certain embodiments, the methods described herein are methods for treating a human subject diagnosed with Mucopolysaccharidosis Type II (MPS II), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding a human IDS to the cerebrospinal fluid of the human subject, thereby forming a depot that secretes glycosylated IDS precursors containing α2,6-sialylated glycans, and the recombinant vector, when used to transduce human neuronal cells in culture, results in the secretion of the glycosylated IDS precursors containing α2,6-sialylated glycans in the cell culture.

[0136] In certain embodiments, the methods described herein are methods for treating a human subject diagnosed with Mucopolysaccharidosis Type II (MPS II), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding a human IDS to the cerebrospinal fluid of the brain of the human subject, thereby forming a depot that secretes a glycosylated IDS precursor containing mannose-6-phosphate, and the recombinant vector, when used to transduce human neuronal cells in culture, results in the secretion of the mannose-6-phosphorylated glycosylated IDS precursor in the cell culture.

[0137] In certain embodiments, the methods described herein are methods for treating a human subject diagnosed with Mucopolysaccharidosis Type II (MPS II), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding a human IDS to the cerebrospinal fluid of the brain of the human subject, thereby forming a depot that secretes a glycosylated IDS precursor containing formylglycine, and the recombinant vector, when used to transduce human neuronal cells in culture, results in the secretion of the glycosylated IDS precursor containing formylglycine in the cell culture.

[0138] 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-deficient AAV vector. In certain embodiments, the expression vector is delivered to the CSF of the subject by intrathecal (e.g., intracisternal, C1-2 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 a subject via intrathecal administration of a solution comprising sodium chloride at a concentration of about 8.77 g / L, magnesium chloride hexahydrate 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 dehydrate at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.001% (vol / vol), sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and disodium phosphate anhydrous at a concentration of about 0.114 g / L.

[0139] In a preferred embodiment, the glycosylated IDS does not contain detectable NeuGc and / or α-Gal. As used herein, the term "detectable NeuGc and / or α-Gal" refers to a NeuGc and / or α-Gal moiety that is detectable by standard assay methods known in the art. For example, NeuGc can be detected by HPLC according to Hara et al., 1989, "Highly Sensitive Determination of N-Acetyl- and N-Glycolylneuraminic Acids in Human Serum, Urine, and Rat Serum by Reversed-Phase Liquid Chromatography with Fluorescence Detection," J. Chromatogr., B:Biomed. 377:111-119, which is incorporated herein by reference, for methods of detecting NeuGc. Alternatively, NeuGc can be detected by mass spectrometry. Alpha-Gal can be detected using ELISA, see e.g., 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 using mass spectrometry, see e.g., Ayoub et al., 2013, "Correct primary structure assessment and extensive glycoprofiling 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 et al., 2015, “Anaphylaxis to the Carbohydrate Side-Chain Alpha-gal” Immunol Allergy Clin North Am. 35(2):247-260.

[0140] In one aspect, provided herein is a method of treating a human subject diagnosed with MPS II, comprising delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or glial cells to the CSF of the human subject, wherein the glycosylated recombinant human IDS precursor is induced by administering a recombinant nucleotide expression vector encoding a human IDS, administering the recombinant nucleotide expression vector at a dose dependent on the brain mass of the human subject, and determining the brain mass via brain MRI of the human subject's brain.

[0141] In another aspect, provided herein is a method of treating a human subject diagnosed with MPS II, comprising determining the brain mass of the human subject by brain MRI of the human subject, and subsequently delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or human glial cells to the CSF of the human subject, wherein the glycosylated recombinant human IDS precursor is delivered by administering a recombinant nucleotide expression vector encoding the human IDS, wherein the recombinant nucleotide expression vector is administered at a dose dependent on the brain mass of the human subject.

[0142] In another aspect, provided herein is a method of treating a human subject diagnosed with MPS II, comprising: (a) determining the brain mass of the human subject from a brain MRI of the human subject; (b) calculating a dose based on the brain mass of the human subject; and (c) subsequently administering the dose of a recombinant nucleotide expression vector encoding a human IDS into the CSF of the subject.

[0143] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising, in order: (a) delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or glial cells to the CSF of the human subject; (b) measuring the 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 the level of heparan sulfate in a reference population; wherein the glycosylated recombinant human IDS precursor is induced by administering a recombinant nucleotide expression vector encoding a human IDS, administering the recombinant nucleotide expression vector at a dose corresponding to the brain mass of the human subject, and determining the brain mass 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 healthy individuals without MPS II, preferably of a similar age, weight, and / or sex as the human subject.

[0144] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising, in order: (a) performing a first measurement of heparan sulfate levels in the human subject's CSF; (b) delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or glial cells to the human subject's CSF; and (c) performing a second measurement of heparan sulfate levels after a period of time, wherein the glycosylated recombinant human IDS precursor is induced by administering a recombinant nucleotide expression vector encoding a human IDS, administering the recombinant nucleotide expression vector at a dose corresponding to the human subject's brain mass, and determining the brain mass 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 months, 4 months, 5 months, 6 months, 7 months, 8 months, 11 months, or 1 year.

[0145] In a preferred embodiment, the glycosylated recombinant human IDS precursor is taken up by cells in the CNS of a human subject. In a preferred embodiment, the glycosylated recombinant human IDS precursor is delivered to the lysosomes of cells in the CNS of a human subject.

[0146] In certain embodiments of the methods of treatment described herein, a human subject's brain volume is adjusted to 1.046 g / cm 3 Multiply by the factor of to obtain the brain volume cm of a human subject. 3 to the brain mass of a human subject, and the brain volume of a human subject is obtained from a brain MRI of the human subject.

[0147] In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered to approximately 1.3 x 10 cells per gram of brain mass as determined by MRI. 10 GC, or approximately 6.5 × 10 per gram of brain mass as determined by MRI. 10 In some embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x 10 10 In some embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by polyA-specific PCR assay). 10 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by transgene-specific PCR assay). 10 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by polyA-specific PCR assay). 10 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by transgene-specific PCR assay).11 In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x 10 GC / g brain mass (e.g., brain mass determined by MRI and genome number determined by polyA-specific PCR assay). 11 The dose is administered at GC / g brain mass (eg, brain mass determined by MRI and genome number determined by transgene-specific PCR assay).

[0148] In various embodiments of the methods of treatment described herein, the human subject is 5 years of age or older and less than 18 years of age. In certain embodiments, the human subject is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age. In certain embodiments, the human subject is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age. In certain embodiments, the human subject is 5-6 years of age, 6-7 years of age, 7-8 years of age, 8-9 years of age, 9-10 years of age, 10-11 years of age, 11-12 years of age, 12-13 years of age, 13-14 years of age, 14-15 years of age, 15-16 years of age, 16-17 years of age, 17-18 years of age, or 18-19 years of age. In certain embodiments, the human subject is about 5-6 years old, 6-7 years old, 7-8 years old, 8-9 years old, 9-10 years old, 10-11 years old, 11-12 years old, 12-13 years old, 13-14 years old, 14-15 years old, 15-16 years old, 16-17 years old, 17-18 years old, or 18-19 years old. In certain embodiments, the recombinant nucleotide expression vector is administered at a concentration of about 6.5 x 10 per gram of brain mass as determined by MRI. 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose listed in Table 7.

[0149] In various embodiments of the methods of treatment described herein, the human subject is at least 4 months old and less than 5 years old. In certain embodiments, the human subject is 4, 5, 6, 7, 8, 9, 10, or 11 months old. In certain embodiments, the human subject is about 4, 5, 6, 7, 8, 9, 10, or 11 months old. In certain embodiments, the human subject is 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months old. In certain 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 certain embodiments, the human subject is 1, 2, 3, 4, or 5 years old. In certain embodiments, the human subject is about 1, 2, 3, 4, or 5 years old. In certain embodiments, the human subject is 1-2 years old, 2-3 years old, 3-4 years old, 4-5 years old, or 5-6 years old. In certain embodiments, the human subject is about 1-2 years old, 2-3 years old, 3-4 years old, 4-5 years old, or 5-6 years old. In certain embodiments, the recombinant nucleotide expression vector is administered at a concentration of about 1.3 x 10 per gram of brain mass as determined by MRI. 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x 10 GC per gram of brain mass as determined by MRI. 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x 10 GC per gram of brain mass as determined by MRI. 11 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x 10 GC per gram of brain mass as determined by MRI. 11 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x 10 GC per gram of brain mass as determined by MRI. 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x 10 GC per gram of brain mass as determined by MRI. 10 In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x 10 GC / g brain mass. 11GC (e.g., brain mass determined by MRI and genome number determined by polyA-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x 10 GC / g brain mass. 11 GC (e.g., brain mass determined by MRI and genome number determined by transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x 10 GC / g brain mass. 10 GC (e.g., brain mass determined by MRI and genome number determined by polyA-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x 10 GC / g brain mass. 10 GC (e.g., brain mass determined by MRI and genome number determined by transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x 10 GC / g brain mass. 10 GC (e.g., brain mass determined by MRI and genome number determined by polyA-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x 10 GC / g brain mass. 10 The recombinant nucleotide expression vector is administered at a dose of GC (e.g., brain mass determined by MRI and genome number determined by transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose selected from Dose 1 or Dose 2 according to Table 5. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose set forth in Table 6.

[0150] In some embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vector is administered via intracisternal (IC) administration. In other embodiments of the methods described herein, the recombinant nucleotide expression vector is administered via intracerebroventricular (ICV) administration.

[0151] In certain embodiments of the methods described herein, the recombinant nucleotide expression vector is administered in a volume that does not exceed 10% of the total volume of cerebrospinal fluid of the human subject.

[0152] In certain embodiments of the therapeutic methods described herein, glycosylated recombinant human IDS precursor is secreted at detectable levels.

[0153] In certain embodiments of the methods of treatment described herein, the human neuronal or human glial cells have at least one mutation in an endogenous gene encoding a human IDS precursor.

[0154] In certain embodiments of the therapeutic methods described herein, human neuronal or glial cells are transduced with a recombinant adeno-associated viral vector (rAAV).

[0155] In a preferred embodiment, the recombinant nucleotide expression vector is an AAV9 or AAVrhlO vector.

[0156] In certain embodiments of the therapeutic methods described herein, a glycosylated recombinant human IDS precursor is expressed under the control of the CB7 promoter.

[0157] In certain embodiments of the therapeutic methods described herein, a glycosylated recombinant human IDS precursor is expressed from a cDNA encoding the human IDS precursor.

[0158] In certain embodiments of the methods of treatment described herein, the glycosylated recombinant human IDS precursor is about 90 kDa as determined by polyacrylamide gel electrophoresis.

[0159] In certain embodiments of the methods of treatment described herein, the glycosylated recombinant human IDS precursor contains formylglycine.

[0160] In certain embodiments of the therapeutic methods described herein, the glycosylated recombinant human IDS precursor (a) is α2,6-sialylated, (b) does not contain detectable NeuGc, (c) does not contain detectable α-Gal antigen, (d) contains tyrosine sulfation, and / or (e) is mannose-6-phosphorylated.

[0161] In certain embodiments of the methods of treatment described herein, the glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO:1.

[0162] In certain embodiments provided herein, the methods further include administering immunosuppressive therapy to the human subject prior to or concurrently with the human IDS precursor treatment, and optionally continuing the immunosuppressive therapy thereafter.

[0163] In some embodiments, the immunosuppressive therapy comprises administering one or more corticosteroids, sirolimus, and / or tacrolimus, hi certain embodiments, the one or more corticosteroids are methylprednisolone and / or prednisone.

[0164] In certain embodiments, the immunosuppressive therapy comprises prednisone at 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, In certain embodiments, the immunosuppressive therapy comprises administering prednisone at a dose ranging from about 0.10 mg / kg to about 0.20 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering prednisone at a dose ranging from about 0.20 mg / kg to about 0.30 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering prednisone at a dose ranging from about 0.30 mg / kg to about 0.40 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering prednisone at a dose ranging from about 0.40 mg / kg to about 0.50 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering prednisone at a dose ranging from about 0.50 mg / kg to about 1 mg / kg. In certain embodiments, these doses are administered daily. In certain embodiments, the immunosuppressive therapy comprises administering prednisone at a dose of 0.5 mg / kg / day. In another specific embodiment, the immunosuppressive therapy comprises administering prednisone at a dose of 0.5 mg / kg / day, followed by tapering and discontinuation.

[0165] In certain embodiments, the immunosuppressive 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, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / 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 certain embodiments, the immunosuppressive therapy comprises administering methylprednisolone at a dose ranging from about 0.50 mg / kg to about 1.0 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering methylprednisolone at a dose ranging from about 1.0 mg / kg to about 2.0 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering methylprednisolone at a dose ranging from about 2.0 mg / kg to about 3.0 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering methylprednisolone at a dose ranging from about 3.0 mg / kg to about 5.0 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering methylprednisolone at a dose ranging from about 5.0 mg / kg to about 10.0 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering methylprednisolone at a dose ranging from about 10.0 mg / kg to about 15.0 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering methylprednisolone at a dose ranging from about 15.0 mg / kg to about 20.0 mg / kg. In certain embodiments, the methylprednisolone is administered once. In certain embodiments, the methylprednisolone is administered intravenously. In certain embodiments, the methylprednisolone is administered at a dose up to 500 mg. In certain embodiments, the methylprednisolone is administered over a period of at least 30 minutes.In a particular embodiment, the immunosuppressive therapy comprises methylprednisolone administered at a dose of 10 mg / kg IV over at least 30 minutes, up to a maximum of 500 mg.

[0166] In certain embodiments, the immunosuppressive therapy comprises administering sirolimus at a dose that maintains a target blood level of 1-3 ng / mL. In certain embodiments, the immunosuppressive therapy comprises administering sirolimus at a dose of about 0.25 mg / m 2 / day, 0.3mg / m 2 / day, 0.4mg / m 2 / day, 0.5mg / m 2 / day, 0.6mg / m 2 / day, 0.7mg / m 2 / day, 0.8mg / m 2 / day, 0.9 mg / m 2 / day, 1mg / m 2 / day, 1.25mg / m 2 / day, 1.5mg / m 2 / day, 1.75mg / m 2 / day, 2mg / m 2 / day, 2.25mg / m 2 / day, 2.5mg / m 2 / day, 2.75mg / m 2 / day, 3mg / m 2 / day, 3.25mg / m 2 / day, 3.5mg / m 2 / day, 3.75mg / m 2 / day, 4mg / m 2 / day, 4.25mg / m 2 / day, 4.5mg / m 2 / day, 4.75mg / m 2 / day, or 5 mg / m 2 In certain embodiments, the immunosuppressive therapy comprises administering sirolimus at a dose of 0.25 mg / m 2 / day~about 0.5mg / m 2 In certain embodiments, the immunosuppressive therapy comprises administering sirolimus at a dose ranging from about 0.50 mg / m 2 / day ~ approx. 1.0 mg / m 2In certain embodiments, the immunosuppressive therapy comprises administering sirolimus at a dose ranging from about 1.0 mg / m 2 / day~about 1.5mg / m 2 In certain embodiments, the immunosuppressive therapy comprises administering sirolimus at a dose ranging from about 1.5 mg / m 2 / day ~ approx. 2mg / m 2 In certain embodiments, the immunosuppressive therapy comprises administering sirolimus at a dose ranging from about 2 mg / m 2 / day~about 5mg / m 2 In certain embodiments, the dose is divided for BID administration. In certain embodiments, the immunosuppressive therapy comprises administering sirolimus at a dose ranging from about 1 mg / m 2 / day every 4 hours. In certain embodiments, the immunosuppressive therapy comprises sirolimus at a dose of about 0.5 mg / m divided for BID administration. 2 1 / day dose.

[0167] In certain embodiments, the immunosuppressive therapy comprises administering tacrolimus at a dose that maintains a target blood level of 2 to 4 ng / mL. In certain embodiments, the immunosuppressive 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 certain embodiments, the immunosuppressive therapy comprises administering tacrolimus at a dose ranging from 0.01 mg / kg to 0.02 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering tacrolimus at a dose ranging from 0.02 mg / kg to 0.03 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering tacrolimus at a dose ranging from 0.03 mg / kg to 0.05 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering tacrolimus at a dose ranging from 0.05 mg / kg to 0.07 mg / kg. In certain embodiments, the immunosuppressive therapy comprises administering tacrolimus at a dose ranging from 0.07 mg / kg to 0.10 mg / kg. In certain embodiments, the dose is administered twice daily. In certain embodiments, the immunosuppressive therapy comprises administering tacrolimus at a dose of about 0.05 mg / kg twice daily.

[0168] In some embodiments, the method further comprises administering one or more antibiotics to the human subject prior to or concurrently with the immunosuppressive therapy. In certain embodiments, 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 certain 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 certain embodiments, the one or more antibiotics are administered at a dose ranging from about 1 mg / kg to 2 mg / kg. In certain embodiments, the one or more antibiotics are administered at a dose ranging from about 2 mg / kg to 3 mg / kg. In certain embodiments, one or more antibiotics are administered at a dose ranging from about 3 mg / kg to 5 mg / kg. In certain embodiments, one or more antibiotics are administered at a dose ranging from about 5 mg / kg to 7 mg / kg. In certain embodiments, one or more antibiotics are administered at a dose ranging from about 7 mg / kg to 10 mg / kg. In certain embodiments, one or more antibiotics are administered in doses about three times per week. In certain embodiments, one or more antibiotics are administered to prevent Pneumocystis carinii pneumonia.

[0169] In some embodiments, the method further comprises administering to the human subject one or more antifungal therapies prior to or concurrently with the immunosuppressive therapy. In certain embodiments, the one or more antifungal therapies are administered to a human subject with an absolute neutrophil count of 500 mmHg or higher. 3 If it is less than that, start.

[0170] In some embodiments, the method further comprises, after administering the recombinant nucleotide expression vector, measuring one or more of the following biomarkers: (a) glycosaminoglycan (GAG) levels in CSF, (b) iduronate-2-sulfatase (IS) levels in CSF; (c) GAG levels in plasma, (d) I2S levels in plasma, (e) leukocyte I2S enzyme activity levels, and (f) GAG levels in urine. In certain embodiments, 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 certain embodiments, the measuring step comprises measuring the level of heparin sulfate in CSF. In another specific embodiment, the measuring step comprises measuring the level of leukocyte I2S enzyme activity.

[0171] In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is a liquid composition. In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is a frozen composition. In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vector is a lyophilized composition or a reconstituted lyophilized composition. In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vectors provided herein may be formulated in various dosage forms for IC or ICV administration. In certain embodiments of the methods of treatment described herein, the recombinant nucleotide expression vectors provided herein may be provided in unit-dose or multi-dose formulations. As used herein, a unit-dose formulation refers to a physically discrete unit suitable for administration to a human or animal subject, packaged individually using packaging methods known in the art. Each unit-dose formulation contains a predetermined quantity of the recombinant nucleotide expression vector and / or other ingredient(s) sufficient to produce the desired therapeutic effect in association with the necessary pharmaceutical carriers or excipients. Examples of unit-dose formulations include ampoules, vials, pre-filled syringes, or cartridges.

[0172] In certain embodiments of the methods of treatment described herein, a unit dose may be administered in fractions or multiples thereof. In certain embodiments of the methods of treatment described herein, a multiple unit dose refers to a single container containing multiple identical unit doses that are administered separately. Examples of multiple unit doses include vials, pre-filled syringes, or cartridges. In certain embodiments, a pre-filled syringe contains 8.5 x 10 12 In certain embodiments, the pre-filled syringe contains 9.8 x 10 recombinant nucleotide expression vectors of GC. 12 In certain embodiments, the pre-filled syringe contains 1.1 x 10 recombinant nucleotide expression vector of GC. 13 In certain embodiments, the pre-filled syringe contains 1.3 x 10 recombinant nucleotide expression vector of GC. 13In certain embodiments, the pre-filled syringe contains 1.5 x 10 recombinant nucleotide expression vector of GC. 13 In certain embodiments, the pre-filled syringe contains 1.7 x 10 recombinant nucleotide expression vector of GC. 13 In certain embodiments, the pre-filled syringe contains 4.2 x 10 recombinant nucleotide expression vectors of GC. 13 In certain embodiments, the pre-filled syringe contains 4.9 x 10 recombinant nucleotide expression vectors of GC. 13 In certain embodiments, the pre-filled syringe contains 5.5 x 10 recombinant nucleotide expression vectors of GC. 13 In certain embodiments, the pre-filled syringe contains 6.3 x 10 recombinant nucleotide expression vectors of GC. 13 In certain embodiments, the pre-filled syringe contains 7.3 x 10 recombinant nucleotide expression vectors of GC. 13 In certain embodiments, the pre-filled syringe contains 8.5 x 10 recombinant nucleotide expression vectors of GC. 13 In certain embodiments, the pre-filled syringe contains 9.0 x 10 recombinant nucleotide expression vectors of GC. 13 In certain embodiments, the pre-filled syringe contains a 1.0 x 10 recombinant nucleotide expression vector of GC. 14 In certain embodiments, the pre-filled syringe contains 1.1 x 10 recombinant nucleotide expression vector of GC. 14 In certain embodiments, the pre-filled syringe contains 1.2 x 10 recombinant nucleotide expression vector of GC. 14 In certain embodiments, the pre-filled syringe contains 1.3 x 10 recombinant nucleotide expression vector of GC. 14 In certain embodiments, the pre-filled syringe contains 1.4 x 10 recombinant nucleotide expression vector of GC. 14 In certain embodiments, the pre-filled syringe contains 1.5 x 10 recombinant nucleotide expression vector of GC. 14In certain embodiments, the pre-filled syringe contains 1.6 x 10 recombinant nucleotide expression vector of GC. 14 In certain embodiments, the pre-filled syringe contains 1.7 x 10 recombinant nucleotide expression vector of GC. 14 In certain embodiments, the pre-filled syringe contains 1.8 x 10 recombinant nucleotide expression vector of GC. 14 In certain embodiments, the pre-filled syringe contains 1.9 x 10 recombinant nucleotide expression vector of GC. 14 In certain embodiments, the pre-filled syringe contains 2.0 x 10 recombinant nucleotide expression vectors of GC. 14 In certain embodiments, the pre-filled syringe contains 2.1 x 10 recombinant nucleotide expression vectors of GC. 14 In certain embodiments, the pre-filled syringe contains 2.2 x 10 recombinant nucleotide expression vectors of GC. 14 In certain embodiments, the pre-filled syringe contains 2.3 x 10 recombinant nucleotide expression vectors of GC. 14 In certain embodiments, the pre-filled syringe contains 2.4 x 10 recombinant nucleotide expression vectors of GC. 14 In certain embodiments, the pre-filled syringe contains 2.5 x 10 recombinant nucleotide expression vectors of GC. 14 In certain embodiments, the pre-filled syringe contains 2.6 x 10 recombinant nucleotide expression vectors of GC. 14 In certain embodiments, the pre-filled syringe contains 1.3 x 10 recombinant nucleotide expression vector of GC. 10 In certain embodiments, the pre-filled syringe contains 1.9 x 10 recombinant nucleotide expression vector of GC. 10 In certain embodiments, the pre-filled syringe contains 6.5 x 10 recombinant nucleotide expression vectors of GC. 10 In certain embodiments, the pre-filled syringe contains 9.6 x 10 recombinant nucleotide expression vectors of GC. 10In certain embodiments, the pre-filled syringe contains 2.0 x 10 recombinant nucleotide expression vectors of GC. 11 In certain embodiments, the pre-filled syringe contains 2.9 x 10 recombinant nucleotide expression vectors of GC. 11 The recombinant nucleotide expression vector of GC is included.

[0173] As used herein, the term "about" means within ±10% of a given value or range. In certain embodiments, the term "about" means within ±1% of a given value or range, and the number is a dose relative to the brain mass of a human subject, and the brain mass is determined by brain MRI of the human subject's brain. In certain embodiments, the term "about" means within ±2% of a given value or range, and the number is a dose relative to the brain mass of a human subject, and the brain mass is determined by brain MRI of the human subject's brain. In certain embodiments, the term "about" means within ±5% of a given value or range, and the number is a dose relative to the brain mass of a human subject, and the brain mass is determined by brain MRI of the human subject's brain. In certain embodiments, the term "about" means within ±7% of a given value or range, and the number is a dose relative to the brain mass of a human subject, and the brain mass is determined by brain MRI of the human subject's brain. In certain embodiments, the term "about" means within ±10% of a given value or range, and this value is the dose according to the brain mass of a human subject, and the brain mass is determined by brain MRI of the brain of the human subject. However, it should be understood that the term "about" herein also indicates the exact numerical value associated with the term. For example, "about 10" accurately provides the detail about the number "10".

[0174] 5.1 Processing, N-Glycosylation, and Tyrosine Sulfation Processing Human IDS contains a 25-amino acid signal sequence that is cleaved during processing. The initial 76 kDa intracellular IDS precursor is converted to a phosphorylated 90 kDa IDS precursor after its oligosaccharide chains are modified in the Golgi apparatus. This precursor is processed to the major 55 kDa form via various intracellular intermediates by glycosylation modification and proteolytic cleavage. In summary, after removal of the 25-amino acid signal sequence, proteolytic processing proceeds to the N 31 downstream of which an N-terminal proteolytic cleavage is performed to remove a propeptide of 8 amino acids (residues 26–33) and 513 A C-terminal proteolytic cleavage occurs upstream of this enzyme, releasing an 18 kDa polypeptide and generating a 62 kDa intermediate, which is converted to the 55 kDa mature form. Further proteolytic cleavage generates the 45 kDa mature form, which is located in the lysosomal compartment. (See Figure 4 for a diagram excerpt 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 herein by reference in its entirety.)

[0175] C required for enzyme activity 84 Formylglycine modification of (shown in bold in Figure 1) most likely occurs in the endoplasmic reticulum, possibly as an early post- or co-translational event. (See Millat 1997a, citing Schmidt et al., 1995, Cell 82:271-278.) Post-translational processing continues in the Golgi apparatus, incorporating complex sialic acid-containing glycans and including the acquisition of mannose-6-phosphate residues that bind enzymes for delivery to the lysosomal compartment. (For a brief discussion, the entire contents of which are incorporated herein by reference, see Clarke, 2008, Expert Opin Pharmacother 9:311-317.)

[0176] In certain embodiments, HuGlyIDS used in accordance with the methods described herein can be a mannose-6-phosphorylated form of 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) enzyme when expressed in neuronal or glial cells in vivo or in vitro. IDS produced from neuronal and glial cells can contain higher amounts of M6P, as reported by Daniele 2002 and Sleat, Proteomics, 2005 (which indicates that the human brain contains more M6P glycoprotein (both quantitatively and qualitatively) than other tissues). Measuring the M6P content of IDS precursors can be performed as described in Daniele 2002. Therefore, in certain embodiments, when HuGlyIDS used according to the methods described herein is expressed in neuronal or glial cells, it undergoes higher levels of mannose-6-phosphorylation in vivo or in vitro than IDS expressed in non-neuronal or glial cells. In particular, when HuGlyIDS used according to the methods described herein is expressed in neuronal or glial cells, it undergoes higher levels of mannose-6-phosphorylation in vivo or in vitro than IDS expressed in HT1080 or CHO cells. In certain embodiments, the mannose-6-phosphorylation level of the expressed IDS is measured by measuring the uptake of the IDS by human neuronal cells in the presence of M6P (e.g., 5 mM M6P). In certain embodiments, when expressed in neuronal or glial cells in vivo or in vitro, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% of the HuGlyIDS molecules used in accordance with the methods described herein are mannose-6-phosphorylated.

[0177] 5.1.2 N-Glycosylation Neurons 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 asparagine ("N") glycosylation sites, as shown in Figure 1 (N 31 S.T., N. 115 F.S., N. 144 H.T., N. 246 IT, N 280 IS,N 325 S.T., N. 513 F.S., N. 537 DS). Two of the eight N-linked glycosylation sites, i.e., N 280 and N 116 is mannose-6-phosphorylated in IDS from human brain (Sleat et al., 2006, Mol & Cell Proeomics 5.4:686-701, reported in Table V). There is no single glycosylation site essential for IDS stability, but N 280 Glycosylation at this position is important for cellular internalization via the mannose-6-phosphate (M6P) receptor and lysosomal targeting (Chung et al., 2014, Glycoconj J 31:309-315, first paragraph of p. 310). Under normal physiological conditions, IDS is produced at very low levels, and little, if any, secretion of the enzyme from cells occurs (Clarke, 2008, supra).

[0178] It is not essential that all molecules produced in a gene therapy or protein therapy approach be fully glycosylated and sulfated, but rather that the population of glycoproteins produced will have sufficient glycosylation and sulfation to be effective.

[0179] In certain embodiments, a HuGlyIDS used in accordance with the methods described herein may be glycosylated at 100% of its N-glycosylation sites when expressed in neuronal or glial cells in vivo or in vitro. However, one of skill in the art will recognize that not all N-glycosylation sites of a HuGlyIDS need to be N-glycosylated in order for the glycosylation benefits to be achieved. Rather, the glycosylation benefits may be realized when only a certain percentage of the N-glycosylation sites are glycosylated and / or when only a certain percentage of the expressed IDS molecules are glycosylated. Thus, in certain embodiments, a HuGlyIDS used in accordance with the methods described herein 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 when expressed in vivo or in vitro in neuronal or glial cells. In certain embodiments, when expressed in vivo or in vitro in neuronal or glial cells, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% of the HuGlyIDS molecules used in accordance with the methods described herein are glycosylated at at least one of their available N-glycosylation sites.

[0180] In certain embodiments, when HuGlyIDS is expressed in vivo or in vitro in neuronal or glial cells, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites present in a HuGlyIDS used in accordance with the methods described herein are glycosylated with an Asn residue (or other relevant residue) present at the N-glycosylation site, i.e., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites of the resulting HuGlyIDS are glycosylated.

[0181] In another specific embodiment, when HuGlyIDS is expressed in vivo or in vitro in neuronal or glial cells, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites present in a HuGlyIDS molecule used in accordance with the methods described herein are glycosylated with the same linked glycan attached to an Asn residue (or other relevant residue) present at the N-glycosylation site, i.e., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites of the resulting HuGlyIDS have the same linked glycan.

[0182] Importantly, when the IDS proteins used according to 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 avoided. Instead, as a result of the therapeutic methods described herein (e.g., the use of neuronal or glial cells to express IDS), the N-glycosylation sites of the IDS proteins are advantageously modified with glycans that are appropriate and beneficial for human therapy, particularly at therapeutic target sites. When CHO cells or E. coli are used for protein production, such advantages are not available because, for example, CHO cells (1) do not express 2,6 sialyltransferase and therefore cannot add 2,6 sialic acid during N-glycosylation, and (2) may add Neu5Gc instead of Neu5Ac as the sialic acid, and E. coli does not naturally contain the components necessary for N-glycosylation. Furthermore, such advantages cannot be achieved by using human cells that are not neuronal or glial cells for protein production.Thus, in one embodiment, the IDS protein that is expressed in neuronal or glial cells to produce the HuGlyIDS used in the therapeutic methods described herein is glycosylated in the manner that proteins are N-glycosylated in human neuronal or glial cells, but is not glycosylated in the manner that proteins are glycosylated in CHO cells.In another embodiment, the IDS protein that is expressed in neuronal or glial cells to produce the HuGlyIDS used in the therapeutic methods described herein is glycosylated in the manner that proteins are N-glycosylated in neuronal or glial cells, but such glycosylation cannot be achieved naturally when using prokaryotic host cells, such as E. coli.In one embodiment, the IDS protein expressed in human neuronal or glial cells to provide the HuGlyIDS used in the therapeutic methods described herein is glycosylated in the manner in which proteins are N-glycosylated in human neuronal or glial cells, but is not glycosylated in the manner in which proteins are glycosylated in human cells that are not neuronal or glial cells.

[0183] 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 associated proteins by incubation with hydrazine (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, releasing the attached glycan. The N-acetyl group is lost during this process and must be reconstituted by re-N-acetylation. The released glycans can be purified on a carbon column and subsequently labeled at the reducing end using the fluorescent dye molecule 2-aminobenzamide. 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 length and number of repeating units of the polysaccharide. Structural information can be gleaned by collecting individual peaks and subsequently performing MS / MS analysis. This allows the monosaccharide composition and sequence of the repeating units to be confirmed, further identifying the homogeneity of the polysaccharide composition. Specific low-molecular-weight peaks can be analyzed by MALDI-MS / MS, and the results can be used to confirm the glycan sequence. Each peak corresponds to a polymer consisting of a specific number of repeating units and their fragments. In this way, the chromatogram can be used to measure the distribution of polymer lengths. The elution time is an indicator of polymer length, while the fluorescence intensity correlates with the molar abundance for each polymer.

[0184] The uniformity of glycan patterns associated with proteins is related to glycan length and the number of glycans present across glycosylation sites, and this uniformity can be assessed using methods known in the art, such as measuring glycan length and hydrodynamic radius. Size-exclusion HPLC allows for the measurement of hydrodynamic radius. The greater the number of glycosylation sites on a protein, the greater the variation in hydrodynamic radius compared to carriers with fewer glycosylation sites. However, when analyzing single glycan chains, they can be more uniform due to a higher degree of control over their length. Glycan length can be measured by hydrazinolysis, SDS-PAGE, and capillary gel electrophoresis. In addition, uniformity can also mean that the usage pattern of a particular glycosylation site varies over a broader or narrower range. These factors can be measured by glycopeptide LC-MS / MS.

[0185] N-glycosylation confers numerous advantages to HuGlyIDS used in the methods described herein. These advantages are not available when proteins are produced in E. coli because E. coli does not naturally possess the components required for N-glycosylation. Furthermore, some advantages are not available when proteins are produced in, for example, CHO cells because CHO cells lack the components necessary for the addition of certain glycans (e.g., 2,6 sialic acid) and can add glycans uncommon in humans, such as Neu5Gc, and the α-Gal antigen, which is immunogenic in most individuals and can induce anaphylaxis at high concentrations. Furthermore, some advantages are not available when proteins are produced in human cells that are not neuronal or glial. Thus, expression of IDS in human neuronal or glial cells produces HuGlyIDS containing beneficial glycans, but this does not correlate with proteins produced in CHO cells, E. coli, or human cells that are not neuronal or glial.

[0186] 5.1.3 Tyrosine sulfation In addition to N-linked glycosylation sites, hIDS also contain tyrosine ("Y") sulfation sites (PSSEKY 165 (See, e.g., Yang et al., 2015, Molecules 20:2138-2164, especially p. 2154, for an analysis of amino acids surrounding tyrosine residues that undergo protein tyrosine sulfation, the entire contents of which are incorporated herein by reference. The "rules" can be summarized as follows: Y residues have E or D within positions +5 to -5 of Y, and position -1 of Y is a neutral or acidic charged amino acid - but not a basic amino acid, e.g., R, K, or H, which stop sulfation).

[0187] Importantly, tyrosine-sulfated proteins cannot be produced in E. coli, which does not naturally possess the enzymes required for tyrosine sulfation. Furthermore, CHO cells are defective in tyrosine sulfation. These cells are not secretory cells and have limited capacity for post-translational tyrosine sulfation. See, e.g., Mikkelsen & Ezban, 1991, Biochemistry 30:1533-1537. Advantageously, the methods provided herein require the expression of an IDS, such as HuGlyIDS, in neuronal or glial cells that are secretory and capable of tyrosine sulfation. Assays for detecting tyrosine sulfation are known in the art. See, e.g., Yang et al., 2015, Molecules 20:2138-2164.

[0188] Tyrosine sulfation of hIDS, a robust post-translational process in human CNS cells, improves the processing and activity of transgene products. The significance of tyrosine sulfation of lysosomal proteins remains unclear, but for other proteins, it has been shown to increase the affinity of protein-protein interactions (antibodies and receptors) and facilitate proteolytic processing (peptide hormones). (See Moore, 2003, J. Biol. Chem. 278:24243-46, and Bundegaard et al., 1995, The EMBO J 14:3073-79.) Tyrosine protein sulfotransferase 1 (TPST1), which is responsible for tyrosine sulfation (a potential final step in IDS processing), is apparently expressed at high levels (based on mRNA) in the brain (gene expression data for TPST1 can be found, for example, in the EMBL-EBI Expression Atlas, accessible at http: / / www.ebi.ac.uk / gxa / home).

[0189] 5.2 Constructs and Formulations Viral vectors or other DNA expression constructs encoding iduronate-2-sulfatase (IDS), e.g., human IDS (hIDS), are provided for use in the methods provided herein. Viral vectors or other DNA expression constructs encoding glycosylated (HuGly)aL-iduronidase (IDUA), e.g., human IDUA (hIDUA), are provided for use in the methods provided herein. The viral vectors and other DNA expression constructs provided herein include any suitable method for delivering a transgene to cerebrospinal fluid (CSF). Means of transgene delivery 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), polymeric 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 that targets neural cells.

[0190] In some aspects, the disclosure provides a nucleic acid for use, the nucleic acid encoding an IDS, e.g., a hIDS, operably linked to a promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, an MMT promoter, an EF-1 alpha promoter, a UB6 promoter, a chicken beta-actin promoter, a CAG promoter, an RPE65 promoter, and an opsin promoter.

[0191] In certain embodiments, provided herein are recombinant vectors comprising 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 sequences selected from the group consisting of a promoter sequence, a gene sequence of interest (transgene, e.g., IDS), an untranslated region, and a termination sequence. In certain embodiments, the viral vectors provided herein comprise a promoter operably linked to a gene of interest.

[0192] In certain embodiments, the nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein can be codon-optimized, for example, via any codon-optimization technique known to those of skill in the art (see, e.g., Quax et al., 2015, Mol Cell 59:149-161).

[0193] In another aspect, the disclosure provides a formulation comprising a recombinant nucleotide expression vector encoding a human IDS, the formulation being suitable for administration to the cerebrospinal fluid of the human brain, whereby a depot is formed in the human central nervous system, the depot secreting a recombinant human IDS glycoprotein precursor that is approximately 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 formylglycine, is α2,6-sialylated, has no detectable NeuGc, is free of α-Gal antigen, and / or is mannose-6-phosphorylated. For example, the formulation can contain a buffer (e.g., a buffer having a specific pH or containing specific components) suitable for administration to the cerebrospinal fluid of the human brain, thereby forming a depot in the human central nervous system that secretes a recombinant human IDS glycoprotein precursor that is approximately 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 formylglycine, is α2,6-sialylated, contains no detectable NeuGc, is free of α-Gal antigen, and / or is mannose-6-phosphorylated. In certain embodiments, the buffer comprises a physiologically compatible aqueous buffer, a surfactant, and optional excipients.

[0194] In another aspect, the disclosure provides a kit comprising a recombinant nucleotide expression vector encoding a human IDS and a pharmaceutically acceptable carrier, wherein the recombinant nucleotide expression vector is suitable for administration into the cerebrospinal fluid (CSF) of the human brain, whereby a depot is formed in the human central nervous system, and the depot secretes a recombinant human IDS glycoprotein precursor that is approximately 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 formylglycine, is α2,6-sialylated, has no detectable NeuGc, is free of α-Gal antigen, and / or is mannose-6-phosphorylated. In another aspect, the disclosure provides a kit comprising a formulation comprising a recombinant nucleotide expression vector encoding human IDS, the formulation being suitable for administration into the CSF of the human brain, whereby a depot is formed in the human central nervous system, the depot secreting a recombinant human IDS glycoprotein precursor that is approximately 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 formylglycine, is α2,6-sialylated, contains no detectable NeuGc, is free of α-Gal antigen, and / or is mannose-6-phosphorylated. The kits described herein comprise a recombinant nucleotide expression vector, or formulation, in one or more containers. Such one or more containers may optionally be accompanied by a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of drugs or biological products, which notice reflects that such agency has approved the manufacture, use, or sale for administration to humans.

[0195] The formulations and kits encompassed herein can be used in accordance with the methods for treating human patients provided in this disclosure.

[0196] 5.2.1.mRNA In certain embodiments, the vectors provided herein are modified mRNAs encoding a gene of interest (e.g., a transgene, e.g., an IDS). Synthesis of modified and unmodified mRNAs for delivery of transgenes to the CSF is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, the entire contents of which are incorporated herein by reference. In certain embodiments, modified mRNAs encoding an IDS, e.g., a hIDS, are described herein.

[0197] 5.2.2. Viral Vectors Viral vectors include adenovirus, adeno-associated virus (AAV, e.g., AAV9), lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus, Sendai virus (hemagglutinin virus of Japan) (HVJ), alphavirus, vaccinia virus, and retroviral vectors. Retroviral vectors include vectors based on murine leukemia virus (MLV) and human immunodeficiency virus (HIV). 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 modified to be replication-deficient in humans. In certain embodiments, the viral vector is an AAV vector configured in a hybrid vector, e.g., a "helpless" adenoviral vector. In certain embodiments, provided herein are viral vectors comprising a viral capsid from a first virus and a viral envelope protein from a second virus. In a particular embodiment, the second virus is vesicular stomatitis virus (VSV). In a more particular embodiment, the envelope protein is VSV-G protein.

[0198] In certain embodiments, the viral vectors provided herein are HIV-based viral vectors. In certain embodiments, the HIV-based vectors provided herein comprise at least two polynucleotides, the gag and pol genes being derived from the HIV genome and the env gene being derived from another virus.

[0199] In certain embodiments, the viral vectors provided herein are herpes simplex virus-based viral vectors. In certain embodiments, the herpes simplex virus-based vectors provided herein are modified to lack one or more immediate-early (IE) genes, thereby rendering them non-cytotoxic.

[0200] In certain embodiments, the viral vectors provided herein are MLV-based viral vectors. In certain embodiments, the MLV-based vectors provided herein contain up to 8 kb of heterologous DNA in place of viral genes.

[0201] In certain embodiments, the viral vectors provided herein are lentivirus-based viral vectors. In certain embodiments, the lentiviral vectors provided herein are derived from human lentiviruses. In certain embodiments, the lentiviral vectors provided herein are derived from non-human lentiviruses. In certain embodiments, the lentiviral vectors provided herein are packaged in lentiviral capsids. In certain embodiments, the lentiviral vectors provided herein comprise one or more of the following elements: a long terminal repeat sequence, a primer binding site, a polypurine tract, an att site, and an encapsidation site.

[0202] In certain embodiments, the viral vectors provided herein are alphavirus-based viral vectors. In certain embodiments, the alphavirus vectors provided herein are recombinant, replication-deficient alphaviruses. In certain embodiments, the alphavirus replicons of the alphavirus vectors provided herein are targeted to specific cell types by displaying functional heterologous ligands on their virion surface.

[0203] 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, the AAV-based vectors provided herein comprise components derived from one or more serotypes of AAV. In certain embodiments, the AAV-based vectors provided herein comprise components derived from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAV10, or AAV11. In preferred embodiments, the AAV-based vectors provided herein comprise components derived 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 for producing recombinant AAV and AAV capsids are taught, for example, in U.S. Patent No. 7,282,199 B2, U.S. Patent No. 7,790,449 B2, U.S. Patent No. 8,318,480 B2, U.S. Patent No. 8,962,332 B2, and International Patent Application No. PCT / EP2014 / 076466, the entire contents of each of which are incorporated herein by reference. In one aspect, provided herein is an AAV (e.g., AAV9 or AAVrhlO)-based viral vector encoding a transgene (e.g., an IDS). In a specific embodiment, provided herein is an AAV9-based viral vector encoding an IDS. In yet a specific embodiment, provided herein is an AAV9-based viral vector encoding a hIDS.

[0204] In certain embodiments, an AAV9 vector is provided that comprises an artificial genome comprising: (i) an expression cassette comprising a transgene under the control of regulatory elements and flanked by ITRs, and (ii) a viral capsid having the amino acid sequence of an AAV9 capsid protein or 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 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 in the sequence of SEQ ID NO: 26, and retains the biological function of the AAV9 capsid. Figure 6 provides a comparative alignment of the amino acid sequences of capsid proteins of different AAV serotypes with potential amino acids that can be substituted at specific positions in the sequences, arranged based on a comparison in the column labeled SUBS. Thus, in certain embodiments, an AAV9 vector comprises an AAV9 capsid variant 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 identified in the SUBS column of Figure 6 that are not present at those positions in the native AAV9 sequence.

[0205] In certain embodiments, the AAV used in the methods described herein is Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6):1056-1068, the entire contents of which are incorporated herein by reference. In certain embodiments, the AAV used in the methods described herein contains one of the following amino acid insertions: LGETTRP or LALGETTRP, as described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323, the entire contents of which are incorporated herein by reference. In certain embodiments, the AAV used in the methods described herein is AAV.7m8, as described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323, the entire contents of each of which are incorporated by reference herein. In certain embodiments, the AAV used in the methods described herein is any AAV disclosed in U.S. Patent No. 9,585,971, e.g., AAV-PHP.B. In certain embodiments, the AAV used in the methods described herein is an AAV disclosed in any of the following patents and patent applications, the entire contents of each of which are incorporated herein by reference: U.S. Patent Nos. 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, 9,284,357, 9,409,953, 9, 169,299, 9,193,956, 9458517, and 9,587,282, U.S. 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.

[0206] In certain embodiments, single-stranded AAV (ssAAV) can be used as described above. In certain embodiments, self-complementary vectors, such as scAAV, can be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16 (pp. 1248-1254); and U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, the entire contents of each of which are incorporated herein by reference).

[0207] In certain embodiments, the viral vector used in the methods described herein is an adenovirus-based viral vector. Recombinant adenovirus vectors can be used to introduce IDS. The recombinant adenovirus can be a first-generation vector with an E1 deletion, with or without an E3 deletion, and an expression cassette inserted into either deleted region. The recombinant adenovirus can be a second-generation vector with a complete or partial deletion of the E2 and E4 regions. Helper-dependent adenoviruses retain only the adenovirus inverted terminal repeats and packaging signal (phi). The transgene is inserted between the packaging signal and the 3' ITR, with or without a stuffer sequence, which maintains the size of the artificial genome close to the wild-type size of approximately 36 kb. Exemplary protocols for the production of adenoviral vectors can be found in Alba et al., 2005, "Gutless adenovirus: last generation adenovirus for gene therapy," Gene Therapy 12:S18-S27, the entire contents of which are incorporated herein by reference.

[0208] In certain embodiments, the viral vector used in the methods described herein is a lentivirus-based viral vector. Recombinant lentivirus vectors can be used to introduce IDS. Four plasmids are used to create the construct: a plasmid containing the Gag / pol sequence, a plasmid containing the Rev sequence, a plasmid containing the envelope protein (i.e., VSV-G), and a Cis plasmid containing packaging elements and the IDS gene.

[0209] For lentiviral vector production, the four plasmids are co-transfected into cells (i.e., HEK293-based cells), whereby polyethylenimine or calcium phosphate, among others, can be used as transfection agents. The lentivirus is then collected in the supernatant (cell harvesting is not necessary / should not be performed, as lentivirus needs to bud from the cells to remain active). The supernatant is filtered (0.45 μm), followed by the addition of magnesium chloride and benzonase. Further downstream processing is highly variable, with the use of TFF and column chromatography being the most GMP-compliant processes. Other processes use ultracentrifugation with or without column chromatography. Exemplary protocols for the production of lentiviral vectors can be found in Lesch et al., 2011, "Production and purification of lentiviral vectors 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 in their entirety.

[0210] In certain embodiments, a vector used in the methods described herein encodes an IDS (e.g., hIDS) that, when transduced into cells of the CNS or relevant cells (e.g., neuronal cells in vivo or in vitro), results in the transduced cells expressing a glycosylated variant of the IDS. In certain embodiments, a vector used in the methods described herein encodes an IDS (e.g., hIDS) that, when transduced into cells of the CNS or relevant cells (e.g., neuronal cells in vivo or in vitro), results in the cells expressing a sulfated variant of the IDS.

[0211] 5.2.3. Promoters and Modifiers of Gene Expression In certain embodiments, the vectors provided herein comprise components that regulate gene delivery or gene expression (e.g., "expression control elements"). In certain embodiments, the vectors provided herein comprise components that regulate gene expression. In certain embodiments, the vectors provided herein comprise components that affect cell binding or targeting. In certain embodiments, the vectors provided herein comprise components that affect localization of a polynucleotide (e.g., a transgene) within a cell after uptake. In certain embodiments, the vectors provided herein comprise components that can be used, for example, as a detectable or selectable marker to detect or select cells that have taken up the polynucleotide.

[0212] In certain embodiments, the viral vectors provided herein comprise one or more promoters. In certain embodiments, the promoter is a constitutive promoter. In alternative embodiments, the promoter is an inducible promoter. Native IDS genes, like most housekeeping genes, primarily use GC-rich promoters. In preferred embodiments, a strong constitutive promoter is used to provide sustained expression of the hIDS. Such promoters include the "CAG" synthetic promoter, which contains the "C" - cytomegalovirus (CMV) early enhancer element, "A" - the promoter and first exon and intron of the chicken beta-actin 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).

[0213] In certain embodiments, the promoter is the CB7 promoter (see Dinculescu et al., 2005, Hum Gene Ther 16:649-663, the entire contents of which are incorporated herein by reference). In some embodiments, the CB7 promoter comprises other expression control elements that enhance vector-driven transgene expression. In certain embodiments, the other expression control elements include a chicken β-actin intron and / or a rabbit β-globin pol A signal. In certain embodiments, the promoter comprises a TATA box. In certain embodiments, the promoter comprises one or more elements. In certain embodiments, one or more promoter elements may be inverted or shifted relative to each other. In certain embodiments, the promoter elements are positioned to function cooperatively. In certain embodiments, the promoter elements 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 the 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., neuronal-specific promoters).

[0214] In certain embodiments, the viral vectors provided herein comprise one or more regulatory elements other than promoters.In certain embodiments, the viral vectors provided herein comprise enhancers.In certain embodiments, the viral vectors provided herein comprise repressors.In certain embodiments, the viral vectors provided herein comprise introns or chimeric introns.In certain embodiments, the viral vectors provided herein comprise polyadenylation sequences.

[0215] 5.2.4. Signal Peptide In certain embodiments, the vectors provided herein include components that regulate protein delivery. In certain embodiments, the viral vectors provided herein include one or more signal peptides. In certain embodiments, the signal peptide enables proper packaging (e.g., glycosylation) of the transgene product (e.g., IDS) within the cell. In certain embodiments, the signal peptide enables proper localization of the transgene product (e.g., IDS) within the cell. In certain embodiments, the signal peptide enables secretion of the transgene product (e.g., IDS) from the cell. Examples of signal peptides for use in connection with the vectors and transgenes provided herein can be found in Table 4. A signal peptide may also be referred to herein as a leader sequence or leader peptide. [Table 7]

[0216] 5.2.5. Untranslated Regions 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 a desired level of protein expression. In certain embodiments, the UTRs are optimized for transgene mRNA half-life. In certain embodiments, the UTRs are optimized for transgene mRNA stability. In certain embodiments, the UTRs are optimized for transgene mRNA secondary structure.

[0217] 5.2.6. Inverted Terminal Repeats In certain embodiments, the viral vectors provided herein comprise one or more inverted terminal repeat (ITR) sequences. The ITR sequences can be used to package a recombinant gene expression cassette into the virion of the viral vector. In certain embodiments, the ITRs are derived from AAV (e.g., AAV9) (see, e.g., Yan et al., 2005, J. Virol., 79(1):364-379; U.S. Patent No. 7,282,199 B2; U.S. Patent No. 7,790,449 B2; U.S. Patent No. 8,318,480 B2; U.S. Patent No. 8,962,332 B2; and International Patent Application No. PCT / EP2014 / 076466, the entire contents of each of which are incorporated herein by reference).

[0218] 5.2.7. Transgene In certain embodiments, the vectors provided herein encode an IDS transgene. In certain embodiments, the IDS is controlled by a suitable expression control element 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.

[0219] The HuGlyIDS encoded by the transgene can include, but is not limited to, a human IDS (hIDS) having the amino acid sequence of SEQ ID NO: 1 (shown in FIG. 1), as well as derivatives of hIDS having amino acid substitutions, deletions, or additions, such as, but not limited to, those derivatives comprising amino acid substitutions selected from corresponding non-conserved residues in orthologs of IDS shown in FIG. 2, provided that such mutations do not include substitution of the cysteine ​​residue at position 84 (C84) required for enzymatic activity (Millat et al., 1997, Biochem J 326:243-247), or, for example, those shown in FIG. 3, or those described in Sukegawa-Hayasaka et al., 2006, J Inherit Metab Dis, each of which is incorporated herein by reference. 29:755-761 (reporting the "weak" variants R48P, A85T, W337R, and truncating variant Q531X, and the "severe" variants P86L, S333L, S349I, R468Q, R468L); Millat et al., 1998, BBA 1406:214-218 (reporting the "weak" variants P480L and P480Q; and the "severe" variant P86L); and Bonucelli et al., 2001, BBA 1537:233-238.

[0220] For example, amino acid substitutions at specific positions in a hIDS can be selected from the corresponding nonconservative amino acid residues found at those positions in the IDS orthologs aligned in Figure 2, provided that such substitutions do not include any of the deleterious mutations shown in Figure 3 or reported in Sukegawa-Hayasaka et al., 2006 (supra), Millat et al., 1998 (supra), or Bonucelli et al., 2001 (supra), each of which is incorporated herein by reference. The resulting transgene product can be tested in in vitro cell culture or in test animals using conventional assays to ensure that the mutations do not impair IDS function. The preferred amino acid substitutions, deletions, or additions selected should maintain or increase the enzymatic activity, stability, or half-life of the IDS as tested by conventional assays for MPS II in in vitro cell culture or animal models. For example, the enzymatic activity of the transgene product can be assessed using a conventional enzyme assay using 4-methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate or 4-methylumbelliferyl sulfate as a substrate (for exemplary IDS enzyme assays that can be used, see, e.g., Lee et al., 2015, Clin. Biochem. 48(18):1350-1353; Dean et al., 2006, Clin. Chem. 52(4):643-649, the entire contents of each of which are incorporated herein by reference). The ability of the transgene product to correct the MPS II phenotype can be assessed in cell culture.For example, MPS II cells in culture can be transduced with a viral vector or other DNA expression construct encoding the rhIDS or derivative, or the transgene or derivative can be added to MPS II cells in culture, or the MPS II cells can be co-cultured with human neuronal / glial host cells engineered to express and secrete the rhIDS or derivative, and correction of the defect in the MPS II cell culture can be assessed, for example, by detecting and determining a decrease in IDS enzyme activity and / or GAG storage in the MPS II cells in culture (see, e.g., Stroncek et al., 1999, Transfusion 39(4):343-350, the entire contents of which are incorporated herein by reference).

[0221] In some embodiments, the dose of a recombinant AAV of the present disclosure is determined using a PCR assay. In some embodiments, the PCR assay is a polyA 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 the dose determined in another assay. For example, the dose determined using a transgene-specific PCR assay is higher (e.g., about 50% higher) than the dose determined using a polyA-specific PCR assay. In some embodiments, the dose determined by the transgene-specific PCR assay is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than about 75% higher than the dose determined by a polyA-specific PCR assay. In some embodiments, the genome copy number is 2.0 x 10 as determined using a polyA-specific PCR assay. 11 The dose of GC / g brain mass was 2.9 × 10, obtained by determining the number of genome copies using a transgene-specific PCR assay. 11 The dose is equivalent to a dose of GC / g brain mass. In some embodiments, the total dose administered to a subject accounts for the subject's estimated brain mass, which can be determined using magnetic resonance imaging (MRI) screening.

[0222] Construct 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 a transgene (e.g., an IDS), h) a fourth linker sequence, i) a polyA sequence, j) a fifth linker sequence, and k) a second ITR sequence.

[0223] 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 a transgene (e.g., an 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 a transgene (e.g., an IDS), wherein the transgene comprises a signal peptide.

[0224] 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 a transgene (e.g., an IDS), i) a second UTR sequence, j) a fourth linker sequence, k) a polyA sequence, l) a fifth linker sequence, and m) a second ITR sequence.

[0225] 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 a transgene (e.g., an IDS), i) a second UTR sequence, j) a fourth linker sequence, k) a polyA sequence, l) a fifth linker sequence, and m) a second ITR sequence, wherein the transgene comprises a signal peptide and wherein the transgene encodes a hIDS.

[0226] In certain embodiments, the viral vectors described herein comprise the elements illustrated in Figure 5 in the illustrated order.

[0227] 5.2.9. Vector Production and Testing The viral vectors provided herein can be produced using host cells. The viral vectors provided herein can be produced using mammalian host cells, such as A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, and myoblasts. The viral vectors provided herein can be produced using host cells derived from humans, monkeys, mice, rats, rabbits, or hamsters.

[0228] Host cells are stably transformed with sequences encoding the transgene and associated elements (i.e., the vector genome) and a means for producing the virus in the host cell, e.g., replication and capsid genes (e.g., the AAV rep and cap genes). For methods of producing recombinant AAV vectors having AAV8 capsids, see Section IV of the detailed description in U.S. Pat. No. 7,282,199 B2, the entire contents of which are incorporated herein by reference. The genome copy titer of the vector can be determined, for example, by TAQMAN® analysis. Virions can be recovered, for example, by CsCl2 sedimentation.

[0229] In vitro assays, e.g., cell culture assays, can be used to measure transgene expression from the vectors described herein, thus, for example, demonstrating the efficacy of the vector. For example, HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11, or ReNcell VM cell lines, or other cell lines derived from neuronal or glial cells, or neuronal or glial progenitor cells, can be used to assess transgene expression. Upon expression, the expression product (i.e., HuGlyIDS) can be characterized, such as by determining the glycosylation and tyrosine sulfation patterns associated with HuGlyIDS.

[0230] 5.2.10. Composition Compositions are described that include a vector encoding a transgene as described herein and a suitable carrier. Suitable carriers (e.g., CSFs and, e.g., for administration to neural cells) may be readily selected by one of skill in the art.

[0231] 5.3 Gene therapy The method for administering a therapeutically effective amount of a transgene construct to a human subject with MPS II is described.More specifically, the method for administering a therapeutically effective amount of a transgene construct to a patient with MPS II is described, particularly the method for administering into CSF.In certain embodiments, this method for administering a therapeutically effective amount of a transgene construct into CSF ​​can be used to treat patients with Hunter syndrome.

[0232] 5.3.1. Target patient population In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to a patient diagnosed with MPS II. In certain embodiments, the patient is diagnosed with mild MPS II. In certain embodiments, the patient is diagnosed with severe MPS II. In certain embodiments, the patient is diagnosed with Hunter syndrome. In certain embodiments, the patient is diagnosed with neuropathic MPS II. In some embodiments, the patient is diagnosed with hepatosplenomegaly, has symptoms associated with hepatosplenomegaly, is suspected of having hepatosplenomegaly, and / or is predisposed to hepatosplenomegaly. Examples of symptoms associated with hepatosplenomegaly include, but are not limited to, dark urine, clay-colored stools, abdominal enlargement or edema, fever, itching, jaundice or yellowing of the eyes and skin, nausea, pain (e.g., in the upper right part of the stomach), fatigue, and / or vomiting. In some embodiments, the patient diagnosed with MPS II has hepatosplenomegaly. In some embodiments, the patient is suffering from hepatosplenomegaly associated with MPS II. In some embodiments, the patient is being treated or has been treated with ERT.

[0233] In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to a patient diagnosed with MPS II, ie, a patient who has been shown to respond to treatment with an IDS, eg, a hIDS.

[0234] In certain aspects, a therapeutically effective amount of the recombinant vector is administered to a pediatric patient. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient under the age of 3. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient between the ages of 2 and 4. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient aged 4 months or older and under the age of 5. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient aged 4 months or older and under the age of 5 who has severe MPS II. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient aged 5 years or older and under the age of 18. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient aged 5 years or older and under the age of 18 who has neuropathic MPS II. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient aged 18 months or older and under the age of 8. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient aged 18 months or older and under the age of 8 who is a male pediatric patient. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient aged 3 to 8 years. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient between 8 and 16 years of age. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient between 5 and 18 years of age. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient under 10 years of age. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient under 10 years of age who has severe MPS II. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient under 18 years of age. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient 5 years of age or older. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient over 10 years of age.

[0235] In certain aspects, a therapeutically effective amount of the recombinant vector is administered to a patient who is 4, 5, 6, 7, 8, 9, 10, or 11 months of age. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient who is about 4, 5, 6, 7, 8, 9, 10, or 11 months of age. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient who is 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months of age. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient who is about 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months of age. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient who is 1, 2, 3, 4, or 5 years of age. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient who is about 1, 2, 3, 4, or 5 years of age. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient aged 1-2 years, 2-3 years, 3-4 years, 4-5 years, or 5-6 years. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient about 1-2 years, 2-3 years, 3-4 years, 4-5 years, or 5-6 years. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient aged 5-6 years, 6-7 years, 7-8 years, 8-9 years, 9-10 years, 10-11 years, 11-12 years, 12-13 years, 13-14 years, 14-15 years, 15-16 years, 16-17 years, 17-18 years, or 18-19 years. In certain embodiments, a therapeutically effective amount of the recombinant vector is administered to a patient who is about 5-6 years old, 6-7 years old, 7-8 years old, 8-9 years old, 9-10 years old, 10-11 years old, 11-12 years old, 12-13 years old, 13-14 years old, 14-15 years old, 15-16 years old, 16-17 years old, 17-18 years old, or 18-19 years old.

[0236] In certain aspects, the therapeutically effective dose of the recombinant vector is administered to an adolescent patient. In certain embodiments, the therapeutically effective dose of the recombinant vector is administered to an adult patient. In some embodiments, the therapeutically effective dose of the recombinant vector is administered to a male patient. In other embodiments, the therapeutically effective dose of the recombinant vector is administered to a female patient.

[0237] In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to a patient diagnosed with MPS II and confirmed to be responsive to treatment with an IDS, e.g., hIDS, infused into the CSF prior to gene therapy treatment.

[0238] 5.3.2. Dosage and Mode of Administration In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to the CSF via intrathecal administration (i.e., injection into the subarachnoid space, allowing the recombinant vector to diffuse through the CSF and transduce cells of the CNS). This can be accomplished in several ways, for example, by intracranial (intracisternal or intraventricular) injection or injection into the lumbar cisterna. In certain embodiments, intrathecal administration is performed by intracisternal (IC) injection (e.g., into the cisterna magna). In certain embodiments, intracisternal injection is performed via CT-guided suboccipital puncture. In certain embodiments, intrathecal injection is performed via lumbar puncture. In certain embodiments, intrathecal injection is performed via C1-2 puncture, if feasible for the patient. Alternatively, the recombinant vector can be injected directly into the ventricles using intracerebroventricular (ICV) administration (a more invasive technique used to introduce anti-infective or anti-cancer drugs that do not penetrate the blood-brain barrier), for example, image-guided ICV injection. In certain embodiments, the recombinant vector is administered via a single image-guided ICV infusion. In further specific embodiments, the recombinant vector is administered via a single image-guided ICV infusion, with the administration catheter immediately removed. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to the CNS via intranasal administration. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to the CNS by intraparenchymal injection. In certain embodiments, the intraparenchymal injection targets the striatum. In certain embodiments, the intraparenchymal injection targets the white matter. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to the CSF by any means known in the art, for example, any means disclosed in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, the entire contents of which are incorporated herein by reference.

[0239] In a preferred embodiment, for intrathecal administration (including IC and ICV administration), a therapeutically effective dose of the recombinant vector is administered into the CSF in an infusion volume not exceeding 10% of the total CSF volume, which is approximately 50 ml in infants and approximately 150 ml in adults. A carrier suitable for intrathecal infusion, such as Elliott's B solution or modified Elliott's B solution, should be used as a vehicle for the recombinant vector. Elliott's B solution (generic name: sodium chloride, sodium bicarbonate, anhydrous dextrose, magnesium sulfate, potassium chloride, calcium chloride, and sodium phosphate) is a sterile, non-pyrogenic, isotonic solution containing no bacteriostatic preservatives and is used as a diluent for intrathecal administration of chemotherapy drugs. Modified Elliot's B solution contains 8.77 g / L sodium chloride, 0.244 g / L magnesium chloride, 0.0278 g / L monosodium phosphate monohydrate, 0.114 g / L disodium phosphate 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 of the disclosure or a composition comprising the AAV is provided in a modified Elliot's B solution for intrathecal administration.

[0240] In one embodiment, a non-replicating recombinant AAV9 vector expressing human iduronate-2-sulfatase (IDS) is used for therapy. In a specific embodiment, 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 a specific embodiment, the transgene contains a chicken beta-actin intron and a rabbit beta-globin polyadenylation (polyA) signal.

[0241] In certain embodiments, the recombinant nucleotide expression vector is administered at a dose that corresponds to the brain mass of the human subject. In a preferred embodiment, the brain mass is determined by brain magnetic resonance imaging (MRI) of the subject's brain. In certain embodiments, the human subject's brain volume cm 3 is multiplied by a factor of 1.046 g / cm3 to convert from the human subject's brain volume to the human subject's brain mass, where the human subject's brain volume is obtained from a brain MRI of the human subject. In some embodiments, the 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., a dose of a recombinant nucleotide expression vector) is determined by a polyA-specific PCR assay. In some embodiments, the number of genome copies in a dose (e.g., a dose of a recombinant nucleotide expression vector) is determined by a transgene-specific PCR assay. In some embodiments, the weight of the brain mass is determined by MRI.

[0242] In certain embodiments, the rAAV9.hIDS is present at 1.4 x 10 in a volume of about 5-20 ml. 13 GC (1.1 × 10 per gram of brain mass) 10 GC) ~ 7.0 × 10 13 GC (5.6 × 10 per gram of brain mass) 10 A single fixed dose in the range of 0.1 mg / kg / day (IC) is administered IC (suboccipital injection). Higher dose ranges may be used if the patient has neutralizing antibodies to AAV. In some embodiments, a single dose of rAAV encoding a hIDS is administered to the central nervous system (e.g., cerebrospinal fluid) of a subject, and surprisingly, a therapeutic effect is observed outside the CNS. For example, changes in organ size are observed outside the CNS (e.g., spleen or liver) after administration of a rAAV of the present disclosure to the CNS of a subject. In some embodiments, changes in biomarker levels (e.g., D2S6, HS, total GAG, and / or anti-IDS antibodies) are detected outside the CNS (e.g., changes in biomarker levels detected in the liver, spleen, urine, plasma, or blood) after administration of a rAAV disclosed herein in the cerebrospinal fluid. In some embodiments, an additional therapy for MPS II is not administered outside the CNS of the subject.

[0243] In certain embodiments, the recombinant vectors described herein deliver approximately 1.3 x 10 per gram of brain mass (e.g., when the human patient is at least 4 months old and less than 5 years old). 10 GC ~ approx. 6.5 × 10 per gram of brain mass 10 In certain embodiments, the recombinant vectors described herein can be administered intrathecally as a single fixed dose in the range of about 1.3 x 10 per gram of brain mass (e.g., for human patients aged 4 months or older and less than 5 years). 10 GCs may be administered intrathecally as a single fixed dose. In certain embodiments, the recombinant vectors described herein are administered intrathecally at a dose of about 1.9×10 per gram of brain mass (e.g., when the human patient is 4 months or older and less than 5 years old). 10 In another specific embodiment, the recombinant vectors described herein are administered intrathecally at a dose of about 6.5 x 10 per gram of brain mass (e.g., when the human patient is 4 months or older and less than 5 years old). 10 GCs may be administered intrathecally as a single fixed dose. In certain embodiments, the recombinant vectors described herein are administered intrathecally at a concentration of about 9.6×10 per gram of brain mass (e.g., when the human patient is 4 months or older and less than 5 years old). 10 GCs may be administered intrathecally as a single fixed dose. In certain embodiments, the recombinant vectors described herein are administered intrathecally at a dose of about 2.0 x 10 per gram of brain mass (e.g., when the human patient is 4 months or older and less than 5 years old). 11 GCs may be administered intrathecally as a single fixed dose. In certain embodiments, the recombinant vectors described herein are administered intrathecally at a dose of about 2.9×10 per gram of brain mass (e.g., when the human patient is 4 months or older and less than 5 years old). 11 In another specific embodiment, the recombinant vectors described herein may be administered intrathecally as a single fixed dose of Dose 1 or Dose 2 as set forth in Table 5 below (e.g., if the human patient is 4 months or older and less than 5 years old).

[0244] In certain embodiments, the recombinant vectors described herein deliver approximately 1.3 x 10 per gram of brain mass (e.g., in a human patient aged 4 months or older and less than 5 years). 10 GC ~ approx. 2.0 × 10 per gram of brain mass 11 In certain embodiments, the recombinant vectors described herein can be administered intrathecally as a single fixed dose in the range of about 1.3 x 10 per gram of brain mass (e.g., for human patients aged 4 months or older and less than 5 years). 10 GC ~ approx. 2.9 × 10 per gram of brain mass 11 In certain embodiments, the recombinant vectors described herein can be administered intrathecally as a single fixed dose in the range of about 2.0 x 10 per gram of brain mass (e.g., for human patients aged 4 months or older and less than 5 years). 11 GCs may be administered intrathecally as a single fixed dose. In certain embodiments, the recombinant vectors described herein are administered intrathecally at a dose of about 2.9×10 per gram of brain mass (e.g., when the human patient is 4 months or older and less than 5 years old). 11 In another specific embodiment, the recombinant vectors described herein may be administered intrathecally as a single fixed dose of GC at dose 3 listed in Table 6 below (e.g., when the human patient is 4 months of age or older and less than 5 years of age).

[0245] In certain embodiments, the recombinant vectors described herein deliver approximately 1.3 x 10 per gram of brain mass (e.g., in a human patient aged 4 months or older and less than 5 years). 10 GC brain mass ~ approx. 6.5×10 10 In certain embodiments, the recombinant vectors described herein can be administered IC as a single fixed dose in the range of about 1.3 x 10 GC / g brain mass (e.g., for human patients aged 4 months or older and less than 5 years). 10 GCs may be administered IC as a single fixed dose of brain mass. In certain embodiments, the recombinant vectors described herein are administered at a dose of about 1.9 x 10 GCs per gram of brain mass (e.g., for human patients aged 4 months or older and less than 5 years).10 GCs can be administered IC as a single fixed dose of brain mass. In another specific embodiment, the recombinant vectors described herein are administered at a dose of about 6.5 x 10 GCs per gram of brain mass (e.g., when the human patient is 4 months or older and less than 5 years old). 10 GCs may be administered IC as a single fixed dose of brain mass. In certain embodiments, the recombinant vectors described herein are administered at a dose of about 9.6 x 10 GCs per gram of brain mass (e.g., for human patients aged 4 months or older and less than 5 years). 10 In another specific embodiment, the recombinant vectors described herein may be administered IC as a single fixed dose of Dose 1 or Dose 2 as set forth in Table 5 below (e.g., when the human patient is 4 months or older and less than 5 years old).

[0246] In certain embodiments, the recombinant vectors described herein deliver approximately 1.3 x 10 per gram of brain mass (e.g., in a human patient aged 4 months or older and less than 5 years). 10 GC ~ approx. 2.0 × 10 per gram of brain mass 11 In certain embodiments, the recombinant vectors described herein can be administered IC as a single fixed dose in the range of GC. In certain embodiments, the recombinant vectors described herein can be administered at a dose of about 1.3 x 10 per gram of brain mass (e.g., for human patients aged 4 months or older and less than 5 years). 10 GC ~ approx. 2.9 × 10 per gram of brain mass 11 In certain embodiments, the recombinant vectors described herein can be administered IC as a single fixed dose in the range of GC. In certain embodiments, the recombinant vectors described herein can be administered at a dose of about 2.0 x 10 per gram of brain mass (e.g., for human patients aged 4 months or older and less than 5 years). 11 GCs may be administered IC as a single fixed dose. In certain embodiments, the recombinant vectors described herein are administered at a dose of about 2.9 x 10 per gram of brain mass (e.g., for human patients aged 4 months or older and younger than 5 years). 11In another specific embodiment, the recombinant vectors described herein may be administered IC as a single fixed dose of dose 3 listed in Table 6 below (e.g., when the human patient is 4 months or older and less than 5 years old).

[0247] In certain embodiments, the recombinant vectors described herein deliver approximately 1.3 x 10 per gram of brain mass (e.g., in a human patient aged 4 months or older and less than 5 years). 10 GC ~ approx. 6.5 × 10 per gram of brain mass 10 In certain embodiments, the recombinant vectors described herein can be administered ICV as a single fixed dose in the range of GC. In certain embodiments, the recombinant vectors described herein can be administered at a dose of about 1.3 x 10 per gram of brain mass (e.g., for human patients aged 4 months or older and less than 5 years). 10 A single fixed dose of GC may be administered via ICV administration. In certain embodiments, the recombinant vectors described herein are administered at a dose of about 1.9 x 10 per gram of brain mass (e.g., for human patients aged 4 months or older and less than 5 years). 10 It may be administered as a single fixed dose of GC via ICV administration. In another specific embodiment, the recombinant vectors described herein are administered at a dose of about 6.5 x 10 per gram of brain mass (e.g., for human patients aged 4 months or older and younger than 5 years). 10 A single fixed dose of GC may be administered via ICV administration. In certain embodiments, the recombinant vectors described herein are administered at a dose of approximately 9.6 x 10 per gram of brain mass (e.g., for human patients aged 4 months or older and less than 5 years). 10 In another specific embodiment, the recombinant vectors described herein may be administered ICV as a single fixed dose of Dose 1 or Dose 2 as set forth in Table 5 below (e.g., if the human patient is 4 months or older and less than 5 years of age).

[0248] In certain embodiments, the recombinant vectors described herein deliver approximately 1.3 x 10 per gram of brain mass (e.g., in a human patient aged 4 months or older and less than 5 years). 10GC ~ approx. 2.0 × 10 per gram of brain mass 11 In certain embodiments, the recombinant vectors described herein can be administered ICV as a single fixed dose in the range of GC. In certain embodiments, the recombinant vectors described herein can be administered at a dose of about 1.3 x 10 per gram of brain mass (e.g., in human patients aged 4 months or older and less than 5 years). 10 GC ~ approx. 2.9 × 10 per gram of brain mass 11 In certain embodiments, the recombinant vectors described herein can be administered ICV as a single fixed dose in the range of GC. In certain embodiments, the recombinant vectors described herein can be administered at a dose of about 2.0 x 10 per gram of brain mass (e.g., for human patients aged 4 months or older and less than 5 years). 11 GCs may be administered as a single fixed dose via ICV administration. In certain embodiments, the recombinant vectors described herein are administered at a dose of about 2.9 x 10 per gram of brain mass (e.g., for human patients aged 4 months or older and less than 5 years). 11 In another specific embodiment, the recombinant vectors described herein may be administered ICV as a single fixed dose of dose 3 listed in Table 6 below (e.g., when the human patient is 4 months or older and less than 5 years old). [Table 8] [Table 9] TIFF2024505739000011.tif148165

[0249] In certain embodiments, the recombinant vectors described herein are administered at a concentration of about 6.5×10 per gram of brain mass (e.g., when the human patient is 5 years of age or older and less than 18 years of age). 10 In another specific embodiment, the recombinant vectors described herein may be administered intrathecally as a single fixed dose as set forth in and according to Table 7 below (e.g., when the human patient is 5 years of age or older and less than 18 years of age).

[0250] In certain embodiments, the recombinant vectors described herein are administered at a concentration of approximately 6.5×10 per gram of brain mass (e.g., for human patients aged 5 years or older and less than 18 years). 10 In another specific embodiment, the recombinant vectors described herein may be administered IC as a single fixed dose as set forth in and according to Table 7 below (e.g., when the human patient is 5 years of age or older and less than 18 years of age).

[0251] In certain embodiments, the recombinant vectors described herein are administered at a concentration of about 6.5×10 per gram of brain mass (e.g., when the human patient is 5 years of age or older and less than 18 years of age). 10 In another specific embodiment, the recombinant vectors described herein may be administered ICV as a single fixed dose as set forth in and according to Table 7 below (e.g., when the human patient is 5 years of age or older and less than 18 years of age). [Table 10]

[0252] 5.4 Combination therapy The methods of the present invention encompass the administration of HuGlyIDS into the CSF in combination with other available treatments. The additional treatments may be administered before, simultaneously with, or after the gene therapy procedure. Available treatments for MPS II that can be combined with the gene therapy of the present invention include, but are not limited to, enzyme replacement therapy (ERT) using idursulfase administered systemically or into the CSF, and / or HSCT therapy. In another embodiment, ERT can be administered using rHuGlyIDS glycoproteins produced by human neuronal / glial cell lines via recombinant DNA technology. Human neuronal / glial cell lines that can be used for such recombinant glycoprotein production include, but are not limited to, HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11, or ReNcell VM, to name a few. To ensure complete glycosylation, particularly sialylation, and tyrosine sulfation, the cell lines used for production can be enhanced by genetically engineering the host cells to co-express the α-2,6-sialyltransferase (or both α-2,3- and α-2,6-sialyltransferases) responsible for tyrosine-O-sulfation, and / or the TPST-1 and TPST-2 enzymes.

[0253] 5.5 Biomarkers / Sample Collection / Efficacy Monitoring Efficacy can be monitored by measuring cognitive function (e.g., prevention or reduction of neurocognitive decline), a decrease in disease biomarkers (e.g., GAGs such as heparan sulfate and dermatan sulfate) in the CSF and / or serum, and / or an increase in IDS enzyme activity in the CSF and / or serum. Signs of inflammation and other safety events may be monitored.

[0254] In one aspect, provided herein is a method for monitoring the effectiveness of an ERT treatment by determining whether a subject undergoing or who has received ERT treatment can discontinue ERT treatment after administering a gene therapy (e.g., an rAAV encoding a hIDS) of the present disclosure. For example, provided herein is a method for treating and / or identifying a subject diagnosed with MPS II (e.g., a subject likely to respond to discontinuing ERT treatment), comprising: (a) administering to the subject a therapeutically effective amount of a gene therapy (e.g., an rAAV encoding a hIDS) of the present disclosure, wherein the subject has undergone or is undergoing ERT treatment; (b) identifying the subject as likely to respond to ERT treatment, the method 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 likely to respond to ERT treatment if the level of the biomarker in the biological sample is different (e.g., higher or lower) from a reference (e.g., the reference level of at least one biomarker); and (c) discontinuing ERT treatment for the subject. In some embodiments, the biomarker is D2S6, HS, total GAG, and / or anti-IDS antibody. In some embodiments, the ERT is recombinant idursulfase. In some embodiments, the subject diagnosed with MPS II has hepatosplenomegaly.

[0255] In another aspect, provided herein is a method for selectively treating a human subject with MPS II, comprising administering a therapeutically effective amount of an rAAV encoding a hIDS to the subject, wherein the subject has received or is undergoing ERT treatment, and the subject has been determined to be likely to respond to discontinuing ERT treatment according to the 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 if the level of the at least one biomarker is different (e.g., higher or lower) from a reference, the subject is determined to be likely to respond to discontinuing ERT treatment. In some embodiments, the biomarkers are D2S6, HS, total GAG, and / or anti-IDS antibodies. In some embodiments, the ERT is recombinant idursulfase. In some embodiments, the subject diagnosed with MPS II has hepatosplenomegaly.

[0256] In another aspect, provided herein are methods for identifying or diagnosing a subject as having neuropathic or non-neuropathic MPS II or MPS I. In some embodiments, the methods involve determining the level of one or more disaccharides (e.g., DOA0, DOS0, DOA6, D2S6) in a biological sample from the subject. In some embodiments, the methods involve determining the level of undegraded glycosaminoglycans (GAGs) in a biological sample from the subject. In some embodiments, a subject is identified or diagnosed as having neuropathic MPS II or MPS I if the level of one or more disaccharides (e.g., DOA0, DOS0, DOA6, and / or D2S6) is elevated compared to baseline levels. In some embodiments, a subject is identified or diagnosed as having neuropathic MPS II or MPS I if the level of the GAG ​​heparan sulfate (HS) is elevated (e.g., in the brain) compared to baseline levels. 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) after enzymatic digestion (e.g., as determined by bioanalytical mass spectrometry). In some embodiments, for example, elevated levels of D2S6 in the CSF of a subject (e.g., a presymptomatic subject) indicate neuropathic MPS II or MPS I. In some embodiments, the level of D2S6 indicates iduronate-2-sulfatase enzyme activity and can be used for treatment monitoring. In some embodiments, the one or more disaccharides include one or more of D0A0, D0S0, D0A6, D2S6, or a combination thereof. In another aspect, provided herein are methods of identifying or diagnosing a subject as having neuropathic or non-neuropathic MPS II or MPS I, wherein the subject is identified or diagnosed as having neuropathic 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 compared to a reference level. In some embodiments, the subject is pre-symptomatic or does not have visible or detectable MPS II or MPS I symptoms. 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., DOA0, DOS0, DOA6, and / or D2S6) in a biological sample from one or more healthy individuals and / or one or more non-neuropathic subjects. In some embodiments, the reference level is the level of D2S6 in a biological sample (e.g., a CSF sample) from one or more healthy individuals and / or one or more non-neuropathic subjects. In some embodiments, the reference level is a predetermined level. In some embodiments, the level of one or more disaccharides (e.g., DOA0, DOS0, DOA6, D2S6) is about or at least about 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, or more 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% 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., DOA0, DOS0, DOA6, 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%, 90%, or more than 90% higher than the level of one or more disaccharides (e.g., DOA0, DOS0, DOA6, and / or D2S6) in a biological sample from a reference (e.g., a healthy individual).In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or 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 greater 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, hi 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 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, L, 175ng / mL, 180ng / mL, 185ng / mL, 190ng / mL, 195ng / mL, 200ng / mL, 210ng / mL, 220ng / mL, 230ng / mL, 240ng / mL, 250ng / mL, 260ng / mL, 270ng / mL, 280ng / m L, 290ng / mL, 300ng / mL, 310ng / mL, 320ng / mL, 330ng / mL, 340ng / mL, 350ng / mL, 360ng / mL, 370ng / mL, 380ng / mL, 390ng / mL, 400ng / mL, or greater than 400ng / 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 greater 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., in the case of 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., in the case of 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., in the case of 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., in the case of 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., in the case of 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 150 ng / mL (e.g., in the case of 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 170 ng / mL (e.g., in the case of 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 180 ng / mL (e.g., in the case of 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 200 ng / mL (e.g., in the case of D2S6). In some embodiments, the level of one or more heparan sulfate disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6), or the level of D2S6, in a biological sample from a subject is 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 compared to a reference level (e.g., the level of one or more heparan sulfate disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6)), or the level of D2S6 in a biological sample from one or more healthy individuals and / or one or more non-neurologically impaired subjects, or a predetermined value). , 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 higher than 500 ng / mL is extremely high.In some embodiments, the total level of heparan sulfate disaccharide(s) (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample from a subject is 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, 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, 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, 400 ng / mL, 420 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, 5 , 60ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 150ng / mL, 160ng / mL, 170ng / mL, 180ng / mL, 190ng / mL, 200ng / mL, 210ng / mL, 220ng / mL, 2 30ng / mL, 240ng / mL, 250ng / mL, 260ng / mL, 270ng / mL, 280ng / mL, 290ng / mL, 300ng / mL, 310ng / mL, 320ng / mL, 330ng / mL, 340ng / mL, 350ng / mL , 360ng / mL, 370ng / mL, 380ng / mL, 390ng / mL, 400ng / mL, 410ng / mL, 420ng / mL, 430ng / mL, 440ng / mL, 450ng / mL, 460ng / mL, 470ng / mL, 480ng / mL, 490ng / mL, 500ng / mL, 510ng / mL, 520ng / mL, 530ng / mL, 540ng / mL, 550ng / mL, 560ng / mL, 570ng / mL, 580ng / mL, 590ng / mL, 600ng / mL, 610 ng / mL, 620ng / mL, 630ng / mL, 640ng / mL, 650ng / mL, 660ng / mL, 670ng / mL, 680ng / mL, 690ng / mL, 700ng / mL, 710ng / mL, 720ng / mL, 730ng / mL, 7 40ng / mL, 750ng / mL, 760ng / mL, 770ng / mL, 780ng / mL, 790ng / mL, 800ng / mL, 850ng / mL, 900ng / mL, 950ng / mL, 1000ng / mL, or greater than 1000ng / mL.

[0257] In some embodiments, a subject is determined to be responsive to a treatment of the present disclosure (e.g., rAAV9 encoding hIDUA for treatment of 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 a subject (e.g., a subject with MPS I) compared to a reference standard indicates that the subject will be responsive to a treatment of the present disclosure (or treatment with an rAAV9 encoding hIDUA for MPS I). In some embodiments, the reference standard is the level of I0S6 in a biological sample from a healthy individual or a population of healthy individuals. In some embodiments, the reference standard is the 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 standard is the level of I0S6 in a biological sample from a subject not having MPS I or not diagnosed with MPS I, or a population of subjects not having MPS I. In some embodiments, the reference standard is the level of I0S6 in a biological sample from the same subject but obtained at a different time point (e.g., obtained at an earlier time point). In some embodiments, the criterion is a predetermined value.

[0258] 5.5.1. Disease Markers In certain embodiments, the effectiveness of treatment using a recombinant nucleotide expression vector is monitored by measuring the level of a patient's disease biomarker. In certain embodiments, the level of the disease biomarker is measured in the patient's CSF. In certain embodiments, the level of the disease biomarker is measured in the patient's serum. In certain embodiments, the level of the disease biomarker is measured in the patient's plasma. In certain embodiments, the level of the disease biomarker is measured in the patient's urine. In certain embodiments, the disease biomarker is a 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. The I2S enzyme cleaves sulfate from HS in lysosomes, and the absence of I2S leads to the accumulation of long chains of fully sulfated D2S6. In some embodiments, quantitative measurement of D2S6 reflects the level of I2S enzyme activity, and elevated levels of HS and D2S6 are closely correlated with the neuropathic phenotype of MPS II. In some embodiments, the level of D2S6 is inversely correlated with neurocognitive development. In some embodiments, the disease biomarker is an anti-AAV antibody (e.g., an 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.

[0259] In certain embodiments, the effectiveness of a treatment using a recombinant nucleotide expression vector is monitored by measuring one or more of the following biomarkers in a patient-derived sample: (a) the level of GAG in CSF, (b) the level of I2S in CSF, (c) the level of GAG in plasma, (d) the level of I2S in plasma, (e) the level of leukocyte I2S enzyme activity, (f) the level of GAG in urine, (g) the level of heparan sulfate in CSF, and (h) the level of dermatan sulfate in CSF. In certain embodiments, the effectiveness of a treatment using a recombinant nucleotide expression vector is monitored by measuring I2S and / or GAG in CSF, urine, and / or plasma. In certain embodiments, the effectiveness of a treatment using a recombinant nucleotide expression vector is monitored by measuring heparan sulfate in CSF, plasma, and / or urine. In certain embodiments, the effectiveness of a treatment using a recombinant nucleotide expression vector is monitored by measuring non-reducing heparan sulfate. In some embodiments, heparan sulfate measured in CSF is the primary endpoint for determining the effectiveness of a treatment. In certain embodiments, the effectiveness of a treatment using a 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 (e.g., CSF, urine, and / or plasma) used to detect D2S6. In certain embodiments, the effectiveness of a treatment using a recombinant nucleotide expression vector is monitored by measuring urinary total GAG, urinary HS, and / or plasma I2S enzyme activity. In some embodiments, urinary GAG indicates a systemic effect and / or is unrelated to ERT treatment. In some embodiments, the effectiveness of a treatment of the present disclosure is determined based on the level of I2S protein concentration in a sample from the subject (e.g., an increase in the level of I2S protein concentration indicates efficacy). In some embodiments, heparan sulfate (HS) and D2S6 (glycosaminoglycan (GAG)) are measured in cerebrospinal fluid (CSF) at baseline and / or after administration of a recombinant vector of the present disclosure.In some embodiments, determining or monitoring the effectiveness of MPS II treatment in a subject involves administering an rAAV of the disclosure to the subject and then measuring (e.g., compared to a reference) 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, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 8 In some embodiments, the level of at least one biomarker (e.g., D2S6) is detected in a biological sample from the subject obtained after 0, 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 to determine MPS in the subject. Determining or monitoring the efficacy of a treatment can include administering an rAAV of the disclosure to a subject and then measuring (e.g., compared to a reference) 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, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 8 , 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 later, the level of at least one biomarker (e.g., I0S6) is detected and determined in a biological sample from the subject obtained after

[0260] 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 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 a baseline, or a predetermined value). In some embodiments, the level of HS is reduced 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 present disclosure, e.g., compared to a baseline before ERT was discontinued (e.g., compared to the level of HS in the subject before administration of a recombinant vector of the present disclosure). In some embodiments, the level of D2S6 is reduced 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 present disclosure, e.g., compared to a baseline before ERT was discontinued (e.g., compared to the level of D2S6 in the subject before administration of a recombinant vector of the present disclosure). In some embodiments, the level of I0S6 is decreased in the subject compared to a reference (e.g., compared to the level of I0S6 in the subject before administration of a recombinant vector of the present disclosure, or compared to a baseline, or a predetermined value).In some embodiments, the level of I0S6 is reduced 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 present disclosure, e.g., compared to a baseline before ERT was discontinued (e.g., compared to the level of I0S6 in the subject before administration of a recombinant vector of the present disclosure). In some embodiments, biomarkers (e.g., HS, D2S6, I0S6, total GAGs, and / or I2S enzyme) are measured before administration of a recombinant vector of the disclosure, on the same day as administration of a recombinant vector of the disclosure, one day after administration of a recombinant vector of the disclosure, and / or at about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks after administration of a recombinant vector of the disclosure. Measurements are taken after 1 year, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 30 weeks, 35 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 ​​weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, 1 year, 2 years, or more than 2 years. In some embodiments, biomarkers (e.g., HS, D2S6, I0S6, total GAGs, and / or I2S enzyme) are measured prior to administration of a recombinant vector of the disclosure, on the same day as administration of a recombinant vector of the disclosure, or 8 weeks, 16 weeks, 24 weeks, 32 weeks, 40 weeks, 48 ​​weeks, 56 weeks, 72 weeks, 104 weeks, 1 year, 2 years, or more than 2 years after administration of a recombinant vector of the disclosure. In some embodiments, HS and / or D2S6 are measured in CSF in ng / ml.In some embodiments, a biomarker (e.g., HS, I0S6, and / or D2S6) is elevated by about or at least about 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 mg / ml, 40 mg / ml, 45 ng / ml, 50 ng / ml, 55 ng / ml, or more after administration of a recombinant vector of the disclosure, compared to a reference (e.g., compared to a baseline amount or an amount before treatment with a recombinant vector of the disclosure). , 60ng / ml, 65ng / ml, 70ng / ml, 75ng / ml, 80ng / ml, 90ng / ml, 95ng / ml, 100ng / ml, 110ng / ml, 120ng / ml, 130ng / ml, 140ng / ml, 150ng / ml, 160ng / ml, 170ng / ml, 180ng / ml, 190ng / ml, 200ng / ml, 220ng / ml, 250ng / ml, 270ng / ml, 300ng / ml, or greater than 300ng / ml. In some embodiments, total GAGs (e.g., in urine) are measured in g / mol CK. In some embodiments, a biomarker (e.g., total GAGs) is reduced by about or at least about 5 g / mol, 10 g / mol, 15 g / mol, 20 g / mol, 25 g / mol, 30 g / mol, 35 mg / ml, 40 mg / ml, 45 g / mol, 50 g / mol, 55 g / mol, 60 g / mol, 65 g / mol, 70 g / mol, 75 g / mol, 80 g / mol, 85 g / mol, 90 g / mol, 95 g / mol, 100 g / mol, 110 g / mol, 120 g / mol, 130 g / mol, 140 g / mol, 150 g / mol, 160 g / mol, 170 g / mol, 200 g / mol, or more than 200 g / mol after administration of a recombinant vector of the present disclosure, as compared to a reference (e.g., as compared to a baseline amount or an amount before treatment with a recombinant vector of the present disclosure).In some embodiments, biomarkers (e.g., HS, D2S6, I0S6, and / or total GAG) and / or hepatosplenomegaly are reduced by about 3%, 5%, 7%, 8%, 9%, 10%, 12%, or more after administration of a recombinant vector of the present disclosure, e.g., compared to a baseline before ERT was discontinued (e.g., compared to a baseline amount or an amount before treatment with a recombinant vector of the present disclosure). , 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 greater than 100% decrease and / or amelioration. In some embodiments, the determination of whether a biomarker is increased or decreased in a subject is determined before discontinuing ERT in the subject.In some embodiments, the biomarkers (e.g., HS, I0S6, D2S6, and / or total GAGs) and / or hepatosplenomegaly are decreased after administration of a recombinant vector of the disclosure relative to a baseline (e.g., compared to the level of the biomarker in the subject prior to administration of a recombinant vector of the disclosure, or compared to the level of the biomarker from a previously obtained biological sample from the subject, or compared to a baseline, or compared to the level of the biomarker in a healthy individual) after about,...

Claims

1. 1. A pharmaceutical composition comprising human iduronate-2-sulfatase (hIDS) for use in a method of treating mucopolysaccharidosis type II (MPS II) in a human subject, the method comprising: (i) delivering a therapeutically effective amount of the hIDS to the central nervous system (CNS) of the human subject; and (ii) measuring the level of D2S6 present in a biological sample from the human subject; The pharmaceutical composition comprising:

2. 1. A pharmaceutical composition comprising human iduronate-2-sulfatase (hIDS) for use in a method of treating MPS II in a human subject, the method comprising: (a) measuring the level of D2S6 present in a biological sample from the human subject; and (b) delivering a therapeutically effective amount of the hIDS to the central nervous system (CNS) of the human subject if the level of D2S6 in the biological sample of the human subject is higher than a reference level; The pharmaceutical composition comprising:

3. 2. The pharmaceutical composition of claim 1, wherein the method comprises calculating the ratio of D2S6 to total heparan sulfate disaccharides (HS) in the biological sample, wherein the total HS comprises the disaccharides D2S6, D0A0, D0S0, and D0A6.

4. 3. The pharmaceutical composition of claim 2, wherein the method comprises calculating the ratio of D2S6 to total heparan sulfate disaccharides (HS) in the biological sample, wherein the total HS comprises the disaccharides D2S6, D0A0, D0S0, and D0A6.

5. 4. The pharmaceutical composition of claim 3, wherein the human subject is determined to be responsive to the hIDS if the level of D2S6 is at least 20% of the total HS.

6. 5. The pharmaceutical composition of claim 4, wherein the human subject is determined to be responsive to the hIDS if the level of D2S6 is at least 20% of the total HS.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the method comprises delivering hIDS to the CNS via an adeno-associated viral vector (AAV) comprising a nucleotide sequence encoding hIDS.

8. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the method comprises delivering hIDS to the CNS via enzyme replacement therapy using recombinant hIDS.

9. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the method comprises delivering hIDS to the CNS via 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 polymeric molecule, naked DNA, a plasmid, a phage, a transposon, a cosmid, or an episome.

10. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the method comprises delivering hIDS to the CNS via intrathecal, intracisternal (IC), intravenous, or intracerebroventricular (ICV) administration.

11. The pharmaceutical composition of any one of claims 1 to 6, wherein the biological sample is obtained from the cerebrospinal fluid (CSF) of the human subject.

12. The criteria are: (i) a human subject with mild MPS II, or (ii) Human subjects not diagnosed with MPS II 7. The pharmaceutical composition of claim 2, 4, or 6, wherein the level of D2S6 in a CSF sample obtained from

13. The pharmaceutical composition according to any one of claims 2, 4 and 6, wherein the standard is a predetermined value.

14. The pharmaceutical composition according to any one of claims 1 to 6, wherein the MPS II is neuropathic MPS II.

15. wherein the human subject (a) an adult human subject; (b) Under 18 years of age; (c) is at least 5 years old but under 18 years old; or (d) 4 months or older and under 5 years of age; The pharmaceutical composition according to any one of claims 1 to 6, wherein

16. The method comprises: (i) after step (b), monitoring the level of D2S6 present in the biological sample from the human subject, wherein a decrease in the level of D2S6 compared to the level of D2S6 measured in step (a) indicates the effectiveness of the treatment; or (ii) monitoring the level of D2S6 present in a biological sample from the human subject at more than one time following delivery of an active hIDS to the human subject, wherein a decrease in the level of D2S6 over time indicates the effectiveness of the treatment; 7. The pharmaceutical composition of claim 2, 4, or 6, further comprising:

17. The pharmaceutical composition of any one of claims 1 to 6, wherein the level of D2S6 in the human subject after treatment with hIDS is reduced to a mild range.

18. 8. The pharmaceutical composition of claim 7, wherein the method comprises delivering hIDS to the CNS via intrathecal, intracisternal (IC), intravenous, or intracerebroventricular (ICV) administration.

19. 8. The pharmaceutical composition of claim 7, wherein the biological sample is obtained from the cerebrospinal fluid (CSF) of the human subject.

20. The criteria are: (i) a human subject with mild MPS II, or (ii) Human subjects not diagnosed with MPS II 8. The pharmaceutical composition of claim 7, wherein the level of D2S6 in a CSF sample obtained from

21. The pharmaceutical composition according to claim 7 , wherein the standard is a predetermined value.

22. The pharmaceutical composition according to claim 7, wherein the MPS II is neuropathic MPS II.

23. wherein the human subject (a) an adult human subject; (b) Under 18 years of age; (c) is at least 5 years old but under 18 years old; or (d) 4 months or older and under 5 years of age; The pharmaceutical composition according to claim 7, wherein

24. The method comprises: (i) after step (b), monitoring the level of D2S6 present in the biological sample from the human subject, wherein a decrease in the level of D2S6 compared to the level of D2S6 measured in step (a) indicates the effectiveness of the treatment; or (ii) monitoring the level of D2S6 present in a biological sample from the human subject at more than one time following delivery of an active hIDS to the human subject, wherein a decrease in the level of D2S6 over time indicates the effectiveness of the treatment; 8. The pharmaceutical composition of claim 7, further comprising:

25. 8. The pharmaceutical composition of claim 7, wherein the level of D2S6 in the human subject after treatment with the hIDS is reduced to a mild range.

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