Treatment of mucopolysaccharidosis ii with recombinant human iduronate-2 sulfatase (IDS) produced by human neural or glial cells

Abstract

To provide compositions and methods for the delivery of recombinant human iduronate-2-sulfatase (IDS) produced by human neuronal or glial cells to the cerebrospinal fluid of the central nervous system (CNS) of a human subject diagnosed with mucopolysaccharidosis II (MPS II).SOLUTION: The present invention provides glycosylated recombinant human iduronate-2-sulfatase (IDS) precursor produced by human neuronal or human glial cells.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 485,659, filed on April 14, 2017, No. 62 / 573,921, filed on October 18, 2017, No. 62 / 574,355, filed on October 19, 2017, and No. 62 / 579,686, filed on October 31, 2017, which are hereby incorporated by reference in their entirety.

[0002] (Reference to Electronically Submitted Sequence Listing) This application incorporates by reference the sequence listing submitted herewith as a text file named "Sequence_List ing_12656 - 104 - 228.TXT", created on April 3, 2018, and having a size of 166,497 bytes.

[0003] (1. Introduction) Described herein are compositions and methods for delivering recombinant human iduronate - 2 - sulfatase ( IDS) produced by human neurons or glial cells to the cerebrospinal fluid (CSF) of the central nervous system (CNS) of a human subject diagnosed with mucopolysaccharidosis type II (MPS II).

Background Art

[0004] (2. Background of the Invention) Hunter syndrome / MPS II is a rare X - linked recessive genetic disorder that occurs in 0.5 - 1.3 out of every 100,000 live male births. This progressive and destructive disease is caused by a gene mutation in the IDS gene, which results in lysosomal catabolism of heparan sulfate and dermatan sulfate. ​​Leads to a deficiency of iduronate-2-sulfatase, a lysosomal storage enzyme that is an essential enzyme These ubiquitous polysaccharides are called GAGs (glycosaminoglycans) and accumulate in the tissues and organs of MPS II patients, resulting in characteristic storage lesions and various disease sequelae. In this patient population the morbidity and mortality rates are high; in patients with severe phenotypes (characterized by neurocognitive decline), the average age is 11.7 years, and in patients with mild or attenuated phenotypes it has been reported that they die at 21.7 years (Young et al., 1982, "Clinical and genetic studies of Hunter syndrome: 2. Differences between the mild and severe forms", J. Medical Genetics 19:408-411). Most (2 / 3) of the patients 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). This disease mainly affects juveniles however, female patients have non-random X chromosome inactivation in both alleles of the gene and / or or ​​​​​​​has been reported as a result of a mutation (Martin et al., 2008, "Recognition and diagnosis of mucopolysaccharidosis II (Hunter Syndrome)", Pediatrics 121:e377). However, MPS II in females is extremely rare, occurring in less than 2% at that time. (Hunter Syndrome)", Pediatrics 121:e377). However, MPS II in females is extremely rare and occurs in less than 2% at that time.

[0005] MPS II patients appear normal at birth, but disease signs and symptoms typically occur in severe forms between 18 months and 4 years, and in attenuated forms between 4 and 8 years. Common signs and symptoms among all affected patients include short stature, coarsening of facial features, macrocephaly, macroglossia, hearing impairment, hepatosplenomegaly, multiple dysostoses, joint contractures, spinal stenosis, and carpal tunnel syndrome. Frequent upper respiratory and ear infections occur in most patients, and progressive airway obstruction is commonly observed, leading to sleep apnea and sometimes death. Heart disease is the leading cause of death in this population and is characterized by valvular insufficiency, leading to left and right ventricular hypertrophy and heart failure. Death is generally due to obstructive airway disease or heart failure. In the severe form of this disease, it is faced at an important stage of early development (milestone), and growth retardation becomes apparent immediately at 18 - 24 months. Some patients fail the hearing screening test at 1 year, and other important milestones including the ability to sit unsupported, walk, and speak are delayed. The progression of development begins to plateau between 3 and 5 years and regression begins around 6.5 years as reported. Approximately 50% of pediatric patients with MPS II who have started toilet training, although not all,

[0006] In the severe form of this disease, it is faced at an important stage of early development (milestone), and growth retardation becomes apparent immediately at 18 - 24 months. Some patients fail the hearing screening test at 1 year, and other important milestones including the ability to sit unsupported, walk, and speak are delayed. The progression of development begins to plateau between 3 and 5 years and regression begins around 6.5 years as reported. Approximately 50% of pediatric patients with MPS II who have started toilet training, although not all, ​​​​​​​however, they lose this ability as the disease progresses (Wraith et al., 2007, supra; Martin et al. (2008, op. cit.).

[0007] Patients with significant neurological involvement exhibit severe behavioral disturbances, including hyperactivity, rigidity, and aggression. They begin to show this behavior at age 2 years and continue until age 8-9 years, when neurodegeneration attenuates this behavior (Muenzer et al., 2013). Presentation, 2009. “Mucopolysaccharidosis I: Management Guidelines” Pediatrics 123(1): 19-29).

[0008] Seizures have been reported in more than half of severely affected patients by age 10 years, with CNS involvement Most patients with PTSD suffer from severe mental handicaps by the time they die and are constantly Nursing care is required (Wraith et al., 2007, supra; Martin et al., 2008, supra). Although patients with the disease exhibit normal intellectual function, MRI imaging shows white matter abnormalities in all patients with MPS II. Demonstrate gross brain abnormalities, including lesions, enlarged ventricles, and brain atrophy (Muenz er et al., 2009, op. cit.

[0009] Enzyme replacement therapy with recombinant idursulfase produced by HT1080 (fibrosarcoma) cells The Elaprase® therapy (ERT) (Shire Human Genetic Therapies) is a treatment for Hunter syndrome. It is the only product approved for this treatment, administered as a once-weekly injection (ELAPRASE). Lusulfase Injection [package insert]. Lexington, MA: Shire Human Genetic Therapies, Inc. ; 2013, available at http: / / pi.shirecontent.com / PI / PDFs / Elaprase_USA_ENG.pdf).

[0010] However, currently administered ERT does not cross the blood-brain barrier and thus does not address the unmet need in patients with severe disease, namely MPS II associated with CNS / neural recognition and behavior. In a recent clinical trial designed to address this issue, idursulfase (Elaprase) formulated for intrathecal administration was administered monthly to pediatric patients using an intrathecal drug delivery device implanted in the spine (insertion of a catheter at level L4 / L5 with an access port implanted via an incision above the lower rib). These patients also received weekly intravenous idursulfase. See Muenzer et al., 2016, Genetics in Med 18: 73- 81, particularly page 74; available at https: / / www.ncbi.nlm.nih.gov / pubmed / 25834948?dopt=Abstract for a possible summary). Malfunctions of the device led to partial revision, complete surgical revision, or removal in 6 out of 12 (50%) of the treated patients. Of note, 12 out of 14 SAEs (serious adverse events) were device-related (complications of device insertion, device dislocation / connection problems, device breakage / malfunction / failure, infections at the implantation site, procedural pain, and wound dehiscence )(Muenzer et al., 2016, page 75, second paragraph and Figure 1). Device breakage and catheter movement out of the spinal canal were exacerbated by the high activity level of this pediatric population (Muenzer et al., 2016, p.78 discussion). SUMMARY OF THE INVENTION

[0011] (3. Summary of the Invention) The present invention relates to mucopolysaccharidosis including, but not limited to, patients diagnosed with Hunter syndrome and to the delivery of recombinant human iduronate-2-sulfatase (rhIDS) produced by human neurons or glial cells to the cerebrospinal fluid (CSF) of the central nervous system (CNS) of human subjects diagnosed with mucopolysaccharidosis II type (MPS II).

[0012] In a preferred embodiment, the treatment is achieved by gene therapy - for example, by administering a viral vector or other DNA expression construct encoding human IDS (hIDS) or a derivative of h IDS to the CSF of a patient (human subject) diagnosed with MPS II, thereby creating a permanent depot of transduced neurons and / or glial cells that continuously supply the transgene product to the CNS - achieved by creating a permanent depot of transduced neurons and / or glial cells in the CNS. The rhIDS secreted from the neuron / glial cell depot into the CSF is endocytosed by cells in the CNS to "cross-correct" the enzyme deficiency in recipient cells. Further unexpectedly, the depot of transduced neurons and glial cells in the CNS can deliver this recombinant enzyme to both the CNS and the whole body which has been found to reduce or eliminate the need for systemic treatment, such as, for example, weekly intravenous injection of this enzyme

[0013] In an alternative embodiment, hIDS is produced by human neurons or glial cells in cell culture (e.g., a bioreactor) and administered as enzyme replacement therapy ("ERT") by, for example, injecting the enzyme directly into the CSF into the CNS and / or systemically. However ​​​​In addition, gene therapy approaches offer several advantages over ERT - since enzymes cannot cross the blood - brain barrier, the CNS is not treated by systemic delivery of enzymes; and unlike the gene therapy approach of the present invention, direct delivery of enzymes to the CSF and / or CNS would not only be a major burden but would also require repeated infusions that carry a risk of infection.

[0014] The hIDS encoded by the transgene is, but 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, derivatives of hIDS containing amino acid substitutions selected from the corresponding non - conserved residues in the orthologs of IDS shown in Figure 2, provided that such mutations do not include substitution of the cysteine residue (C84) at position 84 required for enzyme activity (Millat et al., 1997, Biochem J 326: 243 - 247); or, for example, those shown in Figure 3 or Sukegawa - Hayasaka et al., 2006, J Inhert Metab Dis 29: 755 - 761, the entirety of which is incorporated herein by reference (reporting "attenuated" mutants R48P, A85T, W337R, and truncated mutant Q531X; and "severe" mutants P86L, S333L, S349I, R468Q, R468L); Millat et al., 1998, BBA 1406: 214 - 218 (reporting "attenuated" mutants P480L and P480Q; and "severe" mutant P86L); and reported by Bonucelli et al., 2001, BBA 1537:233 - 238 ; ; and mutations identified in the severe, severe - moderate, moderate, or attenuated MPS II phenotypes including.

[0015] For example, an amino acid substitution at a specific position in hIDS is the corresponding non - conservative amino acid residue found at that position in the IDS ortholog aligned in Figure 2, provided that such a substitution is not shown in Figure 3 or is not included among those described in the literature of Sukegawa - Hayasaka et al., 2006, supra; Millat et al., 1998, supra; or Bonucelli et al., 2001, supra, which are incorporated herein by reference in their entirety; and is selected from those that do not include the deletion mutations reported by Bonucelli et al., 2001, supra. The resulting transgene product can be tested in cell culture or in test animals using conventional in vitro assays to ensure that the mutation does not impair IDS function. The preferred amino acid substitutions, deletions or additions selected should maintain or increase the enzyme activity, stability, or half - life of IDS as tested by conventional in vitro assays in cell culture or in an MPS II animal model. For example, the enzyme activity of the transgene product can be evaluated using a conventional enzyme assay with 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., the literature of which are incorporated herein by reference in their entirety, which maintain or increase the enzyme activity, stability, or half - life of IDS as tested by conventional in vitro assays in cell culture or in an MPS II animal model. For example, the enzyme activity of the transgene product can be evaluated using a conventional enzyme assay with 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., the literature of which are incorporated herein by reference in their entirety, which maintain or increase the enzyme activity, stability, or half - life of IDS as tested by conventional in vitro assays in cell culture or in an MPS II animal model. For example, the enzyme activity of the transgene product can be evaluated using a conventional enzyme assay with 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., (See, e.g., 2006, Clin. Chem. 52(4):643-649). For example, the ability to transfect MPS II cells in culture with a virus encoding hIDS or a derivative. MPS II in culture by transfection with a vector or other DNA expression construct by adding the transgene product or derivative to the cells; or by inducing MPS II cells into Co-culture with human neuronal / glial host cells engineered to express and secrete rhIDS or derivatives By determining the correction of defects in MPS II cells in culture, e.g. By detecting the decrease in IDS enzyme activity and / or GAG storage in cell cultures (See, e.g., Stronce, supra, which is incorporated herein by reference in its entirety). (see K et al., 1999, Transfusion 39(4):343-350).

[0016] An MPS II animal model that can be used to evaluate the therapeutic agents described herein is described herein. For example, the knockout mouse model of MPS II (IDS-knockout) has been reported to be The gene was engineered by replacing exons 4 and 5 with the neomycin resistance gene (G Arcia et al., 2007, J Inherit Metab Dis 30: 924-34). This IDS-knockout mouse has many of the hallmarks of MPS I, including skeletal abnormalities, hepatosplenomegaly, elevated urinary and tissue GAGs, and brain storage lesions. I characteristics (Muenzer et al., 2001, Acta Paediatr Suppl 91:98-99) and ERT The study was used to evaluate the efficacy of enzyme replacement therapy in MPS II in support of clinical trials regarding was used. Therefore, this mouse model is a relevant model for testing the effect of gene therapy that delivers rIDS produced by neurons or glial cells as a treatment for MPS II (see, for example, the literature of Polito and Cosma, 2009, Am. J. Hum. Genet. 85(2):296-301, which is hereby incorporated by reference in its entirety). Preferably, the hIDS transgene produced by human neurons / glial cells should be controlled by expression control elements that function in neurons and / or glial cells, such as the CB7 promoter ( chicken β-actin promoter and CMV enhancer), and may include other expression control elements (such as chicken β-actin intron and rabbit β-globin polyA signal) that enhance the expression of the transgene driven by the vector. The cDNA construct for the hIDS transgene should include a coding sequence for a signal peptide that ensures proper co-translational and post-translational processing (glycosylation and protein sulfation) by the transfected CNS cells. Such signal peptides used by CNS cells include: · Oligodendrocyte-myelin glycoprotein (hOMG) signal peptide:

[0017] · Cellular repressor of E1A-stimulated genes 2 (hCREG2) signal peptide: · V-set and transmembrane domain-containing 2B (hVSTM2B) signal peptide:

Chemical formula

Chemical formula

[0018] The recombinant vector used to deliver the transgene should have tropism for cells of the CNS, including but not limited to neuronal and / or glial cells. Such vectors can include non-replicating recombinant adeno-associated virus vectors ("rAAV"), particularly preferably viral vectors having an AAV9 or AAVrh10 capsid. AAV variant capsids, particularly preferably AAV / hu.31, and capsids having AAV / hu.32; and those described by Wilson in U.S. Patent No. 7,906,111, the entire contents of which are incorporated herein by reference by reference; and those described by Chatterjee in U.S. Patent No. 8,628,966, U.S. Patent No. 8,927,514, and in the literature of Smith et al., 2014, Mol Ther 22:1625-1634, particularly preferably AAV / hu.31, and capsids having AAV / hu.32; and those described by Chatterjee in U.S. Patent No. 8,628,966, U.S. Patent No. 8,927,514, and in the literature of Smith et al., 2014, Mol Ther 22:1625-1634, the entire contents of each of which are incorporated herein by reference Variant capsids can be used. However, other viral vectors including, but not limited to, lentiviral vectors, vaccinia virus vectors or non-viral expression vectors called "naked DNA" constructs can be used. - a vaccinia virus vector or a non-viral expression vector called "naked DNA" construct can be used. 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, where expression is driven by a hybrid of the cytomegalovirus (CMV) enhancer and the chicken β-actin promoter (CB7), where the IDS expression cassette is flanked by inverted terminal repeats (ITRs), and the transgene contains a chicken β-actin intron and a rabbit β-globin polyadenylation (polyA) signal.

[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, where expression is driven by a hybrid of the cytomegalovirus (CMV) enhancer and the chicken β-actin promoter (CB7), where the IDS expression cassette is flanked by inverted terminal repeats (ITRs), and the transgene contains a chicken β-actin intron and a rabbit β-globin polyadenylation (polyA) signal.

[0020] A pharmaceutical composition suitable for administration to the CSF comprises a suspension of the rhIDS vector in a formulation buffer containing a physiologically compatible aqueous buffer, a surfactant, and any excipient. 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 cistern). 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 certain embodiments, the pharmaceutical composition is suitable for intrathecal administration. In certain embodiments, the pharmaceutical composition is suitable for intracisternal administration (injection into the cistern). 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 certain embodiments, the pharmaceutical composition is suitable for intrathecal administration. In certain embodiments, the pharmaceutical composition is suitable for intracisternal administration (injection into the cistern). 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 certain embodiments, the pharmaceutical composition is suitable for intrathecal administration. In certain embodiments, the pharmaceutical composition is suitable for intracisternal administration (injection into the cistern). 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 certain embodiments, the pharmaceutical composition is suitable for intrathecal administration. In certain embodiments, the pharmaceutical composition is suitable for intracisternal administration (injection into the cistern). 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 certain embodiments, the pharmaceutical composition is suitable for intrathecal administration. In certain embodiments, the pharmaceutical composition is suitable for intracisternal administration (injection into the cistern). 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.

[0021] A therapeutically effective dose of the recombinant vector is administered via intrathecal administration (i.e., injection into the subarachnoid space). ​​​​Introduce it so that the recombinant vector is distributed through the CSF and the cells of the CNS are transduced. ) and should be administered to the CSF. This can be achieved in several ways - for example, by intracranial (cisternal or ventricular) injection, or by lumbar cistern injection. For example, intracisternal (IC) (cisternal to the) injection can be performed by posterior suboccipital puncture guided by CT; or, if feasible for the patient, injection into the subarachnoid space via C1-2 puncture can be performed; or, lumbar puncture (a diagnostic procedure typically performed to collect a CSF sample) can be used to access the CSF. Alternatively, intracerebroventricular (ICV) administration (a more invasive technique used for the introduction of anti-infective or anti-cancer agents that do not cross the blood-brain barrier) can be used to directly instill the recombinant vector into the cerebroventricle. Alternatively, intranasal administration can be used to

[0022] deliver the recombinant vector to the CNS. The CSF concentration can be monitored by directly measuring the concentration of rhIDS in the CSF fluid obtained from posterior or lumbar puncture, or estimated by extrapolation from the

[0023] concentration of rhIDS detected in the patient's serum. As background, human IDS is translated as a 550 amino acid polypeptide and contains eight 31 potential N-glycosylation sites (N 115 、N 144 、N 246 、N 280 、N 325 、N 51 3 and N 537 ) shown in Figure 1, and contains a 25 amino acid signal sequence that is cleaved during The initial 76 kDa intracellular precursor is converted to the phosphorylated 90 kDa precursor after modification of its oligosaccharide chains in the Golgi apparatus. This precursor is processed to the major 55 kDa form through various intracellular intermediates by glycosylation modifications and proteolytic cleavages. In summary, after removal of the 25 amino acid signal sequence, proteolytic processing involves removal of an 8 amino acid (residues 26 - 33) propeptide, an N-terminal proteolytic cleavage downstream of N 31 of the protein , and a C-terminal proteolytic cleavage upstream of N 513 that releases an 18 kDa polypeptide and generates a 62 kDa intermediate, which is converted to the 55 kDa mature form. Further proteolytic cleavage yields a 45 kDa mature form that is located in the lysosomal compartment. (See Figure 4 for a schematic prepared from each of the entirety of which is incorporated herein by reference, 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). It proceeds through various intracellular intermediates to the major 55 kDa form by glycosylation modifications and proteolytic cleavages. In summary, after removal of the 25 amino acid signal sequence, proteolytic processing involves removal of an 8 amino acid (residues 26 - 33) propeptide, 31 an N-terminal proteolytic cleavage downstream of N of the protein 513 and a C-terminal proteolytic cleavage upstream of N that releases an 18 kDa polypeptide and generates a 62 kDa intermediate, which is converted to the 55 kDa mature form. Further proteolytic cleavage yields a 45 kDa mature form that is located in the lysosomal compartment. (See Figure 4 for a schematic prepared from each of the entirety of which is incorporated herein by reference, 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). generates a 45 kDa mature form that is located in the lysosomal compartment. (See Figure 4 for a schematic prepared from each of the entirety of which is incorporated herein by reference, 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). (See Figure 4 for a schematic prepared from each of the entirety of which is incorporated herein by reference, 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). ll 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 )(See Figure 4 for a schematic prepared from each of the entirety of which is incorporated herein by reference, 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).

[0024] C 84 (Shown in bold in Figure 1) formylglycine modification presumably occurs as an early post-translational or co-translational event in the cell body and is required for an enzymatic activity. (See Millat, 1997a, which cites Schmidt et al., 1995, Cell 82: 271 - 278). Post-translational processing continues in the Golgi and involves complex sialic acid-containing modifications. ​​Tagging enzymes for addition of glycans and delivery to the lysosomal compartment involves acquisition of mannose-6-phosphate residues. (For a concise review, see Clarke, 2008, Expert Opin Pharmacother 9: 311- 317, which is incorporated herein by reference in its entirety). A single glycosylation site is not essential for IDS stability, but glycosylation at position N is important for cellular internalization and lysosomal targeting via the mannose-6-phosphate (M6 P) receptor. (Chung et al., 2014, Glycoconj J 31:309-315, page 310, paragraph 1). In normal physiological conditions, IDS is produced at very low levels and, if at all, very little enzyme is secreted from cells. (Clarke, 2008, supra). 280 and mannose-6-phosphorylation and tyrosine-O-sulfation, which are robust processes in the CNS. For each of the post-translational modifications performed by human CNS cells, see, for example, Sleat et al., 2005, Proteomics 5: 1520-1532, which describes the human brain mannose-6-phosphate ( M6P) glycoproteome and notes that the brain contains more proteins and more mannose-6-phosphorylated proteins with more individual isoforms than are found in other tissues, and each of which is incorporated herein by reference in its entirety). In normal physiological conditions, IDS is produced at very low levels and, if at all, very little enzyme is secreted from cells. (Clarke, 2008, supra). In normal physiological conditions, IDS is produced at very low levels and, if at all, very little enzyme is secreted from cells. (Clarke, 2008, supra). In normal physiological conditions, IDS is produced at very low levels and, if at all, very little enzyme is secreted from cells. (Clarke, 2008, supra).

[0025] The present invention is based in part on the following principle: (i) Neurons and glial cells of the CNS are secretory cells with 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. For each of the post-translational modifications performed by human CNS cells, see, for example, Sleat et al., 2005, Proteomics 5: 1520-1532, which describes the human brain mannose-6-phosphate ( M6P) glycoproteome and notes that the brain contains more proteins and more mannose-6-phosphorylated proteins with more individual isoforms than are found in other tissues, and each of which is incorporated herein by reference in its entirety). For each of the post-translational modifications performed by human CNS cells, see, for example, Sleat et al., 2005, Proteomics 5: 1520-1532, which describes the human brain mannose-6-phosphate ( M6P) glycoproteome and notes that the brain contains more proteins and more mannose-6-phosphorylated proteins with more individual isoforms than are found in other tissues, and each of which is incorporated herein by reference in its entirety). For each of the post-translational modifications performed by human CNS cells, see, for example, Sleat et al., 2005, Proteomics 5: 1520-1532, which describes the human brain mannose-6-phosphate ( M6P) glycoproteome and notes that the brain contains more proteins and more mannose-6-phosphorylated proteins with more individual isoforms than are found in other tissues, and each of which is incorporated herein by reference in its entirety). the literature of t, 1996, J Biol Chem 271:19191-98; and the literature of Kanan et al., 2009, Exp. Eye Res. 8 9: 559-567, reporting the production of tyrosine-sulfated glycoprotein secreted by neurons, and referring to the literature of Kanan and Al-Ubaidi, 2015 Exp. Eye Res. 133: 126-131 is desired to be referred to. (ii) The human brain produces multiple isoforms of native / undenatured IDS. In particular, the N-terminal sequencing of mannose-6-phosphorylated glycoproteins has revealed that the N-terminal sequence of the mature 42 kDa chain of hIDS varies in the brain and starts from position 34 or 36 as follows: (Sleat's literature, 2005, Proteomics 5: 1520-1532, Table S2). Two of the eight N-linked glycosylation sites, namely N and N were found to be mannose-6-phosphorylated in IDS obtained from the human brain. (Reported in Sleat et al.'s literature, 2006, Mol & Cell Proeomics 5.4: 686-701, Table V).

Chemical formula

Table 1

Chem.

[0026] For the reasons described above, the production of rhIDS by human neurons and / or glial cells is - for example , in patients (human subjects) diagnosed with MPS II diseases (including but not limited to Hunter), administering a viral vector or other DNA expression construct encoding rhIDS to the CSF to create a persistent depot in the CNS that continuously supplies the fully human glycosylated, mannose-6-phosphorylated, sulfated transgene product secreted by the transduced CNS cells - should result in a "bio-better" molecule for the treatment of MPS II achieved by gene therapy. The hIDS transgene product secreted from the depot into the CSF is endocytosed by cells in the CNS, resulting in a "cross-correction" of the enzyme deficiency in MPS II recipient cells .

[0027] It is not essential that all rhIDS molecules produced in either gene therapy or protein therapy approaches be fully glycosylated, phosphorylated, and sulfated . ​​​Rather, the resulting population of glycoproteins should undergo glycosylation (2,6- sialylation and mannose-6-phosphorylation, including) and sulfation sufficient to demonstrate efficacy. The purpose of the gene therapy treatment of the present invention is to slow or stop the progression of the disease. Efficacy can be monitored by measuring cognitive function (e.g., prevention or reduction of decline in neurocognitive function); reduction of disease biomarkers (e.g., GAG) in CSF and / or blood serum; and / or increase in IDS enzyme activity in CSF and / or serum . Inflammatory signs and other safety events can also be monitored.

[0028] As an alternative or additional treatment to gene therapy, rhIDS glycoprotein can be produced by recombinant DNA technology in cell lines of human neurons or glial cells, and the glycoprotein can be administered systemically and / or to patients diagnosed with MPS II in CSF for ERT. Human cell lines that can be used for such recombinant glycoprotein production include, but are not limited to, for example, HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11 , or ReNcell VM (e.g., for a review of human cell lines that can be used for recombinant production of rHuGlyIDS glycoprotein, the entire of which is incorporated by reference, see Dumo nt et al., 2016, Critical Rev in Biotech 36(6):1110-1122, "Human cell lines for biopharmaceutical manufacturing: history, current status, and future perspectives" . nt et al., 2016, Critical Rev in Biotech 36(6):1110-1122, "Human cell lines for biopharmaceutical manufacturing: history, current status, and future perspectives" : History, Current Status, and Future Perspectives (Human cell lines for biopharmaceutical m Refer to “Manufacturing: History, Status, and Future Perspectives”. Complete To ensure glycosylation, particularly sialylation and tyrosine sulfation, the cell line used in production is engineered to co-express α-2,6-sialyltransferase (or both α-2,3- and α-2,6-sialyltransferases) and / or the TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation in the host cell by manipulation, which can be enhanced.

[0029] While delivery of rhIDS should minimize the immune response, the most obvious potential source of toxicity related to CNS gene therapy is the occurrence of immunity to the rhIDS protein expressed in human subjects who are genetically deficient in IDS and thus potentially intolerant to the vectors used to deliver the protein and / or the introduced gene.

[0030] Therefore, in a preferred embodiment, co-treatment of the patient with immunosuppressive therapy is appropriate, particularly when treating patients with severe diseases where the IDS level is close to zero. Tacrolimus or rapamycin (sirolimus), for example, in a regimen combined with mycophenolic acid, or immunosuppressive therapy with 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 some embodiments, pretreatment with immunosuppressive therapy may be preferred. Immunosuppressive therapy can be continued after the gene therapy treatment, based on the judgment of the treating physician, and then discontinued if immune tolerance is induced, for example, after 180 days.

[0031] ​​The combination of delivery of rhIDS to the CSF achieved by delivery of other available therapies is encompassed by the methods of the present invention. The additional therapy 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 Elapras e® administered systemically or to the CSF; and / or HSCT therapy. (3.1 Exemplary Embodiments) (3.1.1 Set 1) 1. A glycosylated recombinant human iduronate-2-sulfatase (IDS) precursor produced by human neurons or human glial cells. 2. The glycosylated recombinant human IDS precursor according to paragraph 1, which 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. 3. The glycosylated recombinant human IDS precursor according to paragraph 1, which is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose-6-phosphorylated. 4. The glycosylated recombinant human IDS precursor according to any one of paragraphs 1 to 3, which is secreted from a depot of cells in the central nervous system genetically engineered to secrete the human IDS glycoprotein precursor. 5. A depot that is a glycosylated recombinant human IDS precursor formed in the brain of a human subject as described in paragraph 4. precursor of IDS. 6. A glycosylated recombinant human IDS precursor according to any one of paragraphs 1 to 5, wherein the human neurons or human glial cells lack IDS activity. precursor of IDS. 7. A glycosylated recombinant human IDS precursor according to any one of paragraphs 1 to 6, wherein the glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO: 1. precursor of IDS. 8. A recombinant nucleotide expression vector encoding human IDS, wherein when the recombinant nucleotide expression vector is used to transduce primary human neurons in culture, it measures by polyacrylamide gel electrophoresis to be 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), contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose-6-phosphorylated, and is a vector that leads to the expression of the secreted glycosylated human IDS precursor. precursor of IDS. precursor of IDS. precursor of IDS. precursor of IDS. precursor of IDS. precursor of IDS. 9. A recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is suitable for administration to the cerebrospinal fluid (CSF) of the human brain, and as a result, it measures by polyacrylamide gel electrophoresis to be 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), contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, contains no detectable α-Gal antigen, precursor of IDS. precursor of IDS. precursor of IDS. precursor of IDS. First, a glycosylated human IDS precursor that is phosphorylated at mannose-6 and / or A vector in which a depot that secretes 10. Secretion of the glycosylated human IDS precursor is confirmed by transfection of a human neuronal cell line in cell culture with the recombinant nucleotide expression vector according to paragraph 9 The recombinant nucleotide expression vector according to paragraph 9. 11. The recombinant nucleotide expression vector according to paragraph 9 or 10, wherein secretion of the glycosylated human IDS precursor is confirmed in the presence and absence of mannose-6-phosphate. The recombinant nucleotide expression vector according to paragraph 9 or 10, wherein secretion of the glycosylated human IDS precursor is confirmed in the presence and absence of mannose-6-phosphate. 12. The recombinant nucleotide expression vector according to any one of paragraphs 8 to 11, wherein human IDS comprises the amino acid sequence of SEQ ID NO: 1. The recombinant nucleotide expression vector according to any one of paragraphs 8 to 11, wherein human IDS comprises the amino acid sequence of SEQ ID NO: 1. 13. A recombinant nucleotide expression vector according to any one of paragraphs 8 to 12, comprising a neuron-specific promoter that controls the expression of the glycosylated human IDS precursor in human neurons or a glia cell-specific promoter that controls the expression of the glycosylated human IDS precursor in human glia cells. A recombinant nucleotide expression vector according to any one of paragraphs 8 to 12, comprising a neuron-specific promoter that controls the expression of the glycosylated human IDS precursor in human neurons or a glia cell-specific promoter that controls the expression of the glycosylated human IDS precursor in human glia cells. The recombinant nucleotide expression vector according to any one of paragraphs 8 to 12, comprising a neuron-specific promoter that controls the expression of the glycosylated human IDS precursor in human neurons or a glia cell-specific promoter that controls the expression of the glycosylated human IDS precursor in human glia cells. The recombinant nucleotide expression vector according to any one of paragraphs 8 to 12, comprising a neuron-specific promoter that controls the expression of the glycosylated human IDS precursor in human neurons or a glia cell-specific promoter that controls the expression of the glycosylated human IDS precursor in human glia cells. 14. A recombinant nucleotide expression vector according to any one of paragraphs 8 to 13, encoding a leader peptide that ensures translation and post-translational processing of the glycosylated human IDS precursor in human neurons or human glia cells. A recombinant nucleotide expression vector according to any one of paragraphs 8 to 13, encoding a leader peptide that ensures translation and post-translational processing of the glycosylated human IDS precursor in human neurons or human glia cells. The recombinant nucleotide expression vector according to any one of paragraphs 8 to 13, encoding a leader peptide that ensures translation and post-translational processing of the glycosylated human IDS precursor in human neurons or human glia cells. 15. The recombinant nucleotide expression vector according to any one of paragraphs 8 to 14, which is an AAV vector. The recombinant nucleotide expression vector according to any one of paragraphs 8 to 14, which is an AAV vector. 16. The recombinant nucleotide expression vector according to paragraph 15, which is a replication-deficient AAV vector. 17. The recombinant nucleotide expression vector according to paragraph 15 or 16, which is an AAV9 or AAVrh10 vector. The recombinant nucleotide expression vector according to paragraph 15 or 16, which is an AAV9 or AAVrh10 vector. 18. A formulation containing a recombinant nucleotide expression vector encoding human IDS, wherein this formulation is suitable for administration to the CSF of the human brain, and as a result, as measured by polyacrylamide gel electrophoresis, it 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), contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose-6-phosphorylated, and a depot that secretes a glycosylated human IDS precursor forms in the human central nervous system. A formulation. 19. A kit containing a recombinant nucleotide expression vector encoding human IDS and a pharmaceutically acceptable carrier, wherein the recombinant nucleotide expression vector is suitable for administration to the cerebrospinal fluid (CSF) of the human brain, and as a result, as measured by polyacrylamide gel electrophoresis , it 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), contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose-6-phosphate ylated, and a depot that secretes a glycosylated human IDS precursor forms in the human central nervous system. A kit. 20. A kit containing a formulation containing a recombinant nucleotide expression vector encoding human IDS, wherein the formulation is suitable for administration to the CSF of the human brain, and as a result, as measured by polyacrylamide amide gel electrophoresis, it is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kD a, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa), containing formylglycine, α2,6-sialylated, not containing detectable NeuGc, not containing detectable α-Gal antigen, and / or mannose-6-phosphorylated, a secreted glycosylated human IDS precursor depot that forms in the human central nervous system, kit. (3.1.2 Set 2) 1. A glycosylated recombinant human iduronate-2-sulfatase (IDS) precursor for use in the treatment of a human subject diagnosed with mucopolysaccharidosis type II (MPS II), where the glycosylated recombinant human IDS precursor is produced by human neurons or human glial cells, and where the treatment comprises delivering a therapeutically effective amount of the glycosylated recombinant human IDS precursor to the cerebrospinal fluid (CSF) of the human subject. 2. The glycosylated recombinant human IDS precursor for use according to paragraph 1, where the glycosylated recombinant human IDS precursor 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. 3. The glycosylated recombinant human IDS precursor for use according to paragraph 1, where the glycosylated recombinant human IDS precursor is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose- 6-phosphorylated. 4. The glycosylated recombinant human IDS precursor for use according to paragraph 1, where the glycosylated recombinant human IDS precursor is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose- 6-phosphorylated, and is secreted by human neurons or human glial cells. 5. The glycosylated recombinant human IDS precursor for use according to paragraph 1, where the glycosylated recombinant human IDS precursor is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose- Glycosylated recombinant human IDS precursor for use according to paragraph 1, phosphorylated at 6 body. 4. A glycosylated recombinant human IDS precursor secreted from a depot of cells in the central nervous system that has been genetically engineered to secrete the glycosylated recombinant human IDS precursor, for use according to any one of paragraphs 1 to 3. A glycosylated recombinant human IDS precursor for use according to any one of paragraphs 1 to 3. 5. A glycosylated recombinant human IDS precursor for use according to paragraph 4, wherein the depot is formed in the brain of a human subject. body. 6. A glycosylated recombinant human IDS precursor for use according to any one of paragraphs 1 to 5, wherein the human subject lacks IDS activity. body. 7. A glycosylated recombinant human IDS precursor for use according to any one of paragraphs 1 to 6, wherein the glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO: 1. body. 8. A recombinant nucleotide expression vector encoding human IDS for use in the treatment of a human subject diagnosed with MPS II, wherein when the recombinant nucleotide expression vector is used to transduce primary human neurons in culture, as measured by polyacrylamide gel electrophoresis, is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa a, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa), contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose-6-phosphorylated, directs the expression of a secreted glycosylated human IDS precursor, and wherein the treatment is into the CSF of the human subject, the recombinant nucleotide expression vector body. A vector comprising administering a cutter. 9. A recombinant nucleotide expression vector encoding a human IDS for use in the treatment of a human subject diagnosed with MPS II, wherein the recombinant nucleotide expression vector is suitable for administration to the cerebrospinal fluid of the human brain, such that as measured by polyacrylamide gel electrophoresis, it 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), contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose- 6-phosphorylated, and a depot that secretes a glycosylated human IDS precursor is formed in the human central nervous system, and wherein the treatment herein comprises administering the recombinant nucleotide expression vector to the CSF of the human subject. A vector. 10. The recombinant nucleotide expression vector for use according to paragraph 9, wherein the secretion of the glycosylated human IDS precursor is confirmed by transduction of a human neuronal cell line in cell culture with the recombinant nucleotide expression vector. 11. The recombinant nucleotide expression vector for use according to paragraph 9 or 10, wherein the secretion of the glycosylated human IDS precursor is confirmed in the presence and absence of mannose-6-phosphate. 12. The recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 11, wherein the human IDS comprises the amino acid sequence of SEQ ID NO: 1. 13. The recombinant nucleotide expression vector is a neuron-specific promoter that controls the expression of a glycosylated human IDS precursor in human neurons, or in human glial cells wherein the secretion of the glycosylated human IDS precursor is confirmed by transduction of a human neuronal cell line in cell culture with the recombinant nucleotide expression vector as described in paragraph 9. 14. The recombinant nucleotide expression vector for use according to any one of paragraphs 9 to 11, wherein the glycosylated human IDS precursor is secreted by a depot formed in the human central nervous system and the treatment comprises administering the recombinant nucleotide expression vector to the CSF of the human subject. 15. The recombinant nucleotide expression vector for use according to any one of paragraphs 9 to 11, wherein the glycosylated human IDS precursor is secreted by a depot formed in the human central nervous system and the treatment comprises administering the recombinant nucleotide expression vector to the CSF of the human subject. 16. The recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 11, wherein the human IDS comprises the amino acid sequence of SEQ ID NO: 1. 17. The recombinant nucleotide expression vector, wherein the recombinant nucleotide expression vector controls the expression of a glycosylated human IDS precursor in human neurons or human glial cells with a neuron-specific promoter or a glial cell-specific promoter. 18. The recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 11, wherein the human IDS comprises the amino acid sequence of SEQ ID NO: 1. A glial cell-specific promoter that controls the expression of the glycosylated human IDS precursor A recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 12, comprising the same. 14. A recombinant nucleotide expression vector that ensures the translation and post-translation processing of the glycosylated human IDS precursor in human neurons or human glial cells A leader peptide encoding the same, for use according to any one of paragraphs 8 to 13. A recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 13, encoding the same. A recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 13. 15. A recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 14, wherein the recombinant nucleotide expression vector is an AAV vector A recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 14. 16. A recombinant nucleotide expression vector for use according to paragraph 15, wherein the recombinant nucleotide expression vector is a replication-deficient AAV vector A recombinant nucleotide expression vector for use according to paragraph 15. 17. A recombinant nucleotide expression vector for use according to paragraph 15 or 16, wherein the recombinant nucleotide expression vector is an AAV9 or AAVrh10 vector A recombinant nucleotide expression vector for use according to paragraph 15 or 16. 18. A recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 17, wherein the recombinant nucleotide expression vector is delivered to the CSF of a human subject by intrathecal, intraventricular, lumbar puncture, or intranasal administration A recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 17. A recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 17. 19. A recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 18, wherein the human subject lacks IDS activity A recombinant nucleotide expression vector for use according to any one of paragraphs 8 to 18. 20. A preparation for use in the treatment of a human subject diagnosed with MPS II, comprising a recombinant nucleotide expression vector encoding human IDS, wherein the preparation is suitable for administration to the CSF of the human brain, and as a result, measured by polyacrylamide gel electrophoresis, about 9 A preparation for use in the treatment of a human subject diagnosed with MPS II, comprising a recombinant nucleotide expression vector encoding human IDS, wherein the preparation is suitable for administration to the CSF of the human brain, and as a result, measured by polyacrylamide gel electrophoresis, about 9 A preparation for use in the treatment of a human subject diagnosed with MPS II, comprising a recombinant nucleotide expression vector encoding human IDS, wherein the preparation is suitable for administration to the CSF of the human brain, and as a result, measured by polyacrylamide gel electrophoresis, about 9 0 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), contains formylglycine, is α2,6-sialylated, and does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is not mannose-6-phosphorylated, a depot that secretes a glycosylated human IDS precursor that forms in the human central nervous system, a pharmaceutical formulation. (3.1.3. Set 3) 1. Use of a glycosylated recombinant human iduronate-2-sulfatase (IDS) precursor for the manufacture of a medicament for the treatment of a human subject diagnosed with mucopolysaccharidosis type II (MPS II), wherein the glycosylated recombinant human IDS precursor is produced by human neurons or human glial cells, and wherein the treatment comprises delivering a therapeutically effective amount of the glycosylated recombinant human IDS precursor to the cerebrospinal fluid (CSF) of the human subject. 2. The use according to paragraph 1, wherein the glycosylated recombinant human IDS precursor measures 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. 3. The glycosylated recombinant human IDS precursor measures 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 Use according to paragraph 1, which is phosphorylated at 6. 4. Use according to any one of paragraphs 1 - 3, wherein the glycosylated recombinant human IDS precursor is secreted from a depot of cells in the central nervous system that have been genetically engineered to secrete the glycosylated recombinant human IDS precursor. 6. Use according to any one of paragraphs 1 - 3. 5. Use according to paragraph 4, wherein the depot is formed in the brain of a human subject. 6. Use according to any one of paragraphs 1 - 5, wherein the human subject lacks IDS activity. 7. Use according to any one of paragraphs 1 - 6, wherein the glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO: 1. 14. Use according to any one of paragraphs 1 - 6. 8. Use of a recombinant nucleotide expression vector encoding human IDS for the manufacture of a medicament for the treatment of a human subject diagnosed with MPS II, wherein when the recombinant nucleotide expression vector is used to transduce primary human neurons in culture, the secreted glycosylated human IDS precursor has an apparent molecular weight of about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose-6-phosphorylated, and wherein the treatment comprises administering the recombinant nucleotide expression vector to the CSF of the human subject. 24. and wherein the treatment comprises administering the recombinant nucleotide expression vector to the CSF of the human subject. 26. does not contain detectable α-Gal antigen, and / or is mannose-6-phosphorylated, and wherein the treatment comprises administering the recombinant nucleotide expression vector to the CSF of the human subject. 8. Use of a recombinant nucleotide expression vector encoding human IDS for the manufacture of a medicament for the treatment of a human subject diagnosed with MPS II, wherein when the recombinant nucleotide expression vector is used to transduce primary human neurons in culture, the secreted glycosylated human IDS precursor has an apparent molecular weight of about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal 9. Use of a recombinant nucleotide expression vector encoding human IDS for the manufacture of a medicament for the treatment of a human subject diagnosed with MPS II, wherein this recombinant nucleotide 9. Use of a recombinant nucleotide expression vector encoding human IDS for the manufacture of a medicament for the treatment of a human subject diagnosed with MPS II, wherein this recombinant nucleotide The depot expression vector is suitable for administration to the cerebrospinal fluid of the human brain, and as a result, polyacrylamide gel measured by electrophoresis 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), contains formylglycine, is α2,6-sialic acid added, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose-6-phosphorylated, and a depot that secretes a glycosylated human IDS precursor is formed in the human central nervous system, and here the treatment is to the CSF of the human subject, including administering a recombinant nucleotide expression vector. Use. 10. The secretion of the glycosylated human IDS precursor is confirmed by transduction of the recombinant nucleotide expression vector into a human neuronal cell line in cell culture, as described in paragraph 9. Use. 11. The secretion of the glycosylated human IDS precursor is confirmed in the presence and absence of mannose-6-phosphate, as described in paragraph 9 or 10. Use. 12. The human IDS contains the amino acid sequence of SEQ ID NO: 1, and the use according to any one of paragraphs 8 to 11. Use. 13. The recombinant nucleotide expression vector contains a neuron-specific promoter that controls the expression of the glycosylated human IDS precursor in human neurons, or a glia cell-specific promoter that controls the expression of the glycosylated human IDS precursor in human glia cells. Use according to any one of paragraphs 8 to 12. 14. The recombinant nucleotide expression vector contains a leader that ensures the translation and post-translational processing of the glycosylated human IDS precursor in human neurons or human glia cells. Use according to any one of paragraphs 8 to 12. 14. The recombinant nucleotide expression vector contains a leader that ensures the translation and post-translational processing of the glycosylated human IDS precursor during and after translation in human neurons or human glia cells. Use according to any one of paragraphs 8 to 12. Use according to any one of paragraphs 8 to 13, encoding a peptide. 15. Use according to any one of paragraphs 8 to 14, wherein the recombinant nucleotide expression vector is an AAV vector. 16. Use according to paragraph 15, wherein the recombinant nucleotide expression vector is a replication-deficient AAV vector. 17. Use according to paragraph 15 or 16, wherein the recombinant nucleotide expression vector is an AAV9 or AAVrh10 vector. 18. Use according to any one of paragraphs 8 to 17, wherein the recombinant nucleotide expression vector is delivered to the CSF of a human subject by intrathecal, intraventricular, lumbar puncture or intranasal administration. 19. Use according to any one of paragraphs 8 to 18, wherein the human subject lacks IDS activity. 20. Use of a formulation for the manufacture of a medicament for the treatment of a human subject diagnosed with MPS II, wherein the formulation contains a recombinant nucleotide expression vector encoding human IDS, and wherein the formulation is suitable for administration to the CSF of the human brain, resulting in the secretion of a glycosylated human IDS precursor that, as measured by polyacrylamide gel electrophoresis, 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), contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose-6-phosphorylated, and forms a depot in the human central nervous system. (3.1.4. Set 4) 1. A method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), the method comprising: ​​​​​​​​​​​Delivering a therapeutically effective amount of a glycosylated recombinant human iduronate-2-sulfatase (IDS) precursor produced by human neurons or human glial cells to cerebrospinal fluid (CSF). A method comprising: 2. The method of paragraph 1, wherein the glycosylated recombinant human IDS precursor measures 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. 3. The method of paragraph 1, wherein the glycosylated recombinant human IDS precursor measures about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains formylglycine, is α2,6-sialylated, contains no detectable NeuGc, contains no detectable α-Gal antigen, and / or is mannose- 6-phosphorylated. 4. The method according to any one of paragraphs 1-3, wherein the glycosylated recombinant human IDS precursor is secreted from a depot of cells in the central nervous system that have been genetically engineered to secrete the glycosylated recombinant human IDS precursor. 5. The method of paragraph 4, wherein the depot is formed in the brain of a human subject. 6. The method according to any one of paragraphs 1-5, wherein the human subject is deficient in IDS activity. 7. The method according to any one of paragraphs 1-6, wherein the glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO: 1. 8. A method for treating a human subject diagnosed with MPS II, the recombinant encoding human IDS ​​​​​​A method comprising administering a recombinant nucleotide expression vector encoding human IDS to the CSF of a human subject, wherein the recombinant nucleotide expression vector, when used to transduce primary human neurons 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, contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose-6-phosphorylated, and leads to the expression of a secreted, glycosylated human IDS precursor. 9. A method of treating a human subject diagnosed with MPS II, comprising administering a recombinant nucleotide expression vector encoding human IDS to the CSF of the human subject, whereby a depot that secretes a glycosylated human IDS precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose-6-phosphorylated, is formed in the human central nervous system. 10. The method of paragraph 9, wherein secretion of the glycosylated human IDS precursor is confirmed by transduction of a human neuronal cell line in cell culture with the recombinant nucleotide expression vector. 11. The method of paragraph 9, wherein secretion of the glycosylated human IDS precursor is in the presence and absence of mannose-6-phosphate and ​​​​​​​The method according to paragraph 9 or 10, as confirmed in the presence of 12. The method according to any one of paragraphs 8 to 11, wherein the human IDS comprises the amino acid sequence of SEQ ID NO: 1 method. 13. The method according to any one of paragraphs 8 to 12, wherein the recombinant nucleotide expression vector comprises a neuron-specific promoter that controls the expression of the glycosylated human IDS precursor in human neurons, or a glia cell-specific promoter that controls the expression of the glycosylated human IDS precursor in human glial cells in which the expression of the glycosylated human IDS precursor is controlled in human glial cells including. 14. The method according to any one of paragraphs 8 to 13, wherein the recombinant nucleotide expression vector encodes a leader peptide that ensures the translation and post-translation processing of the glycosylated human IDS precursor in human neurons or human glial cells in which the expression of the glycosylated human IDS precursor is controlled including. 15. The method according to any one of paragraphs 8 to 14, wherein the recombinant nucleotide expression vector is an AAV vector including. 16. The method according to paragraph 15, wherein the recombinant nucleotide expression vector is a replication-deficient AAV vector method. 17. The method according to paragraph 15 or 16, wherein the recombinant nucleotide expression vector is an AAV9 or AAVrh10 vector or the method according to paragraph 16. 18. The method according to any one of paragraphs 8 to 17, wherein the recombinant nucleotide expression vector is delivered to the CSF of a human subject by intrathecal, intraventricular, lumbar puncture or intranasal administration including. 19. The method according to any one of paragraphs 8 to 18, wherein the human subject lacks IDS activity 20. A method for treating a human subject diagnosed with MPS II, comprising administering a preparation containing a recombinant nucleotide expression vector encoding human IDS to the CSF of the human subject including. , where this formulation is suitable for administration to the CSF of the human brain, and as a result, polyacrylamide gel electrophoresis measures by capillary electrophoresis to be 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), contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain detectable α-Gal antigen, and / or is mannose-6-phosphorylated, and a depot that secretes a glycosylated human IDS precursor, which is formed in the human central nervous system. A method. (3.1.5 Set 5) 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 neurons or human glial cells to the cerebrospinal fluid (CSF) of the human subject. A method. 2. A method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), wherein a therapeutically effective amount of a glycosylated recombinant human iduronate-2-sulfatase (IDS) precursor that, as measured by polyacrylamide gel electrophoresis, is about 9 0 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa a, or 95 kDa), has a formylglycine residue at C 84 (Figure 1), is α2,6-sialylated, does not contain detectable NeuGc, and is mannose-6-phosphorylated, is delivered to the cerebrospinal fluid (CSF) of the human subject. A method. 3. A method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), ​​​​​Measuring in the cerebrospinal fluid (CSF) of the human subject by polyacrylamide gel electrophoresis, about 9 0 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa a, or 95 kDa), and having a formylglycine residue at C 84 (Figure 1), being α2,6-sialylated without detectable NeuGc and / or α-Gal antigen, and being mannose-6-phosphorylated A method comprising delivering a therapeutically effective dose of a glycosylated recombinant human iduronate-2-sulfatase (IDS) precursor. 4. The method according to any one of paragraphs 1 to 3, wherein the glycosylated recombinant human IDS precursor is delivered from a depot of cells in the central nervous system that have been genetically engineered to secrete the glycosylated recombinant human IDS precursor into the CSF. The method according to any one of paragraphs 1 to 3, wherein the depot is formed in the brain of the human subject. 5. The method according to paragraph 4, wherein the depot is formed in the brain of the human subject. 6. The method according to any one of paragraphs 1 to 5, wherein the human subject is deficient in IDS activity. 7. The method according to any one of paragraphs 1 to 6, wherein the glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO: 1. 8. A method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering a recombinant nucleotide expression vector encoding human iduronate-2-sulfatase (IDS) to the cerebrospinal fluid (CSF) of the human subject, wherein when the recombinant nucleotide expression vector is used to transduce primary human neurons in culture, it measures about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa by polyacrylamide gel electrophoresis. a, or 95 kDa) a, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa), and C 84 contains formylglycine residues (Figure 1), is α2,6-sialylated, and is mannose-6-phosphorylated, a method for inducing the expression of a secreted glycosylated human IDS precursor. 9. A method 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 brain of the human subject, whereby a depot secreting α2,6-sialylated and mannose-6-phosphorylated glycosylated human IDS precursor is formed in the central nervous system of the subject. 10. The method according to paragraph 9, wherein the secretion of the α2,6-sialylated glycosylated human IDS precursor is confirmed by transduction of a human neural cell line in cell culture with the recombinant nucleotide expression vector. 11. The method according to paragraph 9, wherein the secretion of the mannose-6-phosphorylated glycosylated human IDS precursor is confirmed by transduction of a human neural cell line in cell culture with the recombinant nucleotide expression vector. 12. The method according to paragraph 10 or 11, wherein the secretion is confirmed in the presence and absence of mannose-6-phosphate. 13. A method 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 brain of the human subject, whereby a depot secreting a glycosylated human IDS precursor containing α2,6-sialylated glycan is formed; 14. The method according to paragraph 13, wherein the glycosylated human IDS precursor is mannose-6-phosphorylated; 15. The method according to paragraph 13 or 14, wherein the secretion of the glycosylated human IDS precursor is confirmed by transduction of a human neural cell line in cell culture with the recombinant nucleotide expression vector; 16. The method according to paragraph 15, wherein the secretion is confirmed in the presence and absence of mannose-6-phosphate; 17. The method according to any one of paragraphs 9 to 16, wherein the recombinant nucleotide expression vector is administered by intracerebroventricular injection; Wherein when the recombinant nucleotide expression vector is used to transduce human nerve cells in culture, in the cell culture, the method comprises producing the secretion of the glycosylated human IDS precursor containing α2,6-sialic acid-added glycan. 14. A method 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 brain of the human subject, resulting in the formation of a depot that secretes the glycosylated human IDS precursor containing mannose-6-phosphate; Wherein when the recombinant nucleotide expression vector is used to transduce human nerve cells in culture, in the cell culture, the method comprises producing the secretion of the glycosylated human IDS precursor phosphorylated with mannose-6-phosphate. 15. A method 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 brain of the human subject, resulting in the formation of a depot that secretes the glycosylated human IDS precursor containing formylglycine; Wherein when the recombinant nucleotide expression vector is used to transduce human nerve cells in culture, in the cell culture, the method comprises producing the secretion of the glycosylated human IDS precursor containing formylglycine. 16. The method according to any one of paragraphs 8 to 15, wherein human IDS comprises the amino acid sequence of SEQ ID NO: 1. 17. The method according to any one of paragraphs 8 to 15, wherein the recombinant nucleotide expression vector encodes a leader peptide. 18. The method according to any one of paragraphs 8 to 15, wherein the recombinant nucleotide expression vector is a replication-deficient AAV vector. 2 19. The method according to any one of paragraphs 8 to 15, wherein the recombinant nucleotide expression vector is delivered to the CSF of a human subject by intrathecal, intraventricular, lumbar puncture or intranasal administration. 2 20. The method according to any one of paragraphs 8 to 15, wherein the human subject lacks IDS activity.

Brief Description of the Drawings

[0032] (4. Brief Description of the Drawings)

Figure 1

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Chemical

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Figure 2

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Figure 3

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Figure 4

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Figure 5

[0037]

Figure 6

Mode for Carrying Out the Invention

[0038] (5. Detailed Description of the Invention) The present invention relates to the delivery of recombinant human iduronate-2-sulfatase (rhIDS) produced by human neurons or glial cells to the cerebrospinal fluid (CSF) of the central nervous system (CNS) of human subjects diagnosed with, among others, Hunter syndrome, mucopolysaccharidosis type II (MPS II). Also, with respect to the compositions and methods that can be used in accordance with the invention described herein, International Patent Application No. PCT / US2017 / 027770, filed on April 14, 2017 (published as WO / 2017 / 181113 on October 19, 2017), which is hereby incorporated by reference in its entirety, is also referred to. incorporated herein by reference in its entirety. See also International Patent Application No. PCT / US2017 / 027770, filed on April 14, 2017 (published as WO / 2017 / 181113 on October 19, 2017), which is hereby incorporated by reference in its entirety. desired.

[0039] In a preferred embodiment, treatment is via gene therapy - for example, a viral vector or other DNA expression construct encoding human IDS (hIDS) or a derivative of h IDS is administered to the CSF of a patient diagnosed with MPS II (human subject), resulting in the generation of a permanent depot of transduced neurons and / or glial cells that continuously supply the transgene product to the CNS - achieved by. RhIDS secreted into the CSF from the neuron / glial cell depot is endocytosed by cells in the CNS, "transversally correcting" the enzyme deficiency in the recipient cells Furthermore, unexpectedly, the depot of transduced neurons and glial cells in the CNS can deliver this recombinant enzyme to both the CNS and the whole body, which can reduce or eliminate the need for systemic treatment, such as weekly intravenous injection of this enzyme It has been found. In an alternative embodiment, hIDS is produced by human neurons or glial cells in cell culture (e.g., a bioreactor) and administered as enzyme replacement therapy ("ERT"), for example, by injecting the enzyme - into the CSF, Directly into the CNS and / or systemically. However, the gene therapy approach offers several advantages over ERT. Since the enzyme cannot cross the blood - brain barrier, the CNS is not treated by systemic delivery of the enzyme; and unlike the gene therapy approach of the present invention, direct delivery of the enzyme to the CSF and / or CNS would not only be a major Burden but also require repeated injections that carry a risk of infection. The hIDS encoded by the transgene is, but not limited to, the amino acid sequence of SEQ ID NO: 1

[0040] In an alternative embodiment, hIDS is produced by human neurons or glial cells in cell culture (e.g., a bioreactor) and administered as enzyme replacement therapy ("ERT"), for example, by injecting the enzyme - into the CSF, Directly into the CNS and / or systemically. However, the gene therapy approach offers several advantages over ERT. Since the enzyme cannot cross the blood - brain barrier, the CNS is not treated by systemic delivery of the enzyme; and unlike the gene therapy approach of the present invention, direct delivery of the enzyme to the CSF and / or CNS would not only be a major Burden but also require repeated injections that carry a risk of infection. However, the gene therapy approach offers several advantages over ERT. Since the enzyme cannot cross the blood - brain barrier, the CNS is not treated by systemic delivery of the enzyme; and unlike the gene therapy approach of the present invention, direct delivery of the enzyme to the CSF and / or CNS would not only be a major Burden but also require repeated injections that carry a risk of infection. However, the gene therapy approach offers several advantages over ERT. Since the enzyme cannot cross the blood - brain barrier, the CNS is not treated by systemic delivery of the enzyme; and unlike the gene therapy approach of the present invention, direct delivery of the enzyme to the CSF and / or CNS would not only be a major Burden but also require repeated injections that carry a risk of infection.

[0041] The hIDS encoded by the transgene is, but not limited to, the amino acid sequence of SEQ ID NO: 1 Human IDS (hIDS) having the amino acid sequence (shown in Figure 1), as well as amino acid substitutions, deletions, or additions Derivatives of hIDS having, for example, but not limited to, amino acid substitutions selected from the corresponding non-conserved residues in the orthologs of IDS shown in Figure 2 Derivatives of hIDS that include amino acid substitutions selected from the corresponding non-conserved residues in the orthologs of IDS shown in Figure 2, provided that such mutations do not include replacement of the cysteine residue (C84) at position 84, which is required for enzyme activity (Millat et al., 1997, Biochem J 326: 243-247); or, for example, those shown in Figure 3 or Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-761, the entirety of each of which is incorporated herein by reference (the "attenuated" mutants R48P, A85T, W337R, and the truncated mutant Q531X; and the "severe" mutants P86L, S333L, S349I, R468Q, R468L reported therein); Millat et al., 1998, BBA 140 6: 214-218 (the "attenuated" mutants P480L and P480Q; and the "severe" mutant P86L reported therein); and mutations identified in severe, severe-moderate, moderate, or attenuated MPS II phenotypes reported by Bonucelli et al., 2001, BBA 1537:233-238 are included; For example, an amino acid substitution at a specific position of hIDS is the corresponding non-conserved amino acid residue found at that position in the IDS ortholog aligned in Figure 2, provided that such a substitution is shown in Figure 3 or the entirety of each of which is incorporated herein by reference or those shown in Figure 3 or Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-761, the entirety of each of which is incorporated herein by reference (the "attenuated" mutants R48P, A85T, W337R, and the truncated mutant Q531X; and the "severe" mutants P86L, S333L, S349I, R468Q, R468L reported therein); Millat et al., 1998, BBA 140 6: 214-218 (the "attenuated" mutants P480L and P480Q; and the "severe" mutant P86L reported therein); and mutations identified in severe, severe-moderate, moderate, or attenuated MPS II phenotypes reported by Bonucelli et al., 2001, BBA 1537:233-238 are included;

[0042] For example, an amino acid substitution at a specific position of hIDS is the corresponding non-conserved amino acid residue found at that position in the IDS ortholog aligned in Figure 2, provided that such a substitution is shown in Figure 3 or the entirety of each of which is incorporated herein by reference or those shown in Figure 3 or Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-761, the entirety of each of which is incorporated herein by reference (the "attenuated" mutants R48P, A85T, W337R, and the truncated mutant Q531X; and the "severe" mutants P86L, S333L, S349I, R468Q, R468L reported therein); Millat et al., 1998, BBA 140 ​from the rare literature of Sukegawa-Hayasaka et al., 2006, supra; Millat et al., 1998, supra; or it can be selected from those that do not contain the deletion mutations reported by Bonucelli et al., 2001, supra. The resulting transgene product can be tested in cell culture or test animals using conventional in vitro assays to ensure that the mutation does not impair the IDS function. The preferred amino acid substitutions, deletions or additions selected should maintain or increase the enzyme activity, stability, or half-life of IDS as tested by conventional assays in vitro in cell culture or in animal models of MPS II. For example, the enzyme activity of the transgene product can be evaluated using conventional enzyme assays with 4-methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate or 4-methylumbelliferyl sulfate as substrates (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, each of which is incorporated herein by reference in its entirety). The ability of the transgene product to correct the MPS II phenotype can be determined, for example, by transducing MPS II cells in culture with a viral vector or other DNA expression construct encoding hIDS or a derivative; adding this transgene product or derivative to MPS I I cells in culture; or co-culturing MPS II cells with human neural / glial host cells engineered to express and secrete rhIDS or a derivative. For possible exemplary IDS enzyme assays, see, for example, Lee et al., 2015, Clin. Biochem. 48(18):1350-1353, Dean et al., 2006, Clin. Chem. 52(4):643-649, each of which is incorporated herein by reference in its entirety. The ability of the transgene product to correct the MPS II phenotype can be determined, for example, by transducing MPS II cells in culture with a viral vector or other DNA expression construct encoding hIDS or a derivative; adding this transgene product or derivative to MPS I I cells in culture; or co-culturing MPS II cells with human neural / glial host cells engineered to express and secrete rhIDS or a derivative. For example, in cultured MPS II cells, by transducing with a viral vector or other DNA expression construct encoding hIDS or a derivative; in cultured MPS I I cells, by adding this transgene product or derivative; or by co-culturing MPS II cells with human neural / glial host cells engineered to express and secrete rhIDS or a derivative. The ability of the transgene product to correct the MPS II phenotype can be determined, for example, by transducing MPS II cells in culture with a viral vector or other DNA expression construct encoding hIDS or a derivative; adding this transgene product or derivative to MPS I By culturing and determining the correction of the deficiency in MPS II cultured cells, for example, MPS II cells during culture by detecting a decrease in IDS enzyme activity and / or GAG storage levels in: cells can be evaluated during cell culture (see, for example, Stron cek et al., 1999, Transfusion 39(4):343-350, which is hereby incorporated by reference in its entirety).

[0043] MPS II animal models that can be used to evaluate the therapeutic agents described herein are described . For example, a knockout mouse model of MPS II (IDS-knockout) was engineered by replacement of exons 4 and 5 of the IDS gene with a neomycin resistance gene (G arcia et al., 2007, J Inherit Metab Dis 30: 924-34). This IDS-knockout mouse exhibits many of the features of MPS I I, including skeletal abnormalities, hepatosplenomegaly, elevated urinary and tissue GAG, and brain storage lesions (Muenzer et al., 2001, Acta Paediatr Suppl 91:98-99), and has been used to evaluate the effect of enzyme replacement therapy in MPS II in order to support clinical trials regarding ERT . Thus, this mouse model is a relevant model for testing the effect of gene therapy that delivers rIDS produced by neurons or glial cells as a treatment for MPS II (see, for example, Polito and Cosma, 2009 , Am. J. Hum. Genet. 85(2):296-301, which is hereby incorporated by reference in its entirety). Preferably, the hIDS transgene produced by human neurons / glial cells is a neuron .

[0044] and / or expression control elements that function in glial cells, e.g., the CB7 promoter ( the chicken β-actin promoter and CMV enhancer) should be controlled by and other expression control elements that enhance the expression of the transgene driven by the vector (e.g., the chicken β-actin intron and the rabbit β-globin polyA signal) can be included . The cDNA construct for the hIDS transgene should include a coding sequence for a signal peptide that ensures proper translational and post-translational processing (glycosylation and protein sulfation) by the transduced CNS cells. Such signal peptides used by CNS cells include: · Oligodendrocyte-myelin glycoprotein (hOMG) signal peptide:

Chem.

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Chem.

Chem.

Chem.

Chem.

[0045] The recombinant vectors used to deliver the transgenes are expressed in neurons and / or glia. The cells should have tropism for cells of the CNS, including but not limited to cells of the CNS. Vectors can include non-replicating recombinant adeno-associated viral vectors ("rAAV"), particularly Preferred are viral vectors having an AAV9 or AAVrh10 capsid in the vector. No. 7,906,111, incorporated herein by reference. AAV variant capsids, particularly preferred AAV capsids having AAV / hu.31, and AAV / hu.32; and Nos. 8,628,966 and 8,927,514, all of which are incorporated herein by reference. and Smith et al., 2014, Mol Ther 22:1625-1634. Variant capsids can be used. However, "naked DNA" capsids can be used. The vectors are lentiviral vectors, called constructs, vaccinia virus vectors, or non-viral vectors. using other viral vectors, including but not limited to viral expression vectors It is possible.

[0046] Pharmaceutical compositions suitable for administration to the CSF include a physiologically compatible aqueous buffer, a surfactant, and In one embodiment, the formulation comprises a suspension of the rhIDS vector in a formulation buffer containing optional excipients. Furthermore, the pharmaceutical composition is suitable for intrathecal administration. In certain embodiments, the pharmaceutical composition is suitable for cisternal administration (injection into the cistern). 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 .

[0047] The therapeutically effective dose of the recombinant vector should be administered to the CSF via intrathecal administration (i.e., injected into the subarachnoid space so that the recombinant vector is distributed through the CSF and transduces the cells of the CNS ). This can be achieved in several ways - for example, by intracranial (cisternal or ventricular) injection, or by injection into the lumbar cistern. For example, cisternal (IC) (into the cistern) injection can be performed by posterior suboccipital puncture guided by CT; or, if feasible for the patient, injection into the subarachnoid space via C1-2 puncture can be performed; or, lumbar puncture (a diagnostic procedure typically performed to collect a sample of CSF) can be used to access the CSF . Alternatively, intraventricular (ICV) administration (a more invasive technique used for the introduction of anti-infective or anti-cancer agents that do not cross the blood-brain barrier) can be used to infuse the recombinant vector directly into the ventricle. Alternatively, intranasal administration can be used to deliver the recombinant vector to the CNS. Due to the fairly rapid brain growth that occurs early in developing children, the total dose of AAV9 .hIDS administered IC is determined by the estimated brain mass across different age groups. The subject ...

[0048] ... ... For age-related brain mass in elephants, see, for example, the literature of AS Dekaban, Ann Neurol, 1978 Oct ; 4(4): 345-56.

[0049] Table: Total dosage administered by age

Table 2

[0050] CSF concentration is monitored by directly measuring the concentration of rhIDS in CSF fluid obtained from the posterior head or lumbar puncture, or estimated by extrapolation from the concentration of rhIDS detected in the patient's serum. or by extrapolation from the concentration of rhIDS detected in the patient's serum. It can be estimated by extrapolation.

[0051] As background, human IDS is translated as a 550 amino acid polypeptide and contains eight potential N-glycosylation sites (N , N 31 , N 115 , N 144 , N 246 , N 280 , N 325 , N 51 3 and N 537 ) shown in Figure 1, 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 modification of its oligosaccharide chains in the Golgi apparatus. This precursor is processed to the major 55 kDa form through various intracellular intermediates by glycosylation modification and proteolytic cleavage. In summary, after removal of the 25 amino acid signal sequence, protein processing removes an 8 amino acid (residues 26-33) propeptide and proteolytically cleaves the N-terminus downstream of N to the major 55 kDa form through various intracellular intermediates by glycosylation modification and proteolytic cleavage. In summary, after removal of the 25 amino acid signal sequence, protein processing removes an 8 amino acid (residues 26-33) propeptide and proteolytically cleaves the N-terminus downstream of N to the major 55 kDa form through various intracellular intermediates by glycosylation modification and proteolytic cleavage. In summary, after removal of the 25 amino acid signal sequence, protein processing removes an 8 amino acid (residues 26-33) propeptide and proteolytically cleaves the N-terminus downstream of N . to remove an 8 amino acid (residues 26-33) propeptide and proteolytically cleaves the N-terminus downstream of N 31 -terminal proteolysis Cleavage and release of the 18 kDa polypeptide 513 Regarding the C-terminal proteolytic cleavage upstream of The enzyme reacts with the ribozyme to produce a 62 kDa intermediate that is converted to the 55 kDa mature form. Catalytic cleavage generates a 45 kDa mature form that is localized to the lysosomal compartment. Millat et al., 1997, Exp Cell Res, which is incorporated herein by reference in its entirety. 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. (See FIG. 4 for a schematic diagram.)

[0052] C 84 The formylglycine modification of (shown in bold in Figure 1) is probably mostly Required for enzymatic activity that may occur as an early post-translational or co-translational event in the plasmolympic reticulum (Millat, citing Schmidt et al., 1995, Cell 82: 271-278) Post-translational processing continues in the Golgi, where the complex sialic acid- Tagged enzymes for the addition of glycans and delivery to the lysosomal compartment (For a brief review, see reference in its entirety.) Clarke, 2008, Expert Opin Pharmacother 9:31, incorporated herein by reference. (See, e.g., J. Am. Soc ... position N 280Glycosylation in is important for cellular internalization and lysosomal targeting via the mannose-6-phosphate ( M6P) receptor. (Chung et al., 2014, Glyc oconj J 31:309-315, page 310, first paragraph). In normal physiological conditions, IDS is produced at very low levels and, if at all, very little enzyme is secreted from the cell. (Clarke, 2008, supra).

[0053] The present invention is based in part on the following principles: (i) Neurons and glial cells in the CNS are secretory cells having 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. For post-translational modifications performed by human CNS cells, the entirety of each of which is incorporated by reference, for example, the human brain mannose-6-phosphate ( M6P) glycoproteome is described, noting that the brain has more proteins and more mannose-6-phosphorylated proteins with many more individual isoforms than found in other tissues, Sleat et al., 2005, Proteomics 5: 1520-1532, and Sleat, 1996, J Biol Chem 271:19191-98; and also, Kanan et al., 2009, Exp. Eye Res. 8 9: 559-567, and Kanan and Al-Ubaidi, 2015 Exp. Eye Res. 133: 126-131, which report the production of tyrosine sulfated glycoproteins secreted by neurons, are incorporated by reference. M6P) glycoproteome is described, noting that the brain has more proteins and more mannose-6-phosphorylated proteins with many more individual isoforms than found in other tissues, Sleat et al., 2005, Proteomics 5: 1520-1532, and Sleat, 1996, J Biol Chem 271:19191-98; and also, Kanan et al., 2009, Exp. Eye Res. 8 more proteins and more mannose-6-phosphorylated proteins with many more individual isoforms than found in other tissues, Sleat et al., 2005, Proteomics 5: 1520-1532, and Sleat, 1996, J Biol Chem 271:19191-98; and also, Kanan et al., 2009, Exp. Eye Res. 8 9: 559-567, and Kanan and Al-Ubaidi, 2015 Exp. Eye Res. 133: 126-131, which report the production of tyrosine sulfated glycoproteins secreted by neurons, are incorporated by reference. sulfated glycoproteins secreted by neurons, are incorporated by reference. See also, Kanan et al., 2009, Exp. Eye Res. 8 9: 559-567, and Kanan and Al-Ubaidi, 2015 Exp. Eye Res. 133: 126-131. See also, Kanan et al., 2009, Exp. Eye Res. 89: 559-567, and Kanan and Al-Ubaidi, 2015 Exp. Eye Res. 133: 126-131. (ii) The human brain produces multiple isoforms of native / undenatured IDS. In particular, the human brain​ N-terminal sequencing of the mannose-6-phosphorylated glycoprotein revealed that the N-terminal sequence of the mature 42 kDa chain of hIDS varies in the brain and starts from position 34 or 36 as follows: [Chemical formula] as revealed (Sleat, 2005, Proteomics 5: 1520-1532, Table S2). Two of the eight N-linked glycosylation sites, namely N 280 and N 116 were found to be mannose-6-phosphorylated in IDS obtained from human brain. (Sleat et al., 2006, Mol & C ell Proeomics 5.4: 686-701, reported in Table V). (iii) During processing of hIDS, two polypeptides of 76 kDa and 90 kDa are secreted from neurons and glial cells, but only the 90 kDa polypeptide is mannose-6-phosphorylated which is necessary for the secreted form of the enzyme to achieve trans-correction. (Millat, 199 7 (Figure 1 shows results for transfected lymphoblastoid cells), and Froissart, 1995 (Figure 4 shows similar results for transfected fibroblasts indicating that only the 90 kDa form is phosphorylated in the culture medium). See also ). Interestingly, recombinant IDS produced by neurons and glia was found to be more avidly endocytosed by recipient CNS cells than recombinant IDS produced by other cells such as the kidney. Daniele, 2002 showed that the M6P-receptor is involved in the conditioning of transfected neuronal and glial cell cultures by the recipient population of non-transfected neurons and glia cells. ​ mediated the endocytosis of the recombinantly produced IDS of placental origin and probably processed this precursor into the 45 kDa mature active form was demonstrated. The uptake of recombinantly produced IDS (74% endocytosis) generated by neuronal and glial cell lines was much higher than that of the enzyme generated by renal cell lines (5.6% endocytosis). In each case, uptake was inhibited by M6P, indicating that the uptake of recombinantly produced IDS is mediated by the M6P-receptor. (See Daniele, 2002, Tables 2 and 4 and the description accompanying the "Results" on pages 205-206, which are summarized in Table 1 below). Table 1. Summary of results reported in Daniele, 2002

Table 3

Chemical formula

[0054] For the reasons described above, the production of rhIDS by human neurons and / or glial cells, for example, from patients diagnosed with MPS II diseases (including but not limited to Hunter), ​​​​​​​​​​​​​​​ administering a viral vector or other DNA expression construct encoding rhIDS to the CSF of the subject to create a persistent depot in the CNS that continuously supplies the fully human glycosylated, mannose-6-phosphorylated, sulfated transgene product secreted by the transduced CNS cells to provide a "bio-beta" molecule for the treatment of MPS II achieved by gene therapy The hIDS transgene product secreted from the depot into the CSF is endocytosed by cells in the CNS, resulting in a "cross-correction" of the enzyme deficiency in MPS II recipient cells.

[0055] It is not essential that all rhIDS molecules produced in either a gene therapy or protein therapy approach be fully glycosylated, phosphorylated, and sulfated. Rather, the resulting population of glycoproteins should undergo glycosylation (including 2,6-sialylation and mannose-6-phosphorylation) and sulfation sufficient to demonstrate efficacy. The goal of the gene therapy treatment of the present invention is to slow or stop the progression of the disease. Efficacy can be monitored by measuring cognitive function (e.g., prevention or reduction of decline in neurocognitive function); reduction of disease biomarkers (e.g., GAG) 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.

[0056] As an alternative or additional treatment to gene therapy, the rhIDS glycoprotein can be produced in cell lines of human neurons or glial cells by recombinant DNA technology, and the glycoprotein can be systemic ​ It can be administered to patients diagnosed with MPS II in CSF for IRT and / or ERT. Thus Human cell lines that can be used for recombinant glycoprotein production, including but not limited to HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11 , or ReNcell VM (see, for example, the review of human cell lines that can be used for recombinant production of rHuGlyIDS glycoprotein, which is incorporated in its entirety by reference, Dumo nt et al., 2016, Critical Rev in Biotech 36(6):1110-1122, "Human Cell Lines for Biopharmaceutical Manufacturing: History, Current Status, and Future Perspectives"). To ensure complete glycosylation, especially sialylation and tyrosine sulfation, the cell line used for production is co-expressed with α-2,6 -sialyltransferase (or both α-2,3- and α-2,6-sialyltransferases) and / or the TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation. The host cells can be engineered to enhance this by operating them to co-express α-2,6 -sialyltransferase (or both α-2,3- and α-2,6-sialyltransferases) and / or the TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation. This can be enhanced.

[0057] While delivery of rhIDS should minimize the immune response, the most obvious potential source of toxicity related to CNS gene therapy is the expression of rhIDS protein in human subjects who are genetically deficient in IDS and thus potentially intolerant of the vectors used to deliver the protein and / or the transgene. This results in the generation of immunity against the rhIDS protein. In human subjects who are genetically deficient in IDS and thus potentially intolerant of the vectors used to deliver the protein and / or the transgene. This results in the generation of immunity against the rhIDS protein.

[0058] Therefore, in a preferred embodiment, co-treating the patient with immunosuppressive therapy is appropriate, especially when treating patients with severe disease with an IDS level close to 0. Tacrolimus Sirolimus or rapamycin, for example, in a regimen combined with mycophenolic acid, or immunosuppressive treatment with other immunosuppressive regimens used in tissue transplantation procedures can be utilized. Such immunosuppressive treatment can be administered during the course of gene therapy, and in certain embodiments, pretreatment with immunosuppressive treatment may be preferred. The immunosuppressive treatment can be continued after the gene therapy treatment, based on the judgment of the treating physician, and then immunosuppression can be induced: for example, after 180 days, it can be discontinued.

[0059] Combinations of delivery of rhIDS to CSF achieved by delivery of other available treatments are encompassed by the methods of the present invention. Additional treatments can be administered before, simultaneously with, or after the gene therapy treatment. Available treatments 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 into the CSF; and / or HSCT therapy.

[0060] In certain embodiments, a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II) comprising delivering a therapeutically effective amount of a recombinant human iduronate-2-sulfatase (IDS) precursor produced by human neurons or human glial cells to the cerebrospinal fluid (CSF) of the brain of the human subject is described herein.

[0061] In certain embodiments, a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II) comprising delivering to the cerebrospinal fluid (CSF) of the brain of the human subject, by polyacrylamide gel electrophoresis, Measure to be 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), and has a formylglycine residue at C 84 (Figure 1), is α2,6-sialylated, contains no detectable NeuGc, and is mannose-6-phosphorylated, and delivers a therapeutically effective amount of recombinant human iduronate-2-sulfatase (IDS) glycoprotein precursor The method is described herein, including. In certain embodiments, a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II) comprising delivering to the cerebrospinal fluid (CSF) of the brain of the human subject, by polyacrylamide gel electrophoresis

[0062] measured to be 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), contains a formylglycine residue at C (Figure 1), is α2,6-sialylated, contains no detectable NeuGc and / or α-Gal antigen, and is mannose-6- phosphorylated, and delivers a therapeutically effective amount of recombinant human iduronate-2-sulfatase (IDS) glycoprotein precursor 84 The method is described herein, including. In certain embodiments, the human IDS precursor is delivered to the CSF from a depot of cells in the central nervous system that have been genetically engineered to secrete the IDS precursor into the CSF. In certain embodiments the depot is formed within the brain of the subject. In certain embodiments, the human subject has a deficiency in IDS activity

[0063] In certain embodiments, the human IDS comprises the amino acid sequence of SEQ ID NO: 1. operated to secrete the IDS precursor into the CSF. In certain embodiments , the depot is formed within the brain of the subject. In certain embodiments, the human subject has a deficiency in IDS activity In certain embodiments, the human IDS comprises the amino acid sequence of SEQ ID NO: 1.

[0064] In one embodiment, administering to the cerebrospinal fluid (CSF) of the brain of the human subject a recombinant nucleotide expression vector encoding human iduronate-2-sulfatase (IDS) is included in a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II) described herein, wherein the expression vector, when used for transduction of primary human neurons in culture, has a molecular weight of 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 sodium dodecyl sulfate polyacrylamide gel electrophoresis, contains formylglycine residues (Figure 1), is α2,6-sialylated, and is mannose-6-phosphorylated 84 and directs the expression of a secreted human IDS glycoprotein precursor. In one embodiment, administering to the cerebrospinal fluid of the brain of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS, resulting in the secretion of a recombinant human IDS glycoprotein precursor that is α2,6-sialylated and mannose-6-phosphorylated, and a depot that forms in the central nervous system of the subject is included in a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II) described herein.

[0065] In one embodiment, the secretion of the α2,6-sialylated recombinant human IDS glycoprotein is confirmed by transduction of a human neuronal cell line with the recombinant nucleotide expression vector in cell culture. In one embodiment, the mannose-6-phosphorylated

[0066] In one embodiment, the recombinant human IDS glycoprotein that is α2,6-sialylated ​​​​​Secretion of the recombinant human IDS glycoprotein is confirmed by transduction of a human neuronal cell line with the recombinant nucleotide expression vector in certain embodiments, secretion is confirmed in the presence or absence of mannose-6-phosphate.

[0067] In certain embodiments, a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II) comprising administering to the cerebrospinal fluid of the brain of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS, whereby a depot is formed that secretes a glycosylated IDS precursor containing α2,6-sialic acid glycans; wherein the recombinant vector, when used to transduce human neurons in culture, results in secretion of the glycosylated IDS precursor containing α2,6-sialic acid glycans in the cell culture, as described herein.

[0068] In certain embodiments, a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II) comprising administering to the cerebrospinal fluid of the brain of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS, whereby a depot is formed that secretes a glycosylated IDS precursor containing mannose-6-phosphate; wherein the recombinant vector, when used to transduce human neurons in culture, results in secretion of the glycosylated IDS precursor phosphorylated with mannose-6-phosphate in the cell culture, as described herein.

[0069] In certain embodiments, a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II) ​​​​​administering to the cerebrospinal fluid of the brain of the human subject a recombinant nucleotide encoding human IDS in a therapeutically effective amount, such that deposits are formed that secrete a glycosylated IDS precursor containing formylglycine; wherein the recombinant vector, when used for transducing human nerve cells in culture, results in the secretion of the glycosylated IDS precursor containing formylglycine in the cell culture, as described herein. In certain embodiments, 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

[0070] is delivered to the subject's CSF by intrathecal (e.g., cisternal, C1-2 puncture if feasible in the patient, or lumbar puncture), intraventricular, or intranasal administration. In certain embodiments, the human subject has a deficiency in IDS activity. In preferred embodiments, the glycosylated IDS is free of detectable NeuGc and / or α-Gal. As used herein, the phrase "detectable NeuGc and / or α-Gal" means that the NeuGc and / or α-Gal moieties are detectable by standard assay methods known in the art. For example, NeuGc is detected by the method of Hara et al., 1989, "N-Acetyl and N-Glycolylneuraminic Acids in Human Serum and Urine and Rat Serum by Reverse-Phase Liquid Chromatography with Fluorescent Detection," which is incorporated herein by reference for methods of detecting NeuGc.

[0071] ​​​​​​​Highly Sensitive Determination of N-Acetyl-and N-Glycolylneur aminic Acids in Human Serum and Urine and Rat Serum by Reversed-Phase Liquid Chr omatography with Fluorescence Detection.)」J. Chromatogr., B: Biomed. 377: 111-1 19 can be detected by HPLC according to. Alternatively, NeuGc can be detected by mass spectrometry . α-Gal can be detected using ELISA (see, for example, the literature of Galili et al., 1998, " A sensitive assay for measuring alpha-Gal epitope expression on cell surface by monoclonal anti-Gal antibody.)」, Transplantation. 65(8):1129-32), or by mass spectrometry (see, for example, the literature of Ayoub et al., 2013, " Combined use of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques for modified primary structure assessment and extensive glyco-profiling of cetuximab ). See also, Transplantation. 65(8):1129-32), or by mass spectrometry (see, for example, the literature of Ayoub et al., 2013, " Combined use of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques for modified primary structure assessment and extensive glyco-profiling of cetuximab ). See also, Transplantation. 65(8):1129-32), or by mass spectrometry (see, for example, the literature of Ayoub et al., 2013, " Combined use of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques for modified primary structure assessment and extensive glyco-profiling of cetuximab rimary structure assessment and extensive glyco-profiling of cetuximab by a comb ination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spect See Landes Bioscience. 5(5): 699-710 and can be detected. Also see the references cited in the literature by Platts-Mills et al., 2015, "Anaphylaxis to the Carbohydrate Side-Chain Alpha-gal", Immunol Allergy Clin North Am. 35(2): 247-260. (5.1 Processing, N-glycosylation and tyrosine sulfation) (5.1.1 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 modification of its oligosaccharide chain 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 involves N-terminal proteolytic cleavage that removes an 8 amino acid (residues 26-33) propeptide downstream of N 31 and C-terminal proteolytic cleavage upstream of N 513 which releases an 18 kDa polypeptide and produces a 62 kDa intermediate that is converted to the 55 kDa mature form. Further proteolytic cleavage results in a 45 kDa mature form located within the lysosomal compartment. (See the literature by Millat et al., 1997, Exp

[0072] each of which is incorporated herein by reference in its entirety.) 31 ​​​​​​​​​Cell Res 230: 362-367(“Millat 1997”); Millat et al., 1997, Biochem J. 326: 24 3-247(“Millat 1997a”); and Froissart et al., 1995, Biochem J. 309: 425-430 (see Figure 4 for a schematic that has been reproduced).

[0073] C 84 (shown in bold in Figure 1), formylglycine modification is probably mostly required for enzymatic activity that may occur as an early post-translational or co-translational event in the cell body. (See Millat, 1997a, citing Schmidt et al., 1995, Cell 82: 271-278). Post-translational processing continues in the Golgi and involves the addition of complex sialic acid-containing glycans and the acquisition of mannose-6-phosphate residues that tag enzymes for delivery to the lysosomal compartment. (For a concise review, see Clarke, 2008, Expert Opin Pharmacother 9: 311-317, which is hereby incorporated herein by reference in its entirety).

[0074] In a specific embodiment, the HuGlyIDS used in accordance with the methods described herein can be in a mannose-6-phosphorylated form of 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) when expressed in vivo or in vitro in neuronal or glial cells. Daniele, 2002, and the literature of Sleat, Proteomics, 2005 (it has been reported that the human brain contains more (both quantitatively and qualitatively) M6P glycoproteins than other tissues) ), as reported , IDS produced from neurons and glial cells may contain a higher M6P content. Dani ele, as performed in the literature in 2002, it is possible to measure the M6P content of the IDS precursor .

[0075] Thus, in certain embodiments, HuGlyIDS used according to the methods described herein, when expressed in vivo or in vitro in neurons or glial cells, is mannose-6-phosphorylated at a higher level than IDS expressed outside neurons or glial cells . In particular, HuGlyIDS used according to the methods described herein, when expressed in vivo or in vitro in neurons or glial cells, is mannose-6-phosphorylated at a higher level than IDS expressed in HT1080 or CHO cells . In certain embodiments, the mannose-6-phosphorylation level of the expressed IDS is measured by the uptake of IDS by human neurons in the presence of M6P (e.g., 5 mM M6P) . In certain embodiments, when expressed in vivo or in vitro in neurons or glial cells, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60% , 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% of the HuGlyIDS molecules used according to the methods described herein are mannose-6-phosphorylated . (5.1.2 N-glycosylation) . . . . .

[0076] (5.1.2 N-glycosylation) Neurons and glial cells of the CNS express secreted proteins that contain glycosylation and tyrosine-O-sulfation. hIDS are secretory cells that possess the cellular machinery for post-translational processing of proteins. Eight asparagine ("N") glycosylation sites identified in 31 ST;N 115 FS;N 144 HT ;N 246 IT;N 280 IS;N 325 ST;N 513 FS;N 537 DS) and eight N-linked glycosylation sites Two of them, namely N 280 and N 116 showed that mannose-6-phosphorylase in IDS from human brain oxidized (reported in Sleat et al., 2006, Mol & Cell Proeomics 5.4: 686-701, Table V). A single glycosylation site is not essential for IDS stabilization, but position N 280 The glycosylation of Mannose-6-phosphate (M6P) receptor mediates cell internalization and lysosomal (Chung et al., 2014, Glycoconj J 31:309-315, p. 310, first paragraph) Under normal physiological conditions, IDS is produced at very low levels, if at all. , and very little of the enzyme is secreted from the cell (Clarke, 2008, supra).

[0077] Any molecule produced in either a gene therapy or protein therapy approach It is not essential that the glycoprotein be fully glycosylated and sulfated. The protein population should be sufficiently glycosylated and sulfatized to demonstrate efficacy. do.

[0078] In certain embodiments, when HuGlyIDS used according to the methods described herein is expressed in vivo or in vitro in neuronal or glial cells, it can be glycosylated at 100% of its N-glycosylation sites. However, one of ordinary skill in the art will appreciate that not necessarily all N-glycosylation sites of HuGlyIDS need to be N-glycosylated in order for the benefits of glycosylation to be achieved. Rather, the benefits of glycosylation can be achieved when only a certain proportion of the N-glycosylation sites are glycosylated and / or when only a certain proportion of the expressed IDS molecules are glycosylated. Thus, in certain embodiments, HuGlyIDS used according to the methods described herein, when expressed in vivo or in vitro in neuronal or glial cells, is glycosylated at 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80 %, 80% - 90%, or 90% - 100% of its available N-glycosylation sites. In certain embodiments, when expressed in vivo or in vitro in neuronal or glial cells, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90% or 90% - 100% of the HuGlyIDS molecules used according to the methods described herein are glycosylated at at least one of their available N-glycosylation sites. In specific embodiments, when HuGlyIDS is expressed in vivo or in vitro in neuronal or glial cells, the HuGlyIDS present in the HuGlyIDS used according to the methods described herein has ... ...

[0079] In a specific embodiment, when HuGlyIDS is expressed in vivo or in vitro in neuronal or glial cells, the HuGlyIDS used according to the methods described herein present in the HuGlyIDS has At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% are glycosylated with the Asn residue (or other related residue) present at the N-glycosylation site. That is, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the resulting N-glycosylation sites of HuGlyIDS are glycosylated.

[0080] In another specific embodiment, when HuGlyIDS is expressed in vivo or in vitro in neurons or glial cells, at least 10%, 20%, 30%, 40%, 50%, 60%, 70% of the N-glycosylation sites present in the HuGlyIDS molecule used according to the methods described herein are glycosylated with the same attached glycan linked to the Asn residue (or other related residue) present at the N-glycosylation site. That is, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the resulting N-glycosylation sites of HuGlyIDS have the same attached glycan.

[0081] Importantly, when the IDS protein used according to the methods described herein is expressed in neurons 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, the results of the methods described herein (e.g., neurons or ​Use of glial cells), the N-glycosylation sites of the IDS protein are advantageously modified with glycans relevant and beneficial to human therapy, particularly at the target site of treatment. When CHO cells or E. coli are used for protein production, such advantages cannot be achieved; this is because, for example, CHO cells (1) do not express the 2,6-sialyltransferase and thus cannot add 2,6-sialic acid during N-glycosylation, and (2) can add Neu5Gc instead of Neu5Ac as sialic acid; and E. coli do not naturally contain the components necessary for N-glycosylation. Furthermore, such advantages cannot be achieved when human cells other than neuronal or glial cells are used for protein production. Thus, in one embodiment, the IDS protein that is expressed in neuronal or glial cells and gives rise to HuGlyIDS used in the treatment methods described herein is glycosylated in a manner in which the protein is N-glycosylated in human neuronal or glial cells, but not in the manner in which the protein is glycosylated in CHO cells. In another embodiment, the IDS protein that is expressed in neuronal or glial cells and gives rise to HuGlyIDS used in the treatment methods described herein is glycosylated in a manner in which the protein is N-glycosylated in neuronal or glial cells, and such glycosylation is not possible without modification when using a prokaryotic host cell, for example, when using E. coli. In one embodiment, the IDS protein that is expressed in human neuronal or glial cells and gives rise to HuGlyIDS used in the treatment methods described herein is a human neuronal or glial cell. ​​​​​​​​​​​​​Proteins are glycosylated in a manner in which they are N-glycosylated in cells, but not in human cells that are not neurons or glial cells. They are not glycosylated.

[0082] Assays for determining the glycosylation pattern of proteins are known in the art. For example, hydrazinolysis can be used to analyze glycans. First, the polysaccharide is released from its associated protein by incubation with hydrazine (the Ludger Release 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 bound glycan. The N-acetyl groups are lost during this process and must be reconstituted by re-N-acetylation. The free glycan can be purified on a carbon column and subsequently labeled at the reducing end with the fluorescent dye molecule 2-aminobenzamide. The labeled polysaccharide 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 fluorescent chromatogram indicates the length of the polysaccharide and the number of repeating units. Structural information can be gathered by collecting the individual peaks and subsequently performing MS / MS analysis. Thereby, the - Glucan corresponds to a polymer consisting of a specific number of repeating units and fragments thereof. Thus, glucan Using a chromatogram, it becomes possible to measure the distribution of polymer length. The elution time is an index of the polymer length while the fluorescence intensity correlates with the molar abundance for each polymer.

[0083] The uniformity of the glycan pattern that associates with a protein is related to the glycan length, the glycosylation sites and the number of glycans present overall. Therefore, this uniformity can be evaluated using methods known in the art , for example, methods for measuring glycan length and hydrodynamic radius. Size exclusion HPLC enables the measurement of hydrodynamic radius. The more glycosylation sites there are in a protein , the greater the diversity of the hydrodynamic radius compared to carriers with fewer glycosylation sites. However, when single glycan chains are analyzed, they can be more uniform because their lengths are more highly controlled. Glycan length can be measured by hydrazine degradation, SDS PAGE, and capillary gel electrophoresis. Furthermore , being uniform can also mean that the usage pattern of specific glycosylation sites varies over a wider / narrower range. These factors can be measured by glycopeptide LC-MS / MS .

[0084] N-glycosylation confers many benefits to HuGlyIDS used in the methods described herein . Such benefits cannot be achieved by protein production in E. coli, which naturally does not have the components required for N-glycosylation. Additionally , some benefits cannot be achieved by protein production in, for example, CHO cells ​Yes, because CHO cells lack the components necessary for the addition of specific glycans (e.g., 2,6-sialic acid), and glycans that are not typical for humans, such as Neu5Gc and α-Gal antigens that are immunogenic in most individuals and can induce anaphylaxis at high concentrations, can be added. Furthermore, some benefits cannot be achieved through protein production in human cells that are not neuronal or glial cells. Thus, expression of IDS in human neurons or glial cells results in the production of HuGlyIDS containing beneficial glycans that would not be associated with the protein if produced in CHO cells or E. coli, or in human cells that are not neuronal or glial cells. and lack glycans that are not typical for humans, such as Neu5Gc and α-Gal antigens that are immunogenic in most individuals and can induce anaphylaxis at high concentrations. Furthermore, some benefits cannot be reached through protein production in human cells that are not neuronal or glial cells. Therefore, expression of IDS in human neurons or glial cells results in the production of HuGlyIDS containing beneficial glycans that would not be associated with the protein if produced in CHO cells or E. coli, or in human cells that are not neuronal or glial cells. (5.1.3 Tyrosine Sulfation) In addition to N-linked glycosylation sites, hIDS contains tyrosine ("Y") sulfation sites

[0085] (5.1.3 Tyrosine Sulfation) (For example, for the analysis of amino acids surrounding tyrosine residues that undergo protein tyrosine sulfation, see the entire reference incorporated by reference: Yang et al., 2015, Molecules 20 [Chemical Formula] : The "rules" can be summarized as follows: The Y residue has an E or D within the range of +5 to -5 positions from Y, and the -1 position of Y is a neutral or acidic charged amino acid but not a basic amino acid that inactivates sulfation, such as R, K, or H). Importantly, tyrosine-sulfated proteins are natural for the enzymes required for tyrosine sulfation and lack glycans that are not typical for humans, such as Neu5Gc and α-Gal antigens that are immunogenic in most individuals and can induce anaphylaxis at high concentrations.

[0086] Importantly, tyrosine-sulfated proteins are natural for the enzymes required for tyrosine sulfation E. coli does not have it and cannot produce it. Furthermore, CHO cells are deficient in tyrosine sulfation - these cells are not secretory cells and have limited ability to perform post-translational tyrosine sulfation. See, for example, the literature of Mikkelsen and Ezban, 1991, Biochemistry 30: 1 533-1537. Advantageously, the methods provided herein are secretory and indeed require the expression of IDS, such as HuGlyIDS, in neurons or glial cells that have the ability for tyrosine sulfation. Assays for detecting tyrosine sulfation are known in the art . See, for example, the literature of Yang et al., 2015, Molecules 20:2138-2164 . The tyrosine sulfation of hIDS - The robust post-translational processes of human CNS cells should improve the processing and activity of the transgene product. The significance of tyrosine sulfation of lysosomal proteins has not been clarified; however, in other proteins, tyrosine sulfation has been shown to increase the binding affinity of protein-protein interactions (antibodies and receptors) and to promote proteolytic processing (peptide hormones). (See the literature of Moore, 2003, J Biol. Chem. 278:24243-46; and the literature of Bundegaard et al., 1995, EMBO J 14: 3073-79). Tyrosine protein sulfotransferase (TPST1), which is responsible for tyrosine sulfation (which may occur as the final step in the processing of IDS), is clearly expressed in the brain (gene expression data for TPST1 is accessible, for example, at http: / / www.ebi.ac.uk / gxa / home ).

[0087] ). The tyrosine sulfation (which may occur as the final step in the processing of IDS) ​​​​​​​​can be found in the EMBL-EBI expression atlas) and are expressed at higher levels (based on mRNA). are expressed.

[0088] (5.2 Constructs and Formulations) Viral vectors or other DNA expression constructs encoding iduronate-2-sulfatase (IDS), such as human IDS (hIDS), are for use in the methods provided herein. The viral vectors and other DNA expression constructs provided herein include any suitable method for delivering the transgene into the cerebrospinal fluid (CSF). Means for delivering the transgene include viral vectors, liposomes, complexes containing other lipids, other polymeric complexes, synthetically modified mRNA, unmodified mRNA, small molecules, non-bioactive 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, such as a vector that targets nerve cells. In some embodiments, the disclosure provides nucleic acids for use encoding IDS, such as hIDS, which are operably linked to a promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter,

[0089] an MMT promoter, an EF-1α promoter, a UB6 promoter, a chicken β-actin promoter, a CAG promoter, an RPE65 promoter, and an opsin promoter.

[0090]

[0090] In certain embodiments, a recombinant vector comprising one or more nucleic acids (e.g., polynucleotides) Nucleic acid can include DNA, RNA, or a combination of DNA and RNA. In one embodiment, the DNA comprises a promoter sequence, a gene sequence of interest (transfection One or more sequences selected from the group consisting of a gene (e.g., an IDS), an untranslated region, and a termination sequence. In some embodiments, the viral vectors provided herein comprise a sequence encoding a gene encoding a target gene of interest. It comprises a promoter operably linked to a gene of interest.

[0091] In certain embodiments, the nucleic acids (e.g., polynucleotides) and The nucleic acid sequence may be codon-optimized, for example, via any codon-optimization technique known to one of skill in the art. (See, e.g., review by Quax et al., 2015, Mol Cell 59:149-161. sea ​​bream).

[0092] In another aspect, the present disclosure provides a recombinant nucleotide expression vector encoding a human IDS. The present invention provides a formulation containing a cerebrospinal fluid of a human brain, the formulation being suitable for administration to the cerebrospinal fluid of a human brain and As a result, the protein was found to be approximately 90 kDa (e.g., 85 kDa, 86 kDa) by polyacrylamide gel electrophoresis. , 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa), Contains milglycine, is α2,6-sialylated, does not contain detectable NeuGc, and is α-Gal antigen recombinant human IDS glycoprotein that is uncoupled and / or mannose-6-phosphorylated Depots that secrete the drug product are formed in the human central nervous system. A buffer that makes the formulation suitable for administration to the cerebrospinal fluid (a buffer having a particular pH or may include a buffer solution containing certain components), and as a result, polyacrylamide gel electrophoresis measured by capillary electrophoresis, 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), contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain assembled α-Gal antigen, and / or is mannose- 6-phosphorylated, and a depot that secretes recombinant human IDS glycoprotein precursor is formed in the human central nervous system. In a specific embodiment, the buffer solution contains physiologically compatible water -based buffer, surfactant, and any excipient.

[0093] In another aspect, the present disclosure provides a kit comprising a recombinant nucleotide expression vector encoding human IDS and a pharmaceutically acceptable carrier, wherein the recombinant nucleotide expression vector is suitable for administration to the cerebrospinal fluid (CSF) of the human brain, and as a result, polyacrylamide gel electrophoresis measured by capillary electrophoresis, 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), contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain assembled detectable α-Gal antigen, and / or is mannose-6-phosphorylated, and a depot that secretes recombinant human IDS glycoprotein precursor is formed in the human central nervous system. In another aspect, the present disclosure provides a kit comprising a formulation containing a recombinant nucleotide expression vector encoding human IDS, wherein the formulation is suitable for administration to the CSF of the human brain, and as a result, polyacrylamide gel electrophoresis measured by capillary electrophoresis, 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), contains formylglycine, is α2,6-sialylated, does not contain detectable NeuGc, does not contain assembled detectable α-Gal antigen, and / or is mannose-6-phosphorylated, and a depot that secretes recombinant human IDS glycoprotein precursor is formed in the human central nervous system. In another aspect, the present disclosure provides a kit comprising a formulation containing a recombinant nucleotide expression vector encoding human IDS, where the formulation is suitable for administration to the CSF of the human brain, and as a result, polyacrylamide gel electrophoresis ​Measured to be 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), contains formylglycine, is α2,6-sialylated, contains no detectable NeuGc, contains no detectable α-Gal antigen, and / or is mannose-6-phosphorylated, a depot that secretes recombinant human IDS glycoprotein precursor is formed in the human central nervous system. The kits described herein contain a recombinant nucleotide expression vector or formulation in one or more containers. Optionally, one or more such containers may be accompanied by a cautionary statement in a form prescribed by a government regulatory agency that regulates the manufacture, use, or sale of pharmaceuticals or biologics, and this cautionary statement reflects approval by a regulatory agency regarding manufacture, use, or sale for human administration. The formulations and kits encompassed herein can be used in accordance with the methods of treatment of human patients as provided in this disclosure. (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., IDS). The synthesis of modified and unmodified mRNAs for delivery of a transgene to the CSF is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is incorporated herein by reference in its entirety. In certain embodiments, a modified mRNA encoding IDS, e.g., hIDS, is provided herein. The kits described herein contain a recombinant nucleotide expression vector or formulation in one or more containers. Optionally, one or more such containers may be accompanied by a cautionary statement in a form prescribed by a government regulatory agency that regulates the manufacture, use, or sale of pharmaceuticals or biologics, and this cautionary statement reflects approval by a regulatory agency regarding manufacture, use, or sale for human administration. The formulations and kits encompassed herein can be used in accordance with the methods of treatment of human patients as provided in this disclosure. In certain embodiments, the vectors provided herein are modified mRNAs encoding a gene of interest (e.g., a transgene, e.g., IDS). The synthesis of modified and unmodified mRNAs for delivery of a transgene to the CSF is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is incorporated herein by reference in its entirety. In certain embodiments, a modified mRNA encoding IDS, e.g., hIDS, is provided herein. The kits described herein contain a recombinant nucleotide expression vector or formulation in one or more containers. Optionally, one or more such containers may be accompanied by a cautionary statement in a form prescribed by a government regulatory agency that regulates the manufacture, use, or sale of pharmaceuticals or biologics, and this cautionary statement reflects approval by a regulatory agency regarding manufacture, use, or sale for human administration. The formulations and kits encompassed herein can be used in accordance with the methods of treatment of human patients as provided in this disclosure.

[0094] The formulations and kits encompassed herein can be used in accordance with the methods of treatment of human patients as provided in this disclosure. (5.2.1. mRNA)

[0095] (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., IDS). The synthesis of modified and unmodified mRNAs for delivery of a transgene to the CSF is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is incorporated herein by reference in its entirety. In certain embodiments, a modified mRNA encoding IDS, e.g., hIDS, is provided herein. For the delivery of the transgene to the CSF, the synthesis of modified and unmodified mRNAs is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is incorporated herein by reference in its entirety. In certain embodiments, a modified mRNA encoding IDS, e.g., hIDS, is provided herein. The synthesis of modified and unmodified mRNAs for delivery of a transgene to the CSF is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is incorporated herein by reference in its entirety. In certain embodiments, a modified mRNA encoding IDS, e.g., hIDS, is provided herein. In certain embodiments, the vectors provided herein are modified mRNAs encoding a gene of interest (e.g., a transgene, e.g., IDS). The synthesis of modified and unmodified mRNAs for delivery of a transgene to the CSF is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is incorporated herein by reference in its entirety. In certain embodiments, a modified mRNA encoding IDS, e.g., hIDS, is provided herein. In certain embodiments, the vectors provided herein are modified mRNAs encoding a gene of interest (e.g., a transgene, e.g., IDS). The synthesis of modified and unmodified mRNAs for delivery of a transgene to the CSF is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is incorporated herein by reference in its entirety. In certain embodiments, a modified mRNA encoding IDS, e.g., hIDS, is provided herein. In certain embodiments, the vectors provided herein are modified mRNAs encoding a gene of interest (e.g., a transgene, e.g., IDS). The synthesis of modified and unmodified mRNAs for delivery of a transgene to the CSF is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is incorporated herein by reference in its entirety. In certain embodiments, a modified mRNA encoding IDS, e.g., hIDS, is provided herein.

[0096] (5.2.2 Virus Vector) Virus vectors include adenovirus, adeno-associated virus (AAV, e.g., AAV9, AAV Vrh10), lentivirus, helper-dependent adenovirus, herpes simplex virus, pox virus, Sendai virus (hemagglutinin virus of Japan) (HVJ), alphavirus, vaccinia virus, and retrovirus vectors. Retrovirus vectors include mouse leukemia virus (MLV) and human immunodeficiency virus (HIV)-based vectors Alphavirus vectors include Semliki Forest virus (SFV) and Sindbis virus (SIN). In certain embodiments, the virus vectors provided herein are recombinant virus vectors. In certain embodiments, the virus vectors provided herein are modified to be replication-deficient in humans. In certain embodiments , the virus vector is a hybrid vector, e.g., an AAV vector disposed in a "helperless" adenovirus vector. In certain embodiments, a virus vector comprising a virus capsid from a first virus and a viral envelope protein from a second virus is provided herein. In a specific embodiment, the second virus is vesicular stomatitis virus (VSV). In a more specific embodiment, the envelope protein is the VSV-G protein. In certain embodiments, the virus vectors provided herein are HIV-based

[0097] virus vectors. In certain embodiments, the HIV-based virus vectors provided herein In certain embodiments, the HIV-based virus vectors provided herein The vector contains at least two polynucleotides, where the gag and pol genes are from the HIV genome, and the env gene is from another virus.

[0098] 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 do not contain one or more immediate early (IE) genes and are modified to be non-cytopathic for this purpose.

[0099] 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 instead of viral genes.

[0100] 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 lentivirus. In certain embodiments, the lentiviral vectors provided herein are derived from non-human lentivirus. In certain embodiments, the lentiviral vectors provided herein are packaged in a lentiviral capsid. In certain embodiments, the lentiviral vectors provided herein contain one or more of the following elements: long terminal repeat sequences, primer binding sites, polypurine tracts, att sites, and encapsidation sites.

[0101] In certain embodiments, the viral vectors provided herein are alpha virus-based viral vectors. In certain embodiments, the alpha virus vectors provided herein are recombinant, replication-deficient alpha viruses. In certain embodiments, the alpha virus envelopes of the alpha virus vectors provided herein are targeted to specific cell types by presenting functional heterologous ligands on their virion surfaces. In certain embodiments, the viral vectors provided herein are AAV-based viral vectors. In preferred embodiments, the viral vectors provided herein are AAV9 or AAVrh10-based viral vectors. In certain embodiments, the AAV9 or AAVrh10-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, AAVrh10, AAV10 or AAV11. In preferred embodiments, the AAV-based vectors provided herein comprise components derived from one or more of the AAV8, AAV9, AAVrh10, AAV10 or AAV11 serotypes. The AAV9-based viral vectors are used in the methods described herein. The AAV-based viral vectors

[0102] ​​​​​​​​​​​​​​The nucleic acid sequences of viral vectors and the 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, each of which is incorporated herein by reference in its entirety. In one aspect, however, an AAV (e.g., AAV9 or AAVrh10)-based viral vector encoding a transgene (e.g., IDS) is provided herein. In a specific embodiment, an AAV9-based viral vector encoding IDS is provided herein. In a more specific embodiment, an AAV9-based viral vector encoding hIDS is provided herein.

[0103] (i) An expression cassette comprising a transgene under the control of a regulatory element and flanked by ITRs; and (ii) a viral capsid having the amino acid sequence of the AAV9 capsid protein or being at least 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of the AAV9 capsid protein (SEQ ID NO: 26) and simultaneously retaining the biological function of the AAV9 capsid: An AAV9 vector comprising an artificial genome is provided in a particular embodiment. In one embodiment, 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 amino acid substitutions in the sequence of SEQ ID NO: 26 and retains the biological function of the AAV9 capsid. ​Retain. Figure 6 shows the characteristics of the aligned sequences based on the comparison in the column labeled SUBS Amino acid sequence comparison alignment of capsid proteins of different AAV serotypes having potential amino acids that can be replaced at specific positions is provided. Thus, in a specific embodiment, the AAV9 vector contains an AAV9 capsid variant having 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 that position in the native AAV9 sequence .

[0104] 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, which is incorporated herein by reference in its entirety. In certain embodiments, the AAV used in the methods described herein is 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, each of which is incorporated herein by reference in its entirety, and includes one of the following amino acid insertions:

Chemical Formula

[0105] 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, 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).

[0106] In one embodiment, the viral vector used in the methods described herein is an adeno virus-based viral vector. Recombinant adenoviral vectors can be used for the introduction of IDS. Recombinant adenoviruses have an E1 deletion and may or may not have an E3 deletion, and can be first-generation vectors with an expression cassette inserted into either deleted region. Recombinant adenoviruses can be second-generation vectors that include a complete or partial deletion of the E2 and E4 regions. Helper-dependent adenoviruses retain only the adenovirus inverted terminal repeats and the packaging signal (φ). The transgene is inserted between the packaging signal and the 3' ITR, and may or may not have stuffer sequences to maintain an artificial genome near the size of the wild-type at approximately 36 kb. An exemplary protocol for the production of adenovirus vectors can be found in Alba et al., 2005, "Gutless adenovirus: last generation adenovirus for gene therapy", Gene Therapy 12:S18 - S27, which is incorporated herein by reference in its entirety.

[0107] In one embodiment, the viral vector used in the methods described herein is a lentivirus -based viral vector. Recombinant lentiviral vectors can be used for the introduction of IDS. Four plasmids are used: a plasmid containing the construct:Gag / pol sequence, a plasmid containing the Rev sequence, a plasmid containing the envelope protein (i.e., Produce a Cis plasmid having (VSV-G), as well as a packaging element and an IDS gene which is used for doing so.

[0108] For lentiviral vector production, especially polyethyleneimine or calcium phosphate can be used as transfection agents to co-transfect four plasmids into cells (i.e., HEK293-based cells). Subsequently, the lentivirus in the supernatant is harvested (lentivirus needs to bud out from cells to show activity, so harvesting from cells is not necessary and should not be done). The supernatant is filtered (0.45 μm) and then magnesium chloride and benzonase are added. Further downstream processes can be very diverse, and the use of TFF and column chromatography is the most GMP-compliant process In other processes, ultracentrifugation can be used, with or without column chromatography. Exemplary protocols for lentiviral vector production are both incorporated herein by reference in their entirety, the literature of Lesch et al., 2011, "Production and purification of lentiviral vector generated in 293T suspension cells with baculoviral vectors", Gene Therapy 18:531-53 8 and the literature of Ausubel et al., 2012, "Production of CGMP-grade lentiviral vectors" ​​​​​​​​found in Bioprocess Int. 10(2):32-43, "of CGMP-Grade Lentiviral Vectors)" be possible

[0109] In a specific embodiment, the vector used in the method described herein is a vector encoding IDS (e.g., hIDS), and when transduced into cells of the CNS or related cells (e.g., in vivo or in vitro neuronal cells), the glycosylated variants of IDS are expressed by the transduced cells. In a specific embodiment, the vector used in the method described herein is a vector encoding IDS (e.g., hIDS), and when transduced into cells of the CNS or related cells (e.g., in vivo or in vitro neuronal cells), the sulfated variants of IDS are expressed by the cells.

[0110] (5.2.3 Promoters and Regulatory Sequences (modifier) of Gene Expression) In certain embodiments, the vectors provided herein include components (e.g., "expression control elements") that regulate gene delivery or gene expression. In certain embodiments, the vectors provided herein include components that regulate gene expression. In certain embodiments, the vectors provided herein include components that affect binding to cells or target cells. In certain embodiments, the vectors provided herein include components that affect the intracellular localization of polynucleotides (e.g., transgenes) after uptake. In certain embodiments, the vectors provided herein include, for example, components for detecting or selecting cells that have taken up polynucleotides. ​​​​​ It includes components that can be used as possible or selectable markers.

[0111] In certain embodiments, the viral vectors provided herein include one or more pro moters. In certain embodiments, the promoter is a constitutive promoter. In alternative embodiments, the promoter is an inducible promoter. The native IDS gene, like most housekeeping genes, mainly uses GC-rich promoters In preferred embodiments, a strong constitutive promoter that provides continuous expression of hIDS is used. Such promoters include the "CAG" synthetic promoter: this consists of the "C" - cytomegalovirus (CMV) early enhancer element; the "A" - chicken β actin gene promoter and the first exon and intron; and the "G" - rabbit β-globin gene splice acceptor (see Miyazaki et al., 1989, Gene 79: 269-277; and Niwa et al., Gene 108: 193-199).

[0112] In certain embodiments, the promoter is the CB7 promoter (see Dinculescu et al., 2005, Hum Gene Ther 16:649-663, which is incorporated herein by reference in its entirety). In some embodiments, the CB7 promoter includes other expression control elements that enhance the expression of the transgene driven by the vector. In certain embodiments, the other expression control elements include the chicken β-actin intron and / or the rabbit β-globin polA signal. In certain embodiments, the promoter includes a TATA box. ​​​ In certain embodiments, the promoter comprises one or more elements. In certain embodiments the one or more promoter elements may be oriented in opposite directions relative to each other or their positions may be changed. In certain embodiments, the elements of the promoter are arranged to function cooperatively. In certain embodiments, the elements of the promoter are arranged 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 LTR. In certain embodiments, the vectors provided herein comprise one or more tissue-specific promoters (e.g., a neuron-specific promoter).

[0113] In certain embodiments, the viral vectors provided herein comprise one or more regulatory elements other than the promoter. In certain embodiments, the viral vectors provided herein comprise an enhancer. In certain embodiments, the viral vectors provided herein comprise a repressor. In certain embodiments, the viral vectors provided herein comprise an intron or a chimeric intron. In certain embodiments, the viral vectors provided herein comprise a polyadenylation sequence.

[0114] (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 intracellular localization of the transgene product (e.g., IDS). In certain embodiments, the signal peptide enables secretion of the transgene product (e.g., IDS) from the cell. Examples of signal peptides used in connection with the vectors and the transgenes provided herein can be found in Table 1. The signal peptide may also be referred to herein as a leader sequence or leader peptide. Table 2. Signal peptides for use with the vectors provided herein.

Table 4

[0115] (5.2.5 Untranslated Region) In certain embodiments, the viral vectors provided herein include one or more untranslated regions (UTRs) (e.g., 3' and / or 5' UTR). In certain embodiments, the UTR is optimized for the desired level of protein expression. In certain embodiments, the UTR is optimized for the mRNA half-life of the transgene. In certain embodiments, the UTR is the m of the transgene. Optimized for RNA stability. In certain embodiments, the UTR is of the mRNA of the transgene Optimized for secondary structure.

[0116] (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 the recombinant gene expression cassette into the virion of the viral vector. In certain embodiments, the IT R is derived from AAV (e.g., AAV9) (each of which is incorporated herein by reference in its entirety, e.g., see the literature of 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. 8,96 2,332 B2 and International Patent Application No. PCT / EP2014 / 076466).

[0117] (5.2.7 Transgene) In certain embodiments, the vectors provided herein encode an IDS transgene. In a specific embodiment, the IDS is controlled by an expression control element suitable for expression in nerve 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.

[0118] ​The HuGlyIDS encoded by the introduced gene includes, but is not limited to, SEQ ID NO: 1 human IDS (hIDS) having the amino acid sequence of (shown in Figure 1), and amino acid substitutions, deletions, or additions, such as, but not limited to, derivatives of hIDS containing amino acid substitutions selected from the corresponding non-conserved residues in the orthologs of IDS shown in Figure 2 (provided that such mutations do not include substitution of the cysteine residue (C84) at position 84 which is necessary for enzyme activity (Millat et al., 1997, Biochem J 326: 243-247); or those shown in Figure 3 or each of which is incorporated herein by reference, Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-761 (the "attenuated" mutants R48P, A85T, W337R, and the truncated mutant Q531X; as well as the "severe" mutants P86L, S333L, S349I, R468Q, R468L reported therein); Millat et al., 1998, BBA 1406: 214-218 (the "attenuated" mutants P480L and P480Q; as well as the "severe" mutant P86L reported therein); and mutations identified in the severe, severe-moderate, moderate or attenuated MPS II phenotypes reported in Bonucelli et al., 2001, BBA 1537:233-238 may be included. For example, amino acid substitutions at specific positions of hIDS can be selected from the corresponding non-conserved amino acid residues found at that position in the IDS ortholog aligned in Figure 2 (provided that such substitutions

[0119] are shown in Figure 3 or each of which is incorporated herein by reference ​​Included are the documents of Sukegawa-Hayasaka et al., 2006, supra; Millat et al., 1998, supra; or Bonucelli et al., 2001, supra, provided that it does not contain any harmful mutations reported therein). The resulting transgene product can be tested in cell culture or in test animals using conventional in vitro assays to ensure that the mutation does not impair the IDS function. The preferred amino acid substitutions, deletions or additions to be selected should maintain or increase the enzyme activity, stability, or half-life of IDS as tested by conventional in vitro assays in cell culture or in the MPS II animal model. For example, the enzyme activity of the transgene product can be evaluated using a conventional enzyme assay with, for example, 4-methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate or 4-methylumbelliferyl sulfate as the 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, each of which is incorporated herein by reference in its entirety). The ability of the transgene product to correct the MPS II phenotype can be determined, for example, by transducing MPS II cells in culture with a virus vector or other DNA expression construct encoding hIDS or a derivative, by adding the transgene product or a derivative to MPS II cells in culture, or by co-culturing MPS II cells with human neural / glial host cells engineered to express and secrete rhIDS or a derivative, to MP none of the harmful mutations reported in; or, Bonucelli et al., 2001, supra). The resulting transgene product can be tested in cell culture or in test animals using conventional in vitro assays to ensure that the mutation does not impair the IDS function. The preferred amino acid substitutions, deletions or additions to be selected should maintain or increase the enzyme activity, stability, or half-life of IDS as tested by conventional in vitro assays in cell culture or in the MPS II animal model. For example, the enzyme activity of the transgene product can be evaluated using a conventional enzyme assay with, for example, 4-methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate or 4-methylumbelliferyl sulfate as the 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, each of which is incorporated herein by reference in its entirety). The ability of the transgene product to correct the MPS II phenotype can be determined, for example, by transducing MPS II cells in culture with a virus vector or other DNA expression construct encoding hIDS or a derivative. By adding the transgene product or a derivative to MPS II cells in culture, or by co-culturing MPS II cells with human neural / glial host cells engineered to express and secrete rhIDS or a derivative. To MP For example, the enzyme activity of the transgene product can be evaluated using a conventional enzyme assay with, for example, 4-methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate or 4-methylumbelliferyl sulfate as the 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, each of which is incorporated herein by reference in its entirety). The ability of the transgene product to correct the MPS II phenotype can be determined, for example, by transducing MPS II cells in culture with a virus vector or other DNA expression construct encoding hIDS or a derivative, by adding the transgene product or a derivative to MPS II cells in culture, or by co-culturing MPS II cells with human neural / glial host cells engineered to express and secrete rhIDS or a derivative, to MP 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, each of which is incorporated herein by reference in its entirety. The ability of the transgene product to correct the MPS II phenotype can be determined, for example, by transducing MPS II cells in culture with a virus vector or other DNA expression construct encoding hIDS or a derivative, by adding the transgene product or a derivative to MPS II cells in culture, or by co-culturing MPS II cells with human neural / glial host cells engineered to express and secrete rhIDS or a derivative, to MP The ability of the transgene product to correct the MPS II phenotype can be determined, for example, by transducing MPS II cells in culture with a virus vector or other DNA expression construct encoding hIDS or a derivative, by adding the transgene product or a derivative to MPS II cells in culture, or by co-culturing MPS II cells with human neural / glial host cells engineered to express and secrete rhIDS or a derivative, to MP For example, by transducing MPS II cells in culture with a virus vector or other DNA expression construct encoding hIDS or a derivative; by adding the transgene product or a derivative to MPS II cells in culture; or by co-culturing MPS II cells with human neural / glial host cells engineered to express and secrete rhIDS or a derivative. To MP For example, by transducing MPS II cells in culture with a virus vector or other DNA expression construct encoding hIDS or a derivative, by adding the transgene product or a derivative to MPS II cells in culture, or by co-culturing MPS II cells with human neural / glial host cells engineered to express and secrete rhIDS or a derivative, to MP To MP By determining the correction of defects in S II cultured cells, for example, ID in MPS II cells during culture can be evaluated in cell culture by detecting a decrease in S enzyme activity and / or GAG storage amount (for example, see the literature of Stroncek et al., 1999, Transfusion 39(4):343-350, which is incorporated herein by reference in its entirety).

[0120] (5.2.8 construct) In certain embodiments, the viral vectors provided herein have the following order and the following elements: 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., IDS), h) a fourth linker sequence, i) a polyA sequence, j) a fifth linker sequence, and k) a second ITR sequence.

[0121] In certain embodiments, the viral vectors provided herein have the following order and the following elements: a) a promoter sequence, and b) a sequence encoding a transgene (e.g., IDS). In certain embodiments, the viral vectors provided herein have the following order and the following elements: a) a promoter sequence, and b) a sequence encoding a transgene (e.g., IDS ) where the transgene includes a signal peptide.

[0122] In certain embodiments, the viral vectors provided herein have the following order and the following elements: a) a first ITR sequence, b) a first linker sequence, c) a promoter sequence, d) a ​​​a linker sequence of 2, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a transgene (for example, IDS), i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, l) a fifth linker sequence, and m) a second ITR sequence.

[0123] In certain embodiments, the viral vectors provided herein are in the following order and contain the following elements: 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 transgene (for example, IDS), i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, l) a fifth linker sequence, and m) a second ITR sequence, wherein the transgene contains a signal peptide and encodes hIDS.

[0124] (5.2.9 Manufacture and testing of vectors) The viral vectors provided herein can be manufactured using host cells. The viral vectors provided herein can be manufactured using mammalian host cells such as A5 49, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa , 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, and myoblasts. The viral vectors provided herein can be manufactured using host cells from humans, monkeys, mice, rats, rabbits, or hamsters. .

[0125] The host cell contains the transgene and associated elements (i.e., the vector genome), and the means to produce the virus in the host cell, e.g., stably transform using sequences encoding replication and capsid genes (e.g., the rep and cap genes of AAV). For methods of producing recombinant AAV vectors having the AAV8 capsid, see Section IV of the detailed description of U.S. Patent No. 7,282,199 B2, which is hereby incorporated by reference in its entirety. The genomic copy titer of the vector can be determined, for example, by TAQMAN® analysis. The virions can be recovered, for example, by CsCl precipitation. In vitro assays, such as cell culture assays, can be used to measure transgene expression from the vectors described herein and thus, for example, demonstrate the efficacy of the vector. For example, cell lines such as HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11, or ReNcell VM, or other cell lines derived from neural or glial cells or progenitor cells of neural or glial cells can be used to evaluate transgene expression. Once expressed, the characteristics of the expressed product (i.e., HuGlyIDS) can be determined, including determination of the pattern of glycosylation and tyrosine sulfation associated with HuGlyIDS. (5.2.10 Compositions) Compositions are described that contain a vector encoding a transgene described herein and a suitable carrier. Suitable carriers (e.g., CSF and, for example, for administration to neural cells) will be known to those skilled in the art. 2

[0126]

[0127] (5.2.10 Compositions) Compositions are described that contain a vector encoding a transgene described herein and a suitable carrier. Suitable carriers (e.g., CSF and, for example, for administration to neural cells) will be known to those skilled in the art. ​​​​​​​​​​​​is easily selected by

[0128] (5.3 Gene Therapy) Describes a method for administering a therapeutically effective amount of a transgene construct to a human subject having MPS II. More specifically, describes a method for administering a therapeutically effective amount of a transgene construct to a patient having MPS II, especially for administration to the CSF. In certain embodiments, the method for administering such a therapeutically effective amount of a transgene construct to the CSF can be used to treat patients having Hurler syndrome. tract for administration, especially for administration to the CSF. In certain embodiments the method for administering such a therapeutically effective amount of a transgene construct to the CSF can be used to treat patients having Hurler syndrome.

[0129] (5.3.1 Target Patient Population) In certain embodiments, a therapeutically effective dose of a recombinant vector is administered to a patient diagnosed with MPS II. In a specific embodiment, the patient is diagnosed with mild MPS II. In a specific embodiment the patient is diagnosed with severe MPS II. In a specific embodiment, the patient is diagnosed with Hunter syndrome.

[0130] In certain embodiments, a therapeutically effective dose of a recombinant vector is administered to a patient diagnosed with MPS II and identified as responsive to treatment with IDS, e.g., hIDS.

[0131] In certain embodiments, a therapeutically effective dose of a recombinant vector is administered to pediatric patients. In certain embodiments, a therapeutically effective dose of a recombinant vector is administered to patients less than 3 years of age. In certain embodiments, a therapeutically effective dose of a recombinant vector is administered to patients 2 - 4 years of age . In certain embodiments, a therapeutically effective dose of a recombinant vector is administered to patients 4 months or older but less than 5 years of age. In a specific embodiment, a therapeutically effective dose of a recombinant vector is administered to severely ​​​Administer to patients having MPS II of degree and being 4 months or more but less than 5 years old. In certain embodiments administer a therapeutically effective dose of the recombinant vector to patients 18 months or more but less than 8 years old . In a specific embodiment, administer a therapeutically effective dose of the recombinant vector to pediatric male patients who are 18 months or more but less than 8 years old. In certain embodiments, administer a therapeutically effective dose of the recombinant vector to patients 3 to 8 years old. In certain embodiments, administer a therapeutically effective dose of the recombinant vector to patients 8 to 16 years old. In certain embodiments, administer a therapeutically effective dose of the recombinant vector to patients less than 10 years old. In a specific embodiment, administer a therapeutically effective dose of the recombinant vector to patients less than 10 years old having severe MPS II. In certain embodiments, administer a therapeutically effective dose of the recombinant vector to patients over 10 years old. In certain embodiments, administer a therapeutically effective dose of the recombinant vector to adolescent patients. In certain embodiments, administer a therapeutically effective dose of the recombinant vector to adult patients.

[0132] In certain embodiments, administer a therapeutically effective dose of the recombinant vector to patients diagnosed with MPS II and identified as responsive to treatment with IDS, such as hIDS, injected into the CSF prior to gene therapy treatment.

[0133] (5.3.2 Dosage and Administration Mode) In certain embodiments, the therapeutically effective dose of the recombinant vector is administered to the CSF via intrathecal administration (i.e., injected into the subarachnoid space so that the recombinant vector is distributed through the CSF and the cells of the CNS are transduced). This can be done in several ways - for example, by It can be achieved by intracisternal (cisterna magna or ventricle) injection, or injection into the lumbar cistern. In one embodiment, intrathecal administration is performed by intracisternal (IC) injection (e.g., into the cisterna magna). Specifically In one embodiment, intracisternal injection is performed by suboccipital puncture guided by CT. Specifically In one embodiment, intrathecal injection is performed by lumbar puncture. In a specific embodiment, when it seems feasible for the patient, injection into the subarachnoid space is performed by C1-2 puncture. In one embodiment, a therapeutically effective dose of the recombinant vector is administered to the CNS via intranasal administration. In one embodiment, a therapeutically effective dose of the recombinant vector is administered to the CNS by parenchymal injection. In one embodiment, parenchymal injection targets the striatum. In one embodiment, parenchymal injection targets white matter. In one embodiment, a therapeutically effective dose of the recombinant vector is administered to the CSF by any means known in the art, such as any means disclosed in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478 -496, which is incorporated herein by reference in its entirety. For intrathecal administration, a therapeutically effective dose of the recombinant vector should be administered to the CSF with an infusion volume preferably up to about

[0134] 20 mL. A carrier suitable for intrathecal injection, such as Elliotts B solution should be used as a vehicle for the recombinant vector. Elliotts B solution (generic name : sodium chloride, sodium bicarbonate, anhydrous dextrose, magnesium sulfate, potassium chloride, calcium chloride, and sodium phosphate) is a sterile solution that does not contain a bacteriostatic preservative. , a non - pyrogenic, isotonic solution, is used as a diluent for intrathecal administration of chemotherapeutic drugs. .

[0135] In one embodiment, a non - replicating recombinant AAV9 vector expressing human iduronate - 2 - sulfatase (IDS) is used for treatment. In certain embodiments, the IDS expression cassette is flanked by inverted terminal repeats (ITRs) and expression is driven by a hybrid of the cytomegalovirus (CMV) enhancer and the chicken β - actin promoter (CB7). In certain embodiments, the transgene contains a chicken β - actin intron and a rabbit β - globin polyadenylation (polyA) signal. rAAV9.hIDS can be administered IC (by suboccipital injection) as a single fixed dose ranging from about 1.4×10 GC (1.1×10 GC / g brain mass) to 7.0×10 GC (5.6×10 GC / g brain mass) in a volume of about 5 - 20 ml. In this instance, the patient has neutralizing antibodies against AAV and higher ranges of doses may be used.

[0136] 13 10 13 10 10 10

[0137] (5.4 Combination therapy) The combination of administration of HuGlyIDS to the CSF and other available therapies associated therewith is encompassed by the methods of the present invention. Additional therapies can be administered before, simultaneously with, or after the gene therapy treatment. Available MPS II therapies that can be combined with the gene therapy of the present invention include, but are not limited to, idursulfase administered systemically or to the CSF. There is enzyme replacement therapy (ERT) using cerzyme; and / or HSCT therapy. In another embodiment, ERT can be administered using rHuGlyIDS glycoprotein produced in human neural and glial cell lines by recombinant DNA technology . Human neural and glial cell lines that can be used for such recombinant glycoprotein production include, but are not limited to, for example, HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11, or ReNcell VM . To ensure full glycosylation, especially sialylation and tyrosine sulfation, the host cells used in production are engineered to co-express α-2,6-sialyltransferase (or both α-2,3- and α-2,6-sialyltransferases ) as well as the TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation .

[0138] (5.5 Biomarker / Sampling / Monitoring Efficiency) Efficacy can be monitored by measuring cognitive function (e.g., prevention or reduction of neurocognitive decline); reduction of disease biomarkers (e.g., GAG) in CSF and / or serum; and / or increase in IDS enzyme activity in CSF and / or serum . Inflammatory signs and other safety events can also be monitored .

[0139] (5.5.1 Disease Marker) In certain embodiments, the efficacy of treatment with a recombinant vector is monitored by measuring the level of disease biomarker in a patient . In certain embodiments, the level of 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 urine. In certain embodiments, the disease biomarker is GAG. 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.

[0140] (5.5.2 Test of Neurocognitive Function) In certain embodiments, the effectiveness of treatment with the recombinant vector is monitored by measuring the level of the patient's cognitive function. Cognitive function can be measured by any method known to those skilled in the art. In certain embodiments, cognitive function is measured via an approved instrument for measuring the intelligence quotient (IQ). In a specific embodiment, the IQ is measured by the "Wechsler Abbreviated Scale of Intelligence" Second Edition (WASI-II). In certain embodiments, cognitive function is measured via an approved instrument for measuring memory. In a specific embodiment, memory is measured by the Hopkins Verbal Learning Test (HVLT). In certain embodiments, cognitive function is measured via an approved instrument for measuring attention. In a specific embodiment, attention is measured by the Test of Variables of Attention (TOVA). In certain embodiments, cognitive function is measured via an approved instrument for measuring one or more of IQ, memory,

[0141] (5.5.3 Physical Changes) In certain embodiments, the efficacy of treatment with the recombinant vector is monitored by measuring physical characteristics associated with the patient's lysosomal storage defects. In certain embodiments, the physical characteristic is a storage lesion. In certain embodiments, the physical characteristic is short stature. In certain embodiments, the physical characteristic is coarsening of facial features. In certain embodiments, the physical characteristic is obstructive sleep apnea. In certain embodiments, the physical characteristic is hearing impairment. In certain embodiments, the physical characteristic is visual impairment such as. In a specific embodiment, the visual impairment is due to corneal clouding. In certain embodiments, the physical characteristic is hydrocephalus. In certain embodiments, the physical characteristic is spinal cord compression syndrome. In certain embodiments, the physical characteristic is hepatosplenomegaly. In certain embodiments, the physical characteristic is bone and joint deformities. In certain embodiments, the physical characteristic is heart valve disease. In certain embodiments, the physical characteristic is recurrent upper respiratory tract infections such as. In certain embodiments, the physical characteristic is carpal tunnel syndrome. In certain embodiments, the physical characteristic is macroglossia (enlarged tongue). In certain embodiments, the physical characteristic is vocal cord hypertrophy and / or voice change. Such physical characteristics can be measured by any method known to those skilled in the art. (Sequence Listing) [Table 5] TIFF2025087702000027.tif236170TIFF2025087702000028.tif230170TIFF2025087702000029.tif229170TIFF2025087702000030.tif227170TIFF2025087702000031.tif229170TIFF2025087702000032.tif237170TIFF2025087702000033.tif238170TIFF2025087702000034.tif229170TIFF2025087702000035.tif228170TIFF2025087702000036.tif229170TIFF2025087702000037.tif235170TIFF2025087702000038.tif131170

Example

[0142] (6. Example) (6.1 Example 1: hIDS cDNA) The hIDS cDNA-based vector is constructed to contain a transgene containing hIDS (SEQ ID NO: 1). In addition, the transgene contains a nucleic acid containing a signal peptide selected from the group listed in Table 2. Optionally, the vector further contains a promoter.

[0143] (6.2 Example 2: Substituted hIDS cDNA) hIDS having an amino acid substitution, deletion, or addition compared to the hIDS sequence of SEQ ID NO: 1, e.g., non-limitingly, an hIDS cDNA-based vector containing a transgene containing an amino acid substitution selected from the corresponding non-conserved residues in the ortholog of IDS shown in Figure 2 is constructed; provided that such mutations do not include substitution of the cysteine residue (C84) at position 84 required for enzyme activity (Millat et al., 1997, Biochem J 326: 243-247); or alternatively , mutations are, for example, as shown in Figure 3, or each of which is incorporated herein by reference, the literature of Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-76 Incorporated, the literature of Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-76 1(“attenuated” mutants R48P, A85T, W337R, and truncated mutant Q531X; and, “severe” mutants P86L, S333L, S349I, R468Q, R468L reported); the literature of Millat et al., 1998, B BA 1406: 214-218(“attenuated” mutants P480L and P480Q; and, “severe” mutants P86L reported); and, as reported by the literature of Bonucelli et al., 2001, BBA 1537:233-238 such that none of the same mutations identified in severe, severe-moderate, moderate, or attenuated MPS II phenotypes are included. Also, the transgene includes nucleic acids containing a signal peptide selected from the group listed in Table 2. Optionally, the vector further includes a promoter Included. Included. Included.

[0144] (6.3 Example 3: Treatment of MPS II in an animal model using hIDS or substituted hIDS) When expressed as a transgene, the hIDS cDNA-based vector is considered useful for the treatment of MPS II is. An MPS II animal model, for example, the literature of Garcia et al., 2007, J Inherit Meta b Dis 30: 924-34, or the mouse model described in the literature of Muenzer et al., 2001, Acta Paediatr Suppl 91:98-99 The recombinant vector encoding hIDS is administered intrathecally at a dose sufficient to deliver the transgene product and maintain a therapeutically effective concentration in the CSF of the animal maintained. Treatment Thereafter, the animal is evaluated for improvement of symptoms consistent with the disease of a specific animal model.

[0145] (6.1 Example 4: Treatment of MPS II with hIDS or substituted hIDS) When expressed as a transgene, an hIDS cDNA-based vector is useful for the treatment of MPS II is considered to be. To a subject presenting with MPS II, a cDNA-based vector encoding hIDS is administered intrathecally at a dose sufficient to deliver the transgene product and maintain therapeutic concentrations in the CSF (e.g., construct 1 (see below, etc.)). After treatment, the subject is evaluated for improvement of the symptoms of MPS II.

[0146] (6.2 Example 5: Phase I / II multicenter open-label study to evaluate the safety, tolerance, and pharmacokinetics of construct 1 in pediatric subjects with MPS II (Hunter syndrome)) (6.2.1 Overview) (Test article, dose, and route of administration)

[0147] Construct 1: AAV9.CB7.hIDS (recombinant adeno-associated virus serotype 9 capsid containing a human iduronate-2-sulfatase expression cassette). See paragraph

[0019] and Figure 5.

[0148] The product is delivered as a single intrathecal (IC) dose.

[0149] Two dose levels, 1.3×10 10 genome copies (GC) / g brain mass (dose 1) and 6.5×10 10 GC / g brain mass (dose 2) are evaluated. The total dose administered addresses the estimated brain size of the subject based on age. The total volume of the product administered does not exceed 5 mL.

[0150] (Purpose)

[0151] Primary purpose: · To evaluate the safety and tolerability of construct 1 over 24 weeks after a single IC dose administered to pediatric subjects with severe MPS II in the gastrointestinal tract 1.

[0152] Secondary purposes: · To evaluate the long-term safety and tolerability of construct 1. · To evaluate the effect of construct 1 on biomarkers in cerebrospinal fluid (CSF), plasma, and urine · To evaluate the effect of construct 1 on parameters of neurodevelopment of cognitive, behavioral, and adaptive functions · To evaluate the vector shedding into CSF, plasma, and urine

[0153] Exploratory purposes: · To evaluate the immunogenicity of construct 1. · To explore the effect of construct 1 on physiological changes in the CNS · To explore the effect of construct 1 on systemic signs of the disease · To explore the effect of construct 1 on auditory ability · To explore the effect of construct 1 on biomarkers in plasma and urine of subjects who have temporarily interrupted IV ERT (ELAPRASE®) · To explore the effect of construct 1 on quality of life (QOL) and sleep measurements

[0154] (Study design and methodology)

[0155] This is a Phase I / II, first-in-human, multi-center, open-label, single-arm, dose-escalation study of construct 1. There is no control group. Approximately 6 pediatric subjects with severe MPS II are included. ​​​A cohort of pediatric subjects is enrolled at two dose levels, 1.3×10 10 GC / g brain mass (dose 1) or 6.5×10 10 GC / g brain mass (dose 2), and receives a single dose of construct 1 administered by IC injection. Safety is the primary focus for the first 24 weeks post-treatment (primary study period). After completion of the primary study period, subjects are followed for up to 104 weeks total post-treatment with construct 1 for evaluation (safety and efficacy). At the end of the trial, subjects are invited to participate in a long-term follow-up study. The first three eligible subjects are enrolled in the dose 1 cohort (1.3×10 GC / g brain mass). An 8-week observation period for safety is conducted after the first subject receives construct 1. The Internal Safety Committee (ISC) verifies the safety data obtained during the first 8 weeks for this subject (including data obtained at week 8 visit), and if there are no safety concerns, a second subject may be enrolled. The same process is used for enrollment of the third subject. If no Safety Review Trigger (SRT ) events are identified, all available safety data for the dose 1 cohort obtained up to 8 weeks including the week 8 visit for the third subject are evaluated by the Independent Data Monitoring Committee (IDMC). If it is decided to proceed to the second dose (6.5×10

[0156] GC / g brain mass), the subsequent two subjects follow the same dosing scheme as the first dose cohort, and dosing of each subsequent subject is done after all safety data obtained during the first 8 weeks (including data obtained at week 8 visit) for the last dosed subject have been verified. The ISC is the second 10 GC / g brain mass). After the first subject receives construct 1, an 8-week observation period for safety is conducted. The Internal Safety Committee (ISC) verifies the safety data obtained during the first 8 weeks for this subject (including data obtained at week 8 visit), and if there are no safety concerns, a second subject may be enrolled. The same process is used for enrollment of the third subject. If no Safety Review Trigger (SRT events are identified, all available safety data for the dose 1 cohort obtained up to 8 weeks including the week 8 visit for the third subject are evaluated by the Independent Data Monitoring Committee (IDMC). If it is decided to proceed to the second dose (6.5×10 GC / g brain mass), the subsequent two subjects follow the same dosing scheme as the first dose cohort, and dosing of each subsequent subject is done after all safety data obtained during the first 8 weeks (including data obtained at week 8 visit) for the last dosed subject have been verified. The ISC is the second subject to be enrolled. If no Safety Review Trigger (SRT ) events are identified, all available safety data for the dose 1 cohort obtained up to 8 weeks including the week 8 visit for the third subject are evaluated by the Independent Data Monitoring Committee (IDMC). If it is decided to proceed to the second dose (6.5×10 GC / g brain mass), the subsequent two subjects follow the same dosing scheme as the first dose cohort, and dosing of each subsequent subject is done after all safety data obtained during the first 8 weeks (including data obtained at week 8 visit) for the last dosed subject have been verified. The ISC is the second GC / g brain mass), the subsequent two subjects follow the same dosing scheme as the first dose cohort, and dosing of each subsequent subject is done after all safety data obtained during the first 8 weeks (including data obtained at week 8 visit) for the last dosed subject have been verified. The ISC is the second 10 GC / g brain mass), the subsequent two subjects follow the same dosing scheme as the first dose cohort, and dosing of each subsequent subject is done after all safety data obtained during the first 8 weeks (including data obtained at week 8 visit) for the last dosed subject have been verified. The ISC is the second subject to be enrolled. If no Safety Review Trigger (SRT events are identified, all available safety data for the dose 1 cohort obtained up to 8 weeks including the week 8 visit for the third subject are evaluated by the Independent Data Monitoring Committee (IDMC). If it is decided to proceed to the second dose (6.5×10 GC / g brain mass), the subsequent two subjects follow the same dosing scheme as the first dose cohort, and dosing of each subsequent subject is done after all safety data obtained during the first 8 weeks (including data obtained at week 8 visit) for the last dosed subject have been verified. The ISC is the second Verify all safety data of all subjects obtained in a maximum of two weeks, including the subject's second visit, and Determine that it is safe to proceed to dosing the third subject immediately after this evaluation. All available safety data regarding dose cohort 2 will be evaluated by the IDMC after the eighth visit of the third subject in dose cohort 2.

[0157] Potential subjects will be screened for a maximum of 35 days prior to dosing to determine their eligibility for this trial. Those subjects who meet the eligibility criteria will be admitted (in accordance with the facility's practice) between Day - 2 and the morning of Day 1 and undergo baseline evaluations prior to dosing. Subjects will receive a single IC dose of compound 1 on Day 1 and will remain in the hospital for observation for approximately 30 - 36 hours after dosing. Subsequent evaluations during the main study period (i.e., up to Week 24) will be conducted weekly up to Week 4 and at Weeks 8, 12, 16, 20, and 24. After the main study period, subjects will visit the hospital at Weeks 28, 32, 40, 48, 52, 56, 64, 78, and 104. Visits at Weeks 12, 40, and 64 may be conducted by a visiting nurse. Evaluations at Weeks 20 and 28 will be limited and will assess AEs and concomitant medications by telephone.

[0158] All subjects will initially receive an immunosuppressant (IS) in this trial based on potential immunogenicity findings in non - clinical safety / toxicity tests conducted in animals. This is corticosteroid (methylprednisolone 10 mg / kg, IV, once before dosing on Day 1 and oral prednisone starting at 0.5 mg / kg / day on Day 2, tapered, and discontinued by Week 12) and tacrolimus (1 mg PO twice daily [BID] starting on Day 2 until Week 24, with blood levels ​​​​​​​​​​​ Targeting 8 ng / mL, during weeks 24 to 32, gradually reduce over 8 weeks), and sirolimus (on the first day of the month, loading dose 1 mg / m 2 administered 3 times every 4 hours; then from the first day of the previous month, sirolimus 0.5 mg / m 2 / day, divided and administered BID, targeting a blood level of 4 - 8 ng / ml by week 48) including . Perform neurological evaluations and tacrolimus / sirolimus blood level monitoring according to Table 3 . Adjust the doses of sirolimus and tacrolimus to maintain blood levels within the target range .

[0159] Do not plan IS therapy after week 48. If IS is required after week 48 to manage clinically relevant immune responses, the principal investigator (PI), in consultation with the medical monitor and the sponsor, will determine an appropriate immunosuppressive regimen as clinically indicated .

[0160] Efficacy evaluations include measurement of neurocognitive function, auditory ability, brain MRI, liver and spleen size, and levels of pharmacodynamic (PD) biomarkers in CSF, plasma, and urine. Neurocognitive or adaptive scales are part of the subject's standard care while also collecting participation in this trial as determined by the sponsor after discussion at the facility .

[0161] (Endpoint)

[0162] Primary endpoint: · Safety up to week 24: AE and serious adverse events (SAE).

[0163] Secondary endpoints: ​​·Safety up to Week 104: AE reports, clinical laboratory evaluations, vital signs, ECG, physical findings, and neurological evaluations. ·Biomarkers in CSF (GAG, I2S activity), plasma (GAG, I2S activity), and urine (GAG). ·Parameters of neurodevelopment of cognition, behavior, and adaptive function: ○Bayley Scales of Infant Development, Third Edition (BSID-III) (Bayley reference, 2005) or Kaufman Assessment Battery for Children, Second Edition (KABC-II) (Kaufman reference, 2004) ○Vineland Adaptive Behavior Scales, Second Edition, Comprehensive Interview Form (VABS-II) (Sparro w et al. reference, 2005). ·Quantitative polymerase chain reaction (PCR) for construct 1 deoxyribonucleic acid (DNA) vector concentrations in CSF, plasma, and urine.

[0164] Exploratory endpoints: ·Immunogenicity measurements ○Neutralizing antibody titers against AAV9 and binding antibody titers against I2S in CSF and serum ○Enzyme-linked immunospot (ELISPOT) assay: T-cell responses against AAV9 and I2S ○Flow cytometry: AAV- and I2S-specific regulatory T cells. ·CNS structural abnormalities evaluated by magnetic resonance imaging (MRI) of the brain. ·Liver and spleen sizes evaluated by abdominal MRI and ultrasound. ·Changes in auditory ability measured by auditory brainstem response (ABR) testing. ·Plasma and urine GAG in subjects temporarily off IV ERT (ELAPRASE®). ·PedsQL (version 4). ·Overall sleep impression scale.

[0165] The entire duration of this trial is 104 weeks after administration, and the main safety assessment point is 24 weeks. S Cleaning is carried out for a maximum of 35 days.

[0166] (Diagnostic and Selection and Exclusion Criteria)

[0167] To be eligible to participate in this trial, the subject must meet all of the following selection criteria: otherwise: 1. The subject's legal guardian provides a signed informed consent in writing after receiving an explanation of the nature of this trial and before any procedure related to the research, and has the ability to do so. 2. Be male. 3. Meet one of the following criteria: a. Have a diagnosis according to the MPS II document, and be over 4 months and under 5 years old, and neurocognitive test score is >55 to ≦77 (BSID-III or KABC-II), or b. Have a diagnosis according to the MPS II document, and be over 4 months and under 5 years old, and continuous neurocognitive test (BSID-III or KABC-II) has a decline of standard deviation ≧1 and a test score >55, or c. Have a relative diagnosed with severe MPS II with the same IDS mutation as the subject, and genetics in the opinion of the geneticist, severe MPS II is inherited. 4. Have sufficient auditory and visual abilities to complete the required protocol tests regardless of the need for assistance, and comply with the wearing of assistive devices if applicable on the test day.

[0168] ​​​​Subjects meeting any of the following exclusion criteria are not eligible to participate in this study: 1. Include any of the following and IC injection is contraindicated: a. Verification of the baseline MRI test by the team of neuroradiologists / neurosurgeons (one per facility) participating in the study indicates that IC injection is contraindicated. b. History of head / neck surgery that results in contraindication of IC injection based on verification of available information by the team of neuroradiologists / neurosurgeons participating in the study. c. Have any contraindication to computed tomography (CT), contrast agents, or general anesthesia. d. Have any contraindication to MRI or gadolinium. e. Have an estimated glomerular filtration rate (eGFR) < 30 mL / min / 1.73m 2 . 2. Have any condition that is contraindicated for treatment with prednisone, tacrolimus, or sirolimus. 3. Have a diagnosis of any neurocognitive impairment not caused by MPS II, or any neuropsychiatric condition that may confound the interpretation of study results according to the opinion of the PI. 4. Have any contraindication to lumbar puncture. 5. Have a ventricular shunt. 6. Have received hematopoietic stem cell transplantation (HSCT). 7. Have previously received treatment with an AAV-based gene therapy product. 8. Have received idursulfase [ELAPRASE (registered trademark)] by intrathecal (IT) administration. 9. Have received idursulfase [ELAPRASE (registered trademark)] IV and have experienced a severe allergic reaction including anaphylaxis considered to be related to idursulfase [ELAPRASE (registered trademark)] IV administration. 10. Have received any investigational drug for a period of either within 30 days from Day 1 or five times the half-life, whichever is longer, prior to signing the Informed Consent Form (ICF). 11. Have a history of lymphoma that has not been in complete remission for at least 3 months prior to screening, or a history of another cancer other than squamous cell or basal cell carcinoma of the skin. 12. Have a platelet count < 100,000 / microliter (μL). 13. Have a known history of Gilbert's syndrome and, except when having fractionated bilirubin with conjugated bilirubin < 35% of total bilirubin, have alanine aminotransferase (ALT) or aspartate aminotransferase (AST) > 3 × ULN or total bilirubin > 1.5 × ULN at screening. 14. Uncontrolled hypertension (systolic blood pressure [BP] > 180 mmHg, diastolic BP > 100 mmHg) despite maximum medical treatment. 15. Have a history of human immunodeficiency virus (HIV) or hepatitis B or C virus infection, or have a positive screening test for hepatitis B surface antigen or hepatitis B core antibody, or hepatitis C or HIV antibodies. 16. Be a first-degree relative of an employee of the clinical facility or any other individual involved in the conduct of the trial, or be an employee of the clinical facility or any other individual involved in the conduct of the trial. 17. Have clinically significant ECG abnormalities that compromise the safety of the subject in the opinion of the PI. 18. Have a severe or unstable medical or mental condition that compromises the safety of the subject, the success of participation in this trial, or the interpretation of the trial results in the opinion of the PI. ​​​​​​​​​​​19. In the opinion of the PI, has seizures that cannot be managed without exposing the subject to excessive risk. Exclusion criteria related to immunosuppressive therapy: 20. Has a history of hypersensitivity to tacrolimus, sirolimus, or prednisone . 21. Has a history of primary immunodeficiency (e.g., unclassified immunodeficiency syndrome), splenectomy, or a condition that predisposes the subject to infections . 22. Has herpes zoster (VZV), cytomegalovirus (CMV), or Epstein - Barr virus (EBV) infection that has not completely resolved at least 12 weeks before screening . 23. Has an infection that requires treatment with hospitalization or parenteral anti - infectious agents and has not resolved at least 8 weeks before the second visit . 24. Has an active infection that requires oral anti - infectious agents (including antiviral agents) within 10 days before the second visit . 25. Has a history of active tuberculosis (TB) or a positive Quantiferon - TB Gold test at screening . 26. Live vaccine inoculation within 8 weeks before ICF signature. 27. Major surgery within 8 weeks before ICF signature or major surgery planned during the study period. 28. Prediction of the need for adenoidectomy or tonsillectomy within 6 months of enrollment. 29. Has an absolute neutrophil count < 1.3×10 3 / μL. 30. Has an abnormality in a condition or clinical examination that the PI considers inappropriate for immunosuppressive therapy.

[0169] (Statistical analysis method)

[0170] All data are presented in the subject data list. Categorical variables are presented using frequencies and percentages Summarize continuous variables using descriptive statistics (n, mean, standard deviation, median, minimum value, and maximum value). Present graphical displays as necessary. Report safety and PD endpoints by dose group and also report for 2 dose groups combined together.

[0171] Sample size and power calculation: No formal calculation was performed to determine the sample size . (6.2.2 Abbreviations and terms)

Table 6

[0172] (6.2.3 Study design) (Endpoints)

[0173] Primary endpoint: · Safety up to Week 24: AE and SAE.

[0174] Secondary endpoints: · Safety up to Week 104: AE reporting, clinical laboratory evaluations, vital signs, electrocardiogram (ECG), physical findings, and neurological evaluations. · Biomarkers in CSF (GAG, I2S activity), plasma (GAG, I2S activity), and urine (GAG). · Neurodevelopmental parameters of cognition, behavior, and adaptive function: ○ Bayley Scales of Infant Development, Third Edition (BSID-III) (Bayley reference, 2005) or Kaufman Assessment Battery for Children, Second Edition (KABC-II) (Kaufman reference, 2004) ○Vineland Adaptive Behavior Scale, Second Edition, Comprehensive Interview Form (VABS-II) (Sp arrow et al., 2005). · Construct 1 Quantification of vector concentration in CSF, plasma , and urine by quantitative polymerase chain reaction (PCR) for DNA.

[0175] Exploratory endpoints: · Immunogenicity measurements ○ Neutralizing antibody titers against AAV9 and binding antibody titers against I2S in CSF and serum ○ Enzyme-linked immunospot (ELISPOT) assay: T-cell responses against AAV9 and I2S ○ Flow cytometry: AAV- and I2S-specific regulatory T cells. · CNS structural abnormalities evaluated by brain MRI. · Sizes of the liver and spleen evaluated by abdominal MRI. · Changes in auditory ability measured by auditory brainstem response (ABR) testing. · Plasma and urinary GAG in subjects temporarily off IV ERT (ELAPRASE®) . · PedsQL (version 4). · Overall sleep impression scale.

[0176] (Study design)

[0177] This is a Phase I / II, first-in-human, multi-center open-label single-arm dose-escalation study of Construct 1. Approximately 6 pediatric subjects with severe MPS II will be enrolled into 2 dose cohorts, 1.3×10 GC / g brain mass (dose 1) or 6.5×10 10 GC / g brain mass (dose 2) and receive a single dose of Construct 1 administered by IC injection. Safety will be evaluated 10 after treatment and Focus on the first 24 weeks (primary study period). After completion of the primary study period, subjects will continue to be evaluated (for safety and efficacy) for up to 104 weeks in total after treatment with construct 1. At the end of the trial, subjects will be invited to participate in a long-term follow-up study.

[0178] Potential subjects will be screened for up to 35 days prior to dosing to determine their eligibility for this trial. Those subjects who meet the eligibility criteria will be admitted (in accordance with the facility's practice) between Day - 2 and the morning of Day 1 and have a baseline evaluation performed prior to dosing. Subjects will receive a single IC dose of construct 1 on Day 1 and will stay in the hospital for observation for approximately 30 - 36 hours after dosing. Subsequent evaluations during the primary study period (i.e., up to Week 24) will be performed weekly up to Week 4 and at Weeks 8, 12, 16, 20, and 24. After the primary study period, subjects will visit the hospital at Weeks 28, 32, 40, 48, 52, 56, 64, 78, and 104. Visits at Weeks 12, 40, and 64 may be performed by a visiting nurse. The evaluations at Weeks 20 and 28 will be limited and will assess AEs and concomitant medications by telephone. All subjects will receive an initial IS in this trial based on the findings in non-clinical studies. The IS therapy consists of corticosteroids (methylprednisolone 10 mg / kg, IV, once before dosing on Day 1 and oral prednisone starting at 0.5 mg / kg / day on Day 2, tapered, and discontinued by Week 12), tacrolimus (oral [PO] 1 mg twice daily [BID] on Day 2, targeting a blood level of 4 - 8 ng / mL up to Week 24 and tapered over 8 weeks between Weeks 24 and 32), and sirolimus ( starting at Week 2).

[0179] All subjects will receive an initial IS in this trial based on the findings in non-clinical studies. The IS therapy consists of corticosteroids (methylprednisolone 10 mg / kg, IV, once before dosing on Day 1 and oral prednisone starting at 0.5 mg / kg / day on Day 2, tapered, and discontinued by Week 12), and tacrolimus (oral [PO] 1 mg twice daily [BID] on Day 2, targeting a blood level of 4 - 8 ng / mL up to Week 24 and tapered over 8 weeks between Weeks 24 and 32), and sirolimus ( starting at Week 2). targeting a blood level of 4 - 8 ng / mL up to Week 24 and tapered over 8 weeks between Weeks 24 and 32), and sirolimus ( - On the second day, a loading dose of 1 mg / m 2 is administered three times every 4 hours, and then from the first day before: sirolimus 0. 5 mg / m 2 / day is administered twice a day in divided doses, aiming for a blood level of 4 - 8 ng / ml by week 48 ). Neurological evaluation and blood level monitoring of tacrolimus / sirolimus are performed according to Table 3 . The doses of sirolimus and tacrolimus are adjusted to maintain the blood level within the target range .

[0180] IS therapy is not planned after week 48. If IS is still required after week 48 to manage clinically relevant immune responses, the principal investigator (PI) of the clinical trial will consult with the follow-up observer and the sponsor of the clinical trial to determine a suitable immunosuppressive regimen as clinically indicated .

[0181] The safety and tolerability of construct 1 are monitored through physical findings including AE and serious adverse events (SAE), chemistry, hematology assessment , urinalysis, CSF inflammation markers, immunogenicity, vector shedding (vector concentration), vital signs, electrocardiogram (ECG), and neurological evaluation .

[0182] Efficacy evaluation includes measurement of neurocognitive function and adaptive function, auditory ability, brain MRI, liver and spleen size , and levels of PD biomarkers in CSF, plasma, and urine

[0183] (6.2.4 Target Population and Selection) (Selection of the Test Population)

[0184] Approximately 6 pediatric subjects, aged 4 months to less than 5 years, with a documented neurocognitive impairment due to MPS II or a genotype and family history consistent with the severe genetic form of MPS II are included It is treated with the investigational product (IP).

[0185] (Selection Criteria)

[0186] To be eligible to participate in this trial, subjects must meet all of the following inclusion criteria: otherwise: 1. The subject's legal guardian provides written, signed informed consent after receiving an explanation of the nature of the trial and before any procedures related to the research, and has the ability to do so. 2. Be male. 3. Meet one of the following criteria: a. Have a diagnosis of MPS II according to the documentation, and be older than 4 months and younger than 5 years, and and have a neurocognitive test score of >55 to ≤77 (BSID-III or KABC-II), or b. Have a diagnosis of MPS II according to the documentation, and be older than 4 months and younger than 5 years, and have a decline in the continuous neurocognitive test (BSID-III or KABC-II) with a standard deviation ≥1 and a test score >55 or or c. Have a relative diagnosed with severe MPS II with the same IDS mutation as the subject, and in the opinion of a geneticist, the severe form of MPS II is inherited. 4. Have sufficient auditory and visual capabilities to complete the required protocol tests regardless of the need for assistance, and comply with wearing assistive devices if applicable on the test day.

[0187] (Exclusion Criteria)

[0188] Subjects who meet any of the following exclusion criteria are not eligible to participate in this trial:​​​​​​ 1. Inclusion of any of the following, where IC injection is contraindicated: a. Verification by a team of neuroradiologists / neurosurgeons (one per site) participating in the trial that baseline line MRI testing indicates that IC injection is contraindicated. b. History of head / neck surgery that would contraindicate IC injection, based on verification of available information by a team of neuroradiologists / neurosurgeons participating in the trial. c. Any contraindication to computed tomography (CT), contrast agents, or general anesthesia. d. Any contraindication to MRI or gadolinium. e. Estimated glomerular filtration rate (eGFR) < 30 mL / min / 1.73m having. e. Having an estimated glomerular filtration rate (eGFR) < 30 mL / min / 1.73m 2 2. Having any condition that is a contraindication to treatment with prednisone, tacrolimus, or sirolimus. 2. Having any condition that is a contraindication to treatment with prednisone, tacrolimus, or sirolimus. 3. Having a diagnosis of any neurocognitive disorder not caused by MPS II, or any neuropsychiatric condition that could confound the interpretation of the trial results in the opinion of the PI. 3. Having a diagnosis of any neurocognitive disorder not caused by MPS II, or any neuropsychiatric condition that could confound the interpretation of the trial results in the opinion of the PI. 4. Having any contraindication to lumbar puncture. 4. Having any contraindication to lumbar puncture. 5. Having a ventriculoperitoneal shunt. 6. Having received hematopoietic stem cell transplantation (HSCT). 7. Having previously received treatment with an AAV-based gene therapy product. 8. Having received idursulfase by intrathecal (IT) administration. 9. Having received idursulfase [ELAPRASE (R)] IV and having experienced a severe hypersensitivity reaction, including anaphylaxis, considered to be related to idursulfase [ELAPRASE (R)] IV administration. 9. Having received idursulfase [ELAPRASE (R)] IV and having experienced a severe hypersensitivity reaction, including anaphylaxis, considered to be related to idursulfase [ELAPRASE (R)] IV administration. 10. Patients who have received any investigational drug within 30 days of Day 1 or within a period five times the half-life, whichever is longer, prior to signing the informed consent form (ICF). 10. Patients who have received any investigational drug within 30 days of Day 1 or within a period five times the half-life, whichever is longer, prior to signing the informed consent form (ICF). 10. Patients who have received any investigational drug within 30 days of Day 1 or within a period five times the half-life, whichever is longer, prior to signing the informed consent form (ICF). 11. A history of lymphoma that has not been in complete remission for at least 3 months prior to screening, or a history of another cancer other than squamous or basal cell carcinoma of the skin. 12. Having a platelet count < 100,000 / microliter (μL). 13. Except when the subject has a history of Gilbert's syndrome and has fractionated bilirubin with conjugated bilirubin < 35% of total bilirubin, at the time of screening, having alanine aminotransferase (ALT) or aspartate aminotransferase (AST) > 3 × ULN or total bilirubin > 1.5 × ULN. 14. Uncontrolled hypertension (systolic blood pressure [BP] > 180 mmHg, diastolic BP > 100 mmHg) despite maximum medical treatment. 15. A history of human immunodeficiency virus (HIV) or hepatitis B or C virus infection, or a positive screening test for hepatitis B surface antigen or hepatitis B core antibody, or hepatitis C or HIV antibody. 16. Being a first-degree relative of an employee of the clinical facility or any other individual involved in the conduct of the trial, or being an employee of the clinical facility or any other individual involved in the conduct of the trial. 17. Having clinically significant ECG abnormalities that compromise the safety of the subject in the opinion of the PI. 18. Having a severe or unstable medical or mental condition that compromises the safety of the subject or the success of participation in the trial or the interpretation of the trial results in the opinion of the PI. 19. Having unmanageable seizures that expose the subject to excessive risk in the opinion of the PI. Exclusion criteria related to immunosuppressive therapy: 20. Having a history of hypersensitivity reaction to tacrolimus, sirolimus, or prednisone 。 21. Have a history of primary immunodeficiency (e.g., unclassified immunodeficiency syndrome), splenectomy, or a condition that predisposes the subject to infection. 22. Have herpes zoster (VZV), cytomegalovirus (CMV), or Epstein - Barr virus (EBV) infection that has not completely resolved at least 12 weeks before screening. 23. Have an infection that requires treatment with hospitalization or parenteral anti - infectious agents and has not resolved at least 8 weeks before the second visit. 24. Have an active infection that requires oral anti - infectious agents (including antiviral agents) within 10 days before the second visit. 25. Have a history of active tuberculosis (TB) or a positive Quantiferon - TB Gold test at the time of screening. 26. Live vaccine inoculation within 8 weeks before ICF signature. 27. Major surgery within 8 weeks before ICF signature or major surgery planned during the study period. 28. Prediction of the need for adenoidectomy or tonsillectomy within 6 months of enrollment. 29. Have an absolute neutrophil count < 1.3×10 3 / μL. 30. Have an abnormality in a condition or clinical test such that the PI considers the subject not suitable for immunosuppressive therapy.

[0189] (6.2.5 Treatment) (Treatment Administration)

[0190] Construct 1 (see Figure 5), which is the investigational product (IP), is administered as a single - dose IC. Two dose levels: 1.3×10 10 GC / g brain mass (dose 1) or 6.5×10 10 GC / g brain mass (dose 2). The total dose administered (total GC) is adjusted to account for differences in brain size by age. Administered ​​​​​​ The total volume of the resulting product does not exceed 5 mL.

[0191] The reference therapy is not administered during this trial period. The IS therapy is given in addition to the IP, as described below. as described below.

[0192] (Investigational Product)

[0193] Construct 1 is a non-replicating recombinant AAV of serotype 9 capsid containing an hIDS expression cassette. See paragraph

[0019] . [Table 7] AAV = adeno-associated virus; CB = chicken beta-actin; hIDS = human iduronate-2-sulfatase sulfatase

[0194] Construct 1 is a non-replicating recombinant AAV9 vector that can efficiently express a human iduronate-2-sulfatase (hIDS) product in the central nervous system (CNS) after intrathecal (IT) administration. The vector genome contains an hIDS expression cassette adjacent to the AAV2 inverted terminal repeat (ITR). Expression from the cassette is driven by the CB7 promoter, which is a hybrid between the cytomegalovirus (CMV) immediate early enhancer and the chicken beta-actin promoter. Transcription from this promoter is enhanced by the presence of the chicken beta-actin intron (CI). The polyadenylation signal of the expression cassette is derived from the rabbit beta-globin (RBG) gene. A schematic representation of Construct 1 is illustrated in Figure 5.

[0195] The final IP is filled into a 2 mL CRYSTAL ZENITH (R) (CZ) vial and latex ​​A 0.001% Pluronic (registered trademark) F68-sealed freeze solution of AAV vector active ingredient (AAV 9.CB7.hIDS) in modified Elliotts B (registered trademark) solution, sealed with a free rubber stopper and an aluminum flip-off seal. The vial must be stored at ≤ -60 °C. The concentration (in GC / mL) of each IP lot is reported in the Certificate of Analysis (CoA). Detailed dosing instructions based on the product concentration are provided in the administration manual.

[0196] (Immunosuppressive therapy)

[0197] (Corticosteroid) · In the morning on the day of vector administration (Day 1, pre-dose), the subject receives 10 mg / kg (maximum 500 mg) of methylprednisolone IV over at least 30 minutes. Methylprednisolone should be administered before IP lumbar puncture and IC injection. Premedication with acetaminophen and antihistamines is optional at the discretion of the principal investigator of the clinical trial. · Aim to discontinue prednisone by week 12. Start oral prednisone on Day 2. The dosage of prednisone is as follows: ○ From Day 2 to the end of week 2: 0.5 mg / kg / day ○ Weeks 3 and 4: 0.35 mg / kg / day ○ Weeks 5 to 8: 0.2 mg / kg / day ○ Weeks 9 to 12: 0.1 mg / kg. · Prednisone is discontinued after week 12. The exact dosage of prednisone can be adjusted to a clinically practical dosage in a stepwise manner.

[0198] (Sirolimus) · Two days before vector administration (Day -2): 1 mg / m every 4 hours 2 ​​​​​​​​​Loading dose of sirolimus at 3× the dose is administered. · From Day - 1: Sirolimus at a dose of 0.5 mg / m 2 / day, divided into twice - daily dosing to achieve a target blood level of 4 - 8 ng / ml. · Sirolimus is discontinued after the visit at Week 48.

[0199] (Tacrolimus) · Tacrolimus is started at a dose of 1 mg / kg twice - daily on Day 2 (the day after IP administration) and adjusted to achieve a blood level of 4 - 8 ng / mL over 24 weeks. · Tacrolimus is started at the visit in Week 24 and tapered over 8 weeks. At Week 24, the dose is reduced by approximately 50%. At Week 28, the dose is further reduced by approximately 50%. Tacro limus is discontinued at Week 32. · Monitoring of the blood levels of tacrolimus and sirolimus is performed according to Table 3. Dose adjustment is considered in paragraphs

[0220] -

[0222] .

[0200] (Method of allocating subjects to treatment)

[0201] Eligible subjects are registered and sequentially allocated to dose cohorts. The first 3 subjects are allocated to receive 1.3×10 10 GC / g brain mass; the next 3 subjects are allocated to receive 6.5×10 GC / g brain mass while the verification of safety data by the IDMC remains undetermined. 10

[0202] (Considerations on dosing)

[0203] (Investigational product)

[0204] Safety data among individual subjects and after each cohort has been dosed at any dose level ​​​Regarding the description of the plan for sequential dosing subjects, including verification, refer to paragraphs

[0175] to

[0186] . Please refer.

[0205] (Immunosuppressive therapy)

[0206] Prednisone dosing starts at 0.5 mg / kg / day and tapers off by the 12th week of the hospital visit.

[0207] Tacrolimus dosage adjustment is to maintain the trough concentration in whole blood within 4 - 8 ng / mL over the first 24 weeks. At the 24th week, the dosage is reduced by approximately 50%. At the 28th week, the dosage is further reduced by approximately 50%. Tacrolimus is discontinued at the 32nd week. Sirolimus dosage adjustment is to maintain the trough concentration in whole blood within 4 - 8 ng / mL. For most subjects, dosage adjustment can be based on the formula: new dosage = current dosage × (target concentration / current concentration). Subjects should continue the new maintenance dosage for at least 7 - 14 days while monitoring the concentration before further adjusting the dosage. For most of the subjects, the dosage adjustment can be based on the formula: new dosage = current dosage × (target concentration / current concentration). The subjects should continue the new maintenance dosage for at least 7 - 14 days while monitoring the concentration before further adjusting the dosage. For most of the subjects, the dosage adjustment can be based on the formula: new dosage = current dosage × (target concentration / current concentration). The subjects should continue the new maintenance dosage for at least 7 - 14 days while monitoring the concentration before further adjusting the dosage. Subjects should continue the new maintenance dosage for at least 7 - 14 days while monitoring the concentration before further adjusting the dosage. Subjects should continue the new maintenance dosage for at least 7 - 14 days while monitoring the concentration before further adjusting the dosage.

[0208] The following dosing and procedures are prohibited: · IT ERT is not permitted within 6 months of screening. · Any investigational product at any time before signing the ICF or during the trial period (through 104 weeks), for either 30 days or 5 times the half-life, whichever is longer. · When on sirolimus and / or tacrolimus treatment, live vaccines must be avoided. · When on sirolimus and / or tacrolimus treatment, live vaccines must be avoided. . · Strong inhibitors of CYP3A4 and / or P-glycoprotein (PgP) (ketoconazole, voriconazole, itraconazole, posaconazole, erythromycin, telithromycin, or clarithromycin). Larisomycin, etc.) or a strong inducer of CYP3A4 and / or Pgp (rifampin or rifabutin, etc.) must be avoided during sirolimus and / or tacrolimus treatment. · Grapefruit juice inhibits CYP3A-enzymes and results in increased whole blood trough concentrations of tacrolimus and sirolimus. Subjects must avoid eating grapefruit or drinking grapefruit juice during treatment with tacrolimus and / or sirolimus. · Grapefruit juice inhibits CYP3A-enzymes and results in increased whole blood trough concentrations of tacrolimus and sirolimus. Subjects must avoid eating grapefruit or drinking grapefruit juice during treatment with tacrolimus and / or sirolimus. · Grapefruit juice inhibits CYP3A-enzymes and results in increased whole blood trough concentrations of tacrolimus and sirolimus. Subjects must avoid eating grapefruit or drinking grapefruit juice during treatment with tacrolimus and / or sirolimus.

[0209] (Approved Dosage and Procedures)

[0210] Subjects are permitted to continue with a stable regimen of IV ERT and any supportive measurements (e.g., physical therapy). In accordance with the standard care of local hospitals, subjects are permitted to receive medications to prevent claustrophobia during MRI, and to receive general anesthesia for lumbar puncture, MRI, and nerve conduction tests (ABR or sensory evoked potential). Subjects are permitted to continue with a stable regimen of IV ERT and any supportive measurements (e.g., physical therapy). In accordance with the standard care of local hospitals, subjects are permitted to receive medications to prevent claustrophobia during MRI, and to receive general anesthesia for lumbar puncture, MRI, and nerve conduction tests (ABR or sensory evoked potential). Subjects are permitted to continue with a stable regimen of IV ERT and any supportive measurements (e.g., physical therapy). In accordance with the standard care of local hospitals, subjects are permitted to receive medications to prevent claustrophobia during MRI, and to receive general anesthesia for lumbar puncture, MRI, and nerve conduction tests (ABR or sensory evoked potential). Subjects are permitted to continue with a stable regimen of IV ERT and any supportive measurements (e.g., physical therapy). In accordance with the standard care of local hospitals, subjects are permitted to receive medications to prevent claustrophobia during MRI, and to receive general anesthesia for lumbar puncture, MRI, and nerve conduction tests (ABR or sensory evoked potential).

[0211] Medications other than those described above, considered necessary for the safety and well-being of the subject (e.g., for hypertension), are given at the discretion of the study physician in accordance with the standard care of local hospitals and recorded in the appropriate CRF items. Medications other than those described above, considered necessary for the safety and well-being of the subject (e.g., for hypertension), are given at the discretion of the study physician in accordance with the standard care of local hospitals and recorded in the appropriate CRF items. Medications other than those described above, considered necessary for the safety and well-being of the subject (e.g., for hypertension), are given at the discretion of the study physician in accordance with the standard care of local hospitals and recorded in the appropriate CRF items.

[0212] (Equivalents) Although the present invention has been described in detail with reference to its specific embodiments, it will be understood that modifications which are functionally equivalent are within the scope of the present invention. Indeed, various modifications of the invention shown and described herein will be apparent to those skilled in the art from the foregoing specification and attached drawings. Such modifications are within the scope of the appended claims. Although the present invention has been described in detail with reference to its specific embodiments, it will be understood that modifications which are functionally equivalent are within the scope of the present invention. Indeed, various modifications of the invention shown and described herein will be apparent to those skilled in the art from the foregoing specification and attached drawings. Such modifications are within the scope of the appended claims. is intended to be. One of ordinary skill in the art will recognize or be able to use and confirm many equivalents to the specific embodiments of the invention described herein within the scope of routine experimentation. Such equivalents are intended to be encompassed by the following claims. Many equivalents to the specific embodiments of the invention described herein will be recognized or can be used and confirmed within the scope of routine experimentation. Such equivalents are intended to be encompassed by the following claims. It is possible. Such equivalents are intended to be encompassed by the following claims.

[0213] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated as being incorporated herein by reference. As if each individual publication, patent, or patent application were specifically and individually indicated as being incorporated herein by reference, they are hereby incorporated by reference into this specification to the same extent. into this specification.

Claims

1. Glycosylated recombinant human antibodies produced by human neuronal or glial cells Precursor of duronate-2-sulfatase (IDS).

2. The glycosylation product of claim 1, which is determined to be about 90 kDa by polyacrylamide gel electrophoresis. lysed recombinant human IDS precursor.

3. It was determined to be approximately 90 kDa by polyacrylamide gel electrophoresis and contains formylglycine. , α2,6-sialylated, contains no detectable NeuGc, and contains no detectable α-Gal antigen and / or mannose-6-phosphorylated. Human IDS precursor.

4. Cells in the central nervous system genetically engineered to secrete the human IDS glycoprotein precursor The glycosylated recombinant protein according to any one of claims 1 to 3, which is secreted from a cellular depot. Human IDS precursor.

5. The glycosylated recombinant human IgG1 vector according to claim 4, wherein the depot is formed in the brain of a human subject. Human IDS precursor.

6. Any one of claims 1 to 5, wherein the human neuronal or human glial cell is deficient in IDS activity. A glycosylated recombinant human IDS precursor according to claim 1.

7. The glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO:

1. A glycosylated recombinant human IDS precursor according to any one of claims 1 to 6.

8. 1. A method of treating a human subject diagnosed with Mucopolysaccharidosis Type II (MPS II), comprising: Into the cerebrospinal fluid (CSF) of the human subject, a granulocyte produced by human neuronal cells or human glial cells is administered. Delivering therapeutically effective amounts of glycosylated recombinant human iduronate-2-sulfatase (IDS) precursor The method comprising:

9. The glycosylated recombinant human IDS precursor was subjected to polyacrylamide gel electrophoresis. The method of claim 8, wherein the nucleic acid is measured to be about 90 kDa or less.

10. The glycosylated recombinant human IDS precursor was subjected to polyacrylamide gel electrophoresis. It is approximately 90 kDa, contains formylglycine, is α2,6-sialylated, and is detectable contains no significant NeuGc, no detectable α-Gal antigen, and / or is mannose-6-phosphorylated.

9. The method of claim 8, wherein

11. The glycosylated recombinant human IDS precursor is secreted from a depot of cells in the central nervous system that have been genetically engineered to secrete precursors, The method according to any one of claims 8 to 10.

12. The method of claim 11 , wherein the depot is formed in the brain of a human subject.

13. The method of any one of claims 8 to 12, wherein the human subject is deficient in IDS activity.

14. The glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO:

1. The method of any one of claims 8 to 13.

15. A recombinant nucleotide expression vector encoding a human IDS is administered into the CSF of the human subject.

1. A method of treating a human subject diagnosed with MPS II comprising administering to a subject a therapeutically effective amount of: wherein the recombinant nucleotide expression vector is used to transduce primary human neuronal cells in culture. When used to measure the molecular weight of the protein, it is estimated to be approximately 90 kDa by polyacrylamide gel electrophoresis. , contains formylglycine, is α2,6-sialylated, contains no detectable NeuGc, and is secreted, free of possible α-Gal antigens and / or mannose-6-phosphorylated The method leads to the expression of a glycosylated human IDS precursor.

16. A recombinant nucleotide expression vector encoding a human IDS is administered into the CSF of the human subject. The resulting depot was determined to be approximately 90 kDa by polyacrylamide gel electrophoresis. Contains formylglycine, is α2,6-sialylated, contains no detectable NeuGc, and is detectable glycosylated, does not contain a competent α-Gal antigen, and / or is mannose-6-phosphorylated The results showed that the pulmonary pulmonary edema (PED) was associated with the formation of a pulmonary edema (PED) in the human central nervous system that secretes a human IDS precursor. Methods for treating a human subject suffering from a tumor.

17. The secretion of the glycosylated human IDS precursor comprises the step of:

17. The method of claim 16, wherein the method is confirmed by transduction of a human neuronal cell line with the expression vector.

18. The secretion of the glycosylated human IDS precursor is characterized by the presence and absence of mannose-6-phosphate.

18. The method of claim 16 or 17, wherein the detection is performed under the presence of

19. 19. The method of claim 15, wherein the human IDS comprises the amino acid sequence of SEQ ID NO:

1. How to.

20. The recombinant nucleotide expression vector is capable of expressing glycosylated human nucleotides in human neuronal cells. Neuronal-specific promoters controlling expression of human IDS precursors or in human glial cells We have identified a glial cell-specific promoter that controls the expression of a glycosylated human IDS precursor. The method of any one of claims 15 to 19, comprising:

21. The recombinant nucleotide expression vector is capable of expressing a gene in a human neuronal cell or a human glial cell. Proper co-translational and post-translational processing of glycosylated human IDS precursors The method according to any one of claims 15 to 20, further comprising encoding a leader peptide that ensures transcription of the How to.

22. 22. Any of claims 15 to 21, wherein the recombinant nucleotide expression vector is an AAV vector.

3. The method according to claim 1 .

23. 23. The method of claim 22, wherein the recombinant nucleotide expression vector is a replication-deficient AAV vector. How to.

24. 22. The recombinant nucleotide expression vector of claim 21, wherein the recombinant nucleotide expression vector is an AAV9 or AAVrhlO vector. Or the method described in 23.

25. The recombinant nucleotide expression vector is administered intrathecally, intracerebroventricularly, via lumbar puncture, or intranasally. The method of any one of claims 15 to 24, wherein the therapeutic agent is delivered to the CSF of a human subject by

26. The method of any one of claims 15 to 25, wherein the human subject is deficient in IDS activity.

27. 12. A method of treating a human subject diagnosed with MPS II as described above, comprising: a recombinant nucleotide expression vector encoding a human IDS into the CSF of the human subject; and administering to the CSF of the human brain a formulation suitable for administration to the CSF of the human brain. The resultant depot was determined to be approximately 90 kDa by polyacrylamide gel electrophoresis, and the formylglutamate was Contains lysine, is α2,6-sialylated, contains no detectable NeuGc, and contains no detectable α-Gal Glycosylated human I, antigen-free and / or mannose-6-phosphorylated The method comprises forming in the human central nervous system secreting DS precursors.

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