Methods and formulations for treating mucopolysaccharidosis ii-associated hearing loss with recombinant human iduronate-2-sulfatase

Abstract

The present disclosure provides methods and formulations for treating or preventing mucopolysaccharidosis II (MPS II)-associated hearing loss using recombinant human iduronate-2-sulfatase (IDS).

Description

METHODS AND FORMULATIONS FOR TREATING MUCOPOLYSACCHARIDOSIS II-ASSOCIATED HEARING LOSS WITH RECOMBINANT HUMAN IDURONATE-2-SULFATASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application No. 63 / 587,164 filed October 2, 2023, and United States Provisional Application No. 63 / 623,697 filed January 22, 2024, the content of each of which is incorporated by reference in its entirety herein.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] This application contains a computer readable Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “12656-206-228_SEQLISTING.xml”, was created on September 23, 2024, and is 93,406 bytes in size.1. INTRODUCTION

[0003] The present disclosure provides methods and formulations for treating or preventing mucopolysaccharidosis II (MPS II)-associated hearing loss using recombinant human iduronate-2-sulfatase (IDS).2. BACKGROUND OF THE INVENTION

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

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

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

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

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

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

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

[0011] The present disclosure provides methods and formulations for preventing or treating MPS Il-associated hearing loss in a subject in need thereof using recombinant human iduronate-2-sulfatase (IDS). In certain embodiments, the recombinant human IDS is delivered to the subject by a recombinant vector.

[0012] In one aspect, the present disclosure provides a method for preventing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear.

[0013] In another aspect, the present disclosure provides a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2- sulfatase (hIDS) for use in preventing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear.

[0014] In another aspect, the present disclosure provides use of a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2- sulfatase (hIDS) in preventing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear.

[0015] In certain embodiments, the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 24 weeks, at least 48 weeks, at least about 1 year, at least about 52 weeks, at least about 1.5 years, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after the administration of the rAAV.

[0016] In certain embodiments, the onset of hearing loss is indicated by an increase in a hearing threshold as measured by an auditory brain response (ABR) testing in the at least one ear after the administration of the rAAV as compared to a hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV. In certain embodiments, the hearing threshold is an estimate of behavioral threshold (eHL) derived from an ABR threshold (nHL). In certain embodiments, the method or the use disclosed herein further comprises performing the ABR testing in the at least one ear prior to and afterthe administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV.

[0017] In certain embodiments, the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural as determined by a tympanometric testing. In certain embodiments, the method or the use disclosed herein further comprises performing the tympanometric testing in the at least one ear prior to and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.

[0018] In another aspect, the present disclosure provides a method for stabilizing mucopolysaccharidosis II (MPS II)-associated hearing loss or preventing the further loss of hearing associated with MPS II in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents a further loss of the hearing in the at least one ear.

[0019] In another aspect, the present disclosure provides a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2- sulfatase (hIDS) for use in stabilizing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents a further loss of the hearing in the at least one ear.

[0020] In another aspect, the present disclosure provides use of a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2- sulfatase (hIDS) in stabilizing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents a further loss of the hearing in the at least one ear.

[0021] In certain embodiments, the therapeutically effective amount of the rAAV stabilizes the hearing loss or prevents the further loss of the hearing in the at least one ear for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 24 weeks, at least 48 weeks, atleast about 1 year, at least about 52 weeks, at least about 1.5 years, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after the administration of the rAAV.

[0022] In certain embodiments, stabilizing the hearing loss or preventing the further loss of the hearing is indicated by no change or a small change in a hearing threshold as measured by an auditory brain response (ABR) testing in the at least one ear after the administration of the rAAV as compared to a hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV. In certain embodiments, the hearing threshold is an estimate of behavioral threshold (eHL) derived from an ABR threshold (nHL). In certain embodiments, stabilizing the hearing loss or preventing the further loss of the hearing is indicated by an about 10 dB or less change of the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV.

[0023] In certain embodiments, the method or the use disclosed herein further comprises performing the ABR testing in the at least one ear prior to and after the administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV.

[0024] In certain embodiments, the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural as determined by a tympanometric testing. In certain embodiments, the method or the use disclosed herein further comprises performing the tympanometric testing in the at least one ear prior to and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.

[0025] In another aspect, the present disclosure provides a method for improving mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves the hearing in the at least one ear.

[0026] In another aspect, the present disclosure provides a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2- sulfatase (hIDS) for use in improving mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has developed hearing loss in at least one earprior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves the hearing in the at least one ear.

[0027] In another aspect, the present disclosure provides use of a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2- sulfatase (hIDS) in improving mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves the hearing in the at least one ear.

[0028] In certain embodiments, the therapeutically effective amount of the rAAV improves the hearing in the at least one ear in about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 24 weeks, about 6 months, about 48 weeks, about 52 weeks, or about 1 year after the administration of the rAAV.

[0029] In certain embodiments, improving the hearing is indicated by a decrease in a hearing threshold as measured by an auditory brain response (ABR) testing in the at least one ear after the administration of the rAAV as compared to a hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV. In certain embodiments, the therapeutically effective amount of the rAAV decreases the hearing threshold in the at least one ear for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the hearing threshold in the at least one ear prior to the administration of the rAAV. In certain embodiments, the hearing threshold is an estimate of behavioral threshold (eHL) derived from an ABR threshold (nHL). In certain embodiments, improving the hearing is indicated by a more than about 10 dB decrease of the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV. In certain embodiments, the method or the use disclosed herein further comprises performing the ABR testing in the at least one ear prior to and after the administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV.

[0030] In certain embodiments, the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural as determined by a tympanometric testing. In certain embodiments, the method or the use disclosed herein further comprises performing the tympanometric testing in the at least one ear prior to and after the administration of the rAAVto determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.

[0031] In certain embodiments, the at least one ear comprises the left ear or the right ear. In certain embodiments, the at least one ear comprises the left ear and the right ear.

[0032] In certain embodiments, the method or the use disclosed herein further comprises administering an additional treatment to the subject. In certain embodiments, the additional treatment is an enzyme replacement therapy comprising a recombinant idursulfase. In certain embodiments, the additional treatment is a second administration of the rAAV. In certain embodiments, the second administration of the rAAV is at a higher dose as compared to the first administration of the rAAV.

[0033] In certain embodiments, the rAAV is administered to the cerebrospinal fluid (CSF) of the subject. In certain embodiments, the rAAV is administered via intraci sternal (IC) administration. In certain embodiments, the rAAV is administered via intracerebroventricular (ICV) administration. In certain embodiments, the rAAV is administered at a volume that does not exceed 10% of the total cerebrospinal fluid volume of the subject.

[0034] In certain embodiments, the rAAV is administered intravenously to the subject.

[0035] In certain embodiments, the rAAV is administered intrathecally to the subject.

[0036] In certain embodiments, the rAAV is administered to the subject in a solution comprising:(a) sodium chloride at a concentration of about 8.77 g / L,(b) magnesium chloride, at a concentration of about 0.244 g / L,(c) potassium chloride at a concentration of about 0.224 g / L,(d) calcium chloride at a concentration of about 0.206 g / L,(e) dextrose at a concentration of about 0.793 g / L,(f) poloxamer 188 at a concentration of about 0.010 g / L,(g) sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and(h) sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L.

[0037] In certain embodiments, the rAAV is administered at a dose of about 1.3 x 1010GC / g brain mass, about 6.5 x io10GC / g brain mass, or about 2 x io11GC / g brain mass, wherein the brain mass is determined by MRI.

[0038] In certain embodiments, the rAAV is administered to the at least one ear.

[0039] In certain embodiments, the subject is 5 years old or older and less than 18 years old. In certain embodiments, the subject is 4 months old or older and less than 5 years old.

[0040] In certain embodiments, the subject is receiving an enzyme replacement therapy (ERT) at the time of the administration of the rAAV. In certain embodiments, the subject is not responsive to the ERT. In certain embodiments, the ERT comprises a recombinant idursulfase.

[0041] In certain embodiments, the ERT is discontinued about 6 months, about 24 weeks, about 9 months, about 12 months, about 52 weeks, about 15 months, about 18 months, about 21 months, or about 24 months after the administration of the rAAV.

[0042] In certain embodiments, the rAAV is a recombinant adeno-associated virus serotype 9. In certain embodiments, the rAAV comprises a human IDS expression cassette, wherein the expression of the human IDS is driven by a hybrid of a cytomegalovirus (CMV) enhancer and a chicken beta actin promoter (CB7). In certain embodiments, the human IDS expression cassette comprises (i) an IDS transgene flanked by inverted terminal repeats (ITRs), (ii) the chicken beta actin intron, and (iii) a rabbit beta-globin polyadenylation (poly A) signal. In certain embodiments, the ITRs are AAV2 ITRs. In certain embodiments, the human IDS expression cassette comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 45.

[0043] In certain embodiments, the method or the use disclosed herein further comprises administering an immune suppression therapy to the subject before or concurrently with the rAAV, and optionally continuing the immune suppression therapy thereafter. In certain embodiments, the immune suppression therapy comprises administering at least one corticosteroid, sirolimus, and / or tacrolimus. In certain embodiments, the at least one corticosteroid is methylprednisolone and / or prednisone.

[0044] In certain embodiments, the method or the use disclosed herein further comprises administering at least one antibiotics to the subject before or concurrently with the rAAV. In certain embodiments, the at least one antibiotics is trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atovaquone.

[0045] In certain embodiments, the method or the use disclosed herein further comprises administering at least one antifungal therapy to the subject before or concurrently with rAAV.

[0046] In another aspect, the present disclosure provides a formulation suitable for administration to the ear comprising a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS).

[0047] In certain embodiments, the rAAV comprises a human IDS expression cassette, wherein the expression of the human IDS is driven by a hybrid of a cytomegalovirus (CMV) enhancer and a chicken beta actin promoter (CB7). In certain embodiments, the human IDS expression cassette comprises (i) an IDS transgene flanked by inverted terminal repeats (ITRs), (ii) the chicken beta actin intron, and (iii) a rabbit beta-globin polyadenylation (poly A) signal.

[0048] In certain embodiments, the ITRs are AAV2 ITRs. In certain embodiments, the human IDS expression cassette comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 45.3.1 ILLUSTRATIVE EMBODIMENTS1. A method for preventing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2- sulfatase (hIDS), wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear.2. The method of embodiment 1, wherein the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 24 weeks, at least 48 weeks, at least about 1 year, at least about 52 weeks, at least about 1.5 years, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after the administration of the rAAV.3. The method of embodiment 1 or 2, wherein the onset of hearing loss is indicated by an increase in a hearing threshold as measured by an auditory brain response (ABR) testing in the at least one ear after the administration of the rAAV as compared to a hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV.4. The method of embodiment 3, wherein the hearing threshold is an estimate of behavioral threshold (eHL) derived from an ABR threshold (nHL).5. The method of embodiment 3 or 4, wherein the method further comprises performing the ABR testing in the at least one ear prior to and after the administration of the rAAV todetermine hearing thresholds in the at least one ear prior to and after the administration of the rAAV.6. The method of any one of embodiments 1-5, wherein the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural as determined by a tympanometric testing.7. The method of embodiment 6, wherein the method further comprises performing the tympanometric testing in the at least one ear prior to and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.8. A method for stabilizing mucopolysaccharidosis II (MPS II)-associated hearing loss or preventing the further loss of hearing associated with MPS II in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents a further loss of the hearing in the at least one ear.9. The method of embodiment 8, wherein the therapeutically effective amount of the rAAV stabilizes the hearing loss or prevents the further loss of the hearing in the at least one ear for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 24 weeks, at least 48 weeks, at least about 1 year, at least about 52 weeks, at least about 1.5 years, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after the administration of the rAAV.10. The method of embodiment 9, wherein stabilizing the hearing loss or preventing the further loss of the hearing is indicated by no change or a small change in a hearing threshold as measured by an auditory brain response (ABR) testing in the at least one ear after the administration of the rAAV as compared to a hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV.11. The method of embodiment 10, wherein the hearing threshold is an estimate of behavioral threshold (eHL) derived from an ABR threshold (nHL).12. The method of embodiment 11, wherein stabilizing the hearing loss or preventing the further loss of the hearing is indicated by an about 10 dB or less change of the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV.13. The method of any one of embodiments 10-12, wherein the method further comprises performing the ABR testing in the at least one ear prior to and after the administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV.14. The method of any one of embodiments 8-13, wherein the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural as determined by a tympanometric testing.15. The method of embodiment 14, wherein the method further comprises performing the tympanometric testing in the at least one ear prior to and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.16. A method for improving mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2- sulfatase (hIDS), wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves the hearing in the at least one ear.17. The method of embodiment 16, wherein the therapeutically effective amount of the rAAV improves the hearing in the at least one ear in about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 24 weeks, about 6 months, about 48 weeks, about 52 weeks, or about 1 year after the administration of the rAAV.18. The method of embodiment 16 or 17, wherein improving the hearing is indicated by a decrease in a hearing threshold as measured by an auditory brain response (ABR) testing in the at least one ear after the administration of the rAAV as compared to a hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV.19. The method of embodiment 18, wherein the therapeutically effective amount of the rAAV decreases the hearing threshold in the at least one ear for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the hearing threshold in the at least one ear prior to the administration of the rAAV.20. The method of embodiment 18 or 19 , wherein the hearing threshold is an estimate of behavioral threshold (eHL) derived from an ABR threshold (nHL).21. The method of embodiment 20, wherein improving the hearing is indicated by a more than about 10 dB decrease of the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV.22. The method of any one of embodiments 18-21, wherein the method further comprises performing the ABR testing in the at least one ear prior to and after the administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV.23. The method of any one of embodiments 8-22, wherein the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural as determined by a tympanometric testing.24. The method of embodiment 23, wherein the method further comprises performing the tympanometric testing in the at least one ear prior to and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.25. The method of any one of embodiments 1-24, wherein the at least one ear comprises the left ear or the right ear.26. The method of any one of embodiments 1-25, wherein the at least one ear comprises the left ear and the right ear.27. The method of any one of embodiments 1-26, wherein the method further comprises administering an additional treatment to the subject.28. The method of embodiment 27, wherein the additional treatment is an enzyme replacement therapy comprising a recombinant idursulfase.29. The method of embodiment 27, wherein the additional treatment is a second administration of the rAAV.30. The method of embodiment 29, wherein the second administration of the rAAV is at a higher dose as compared to the first administration of the rAAV.31. The method of any one of embodiments 1-30, wherein the rAAV is administered intravenously to the subject or to the cerebrospinal fluid (CSF) of the subject.32. The method of any one of embodiments 1-31, wherein the rAAV is administered via intraci sternal (IC) administration.33. The method of any one of embodiments 1-31, wherein the rAAV is administered via intracerebroventricular (ICV) administration.34. The method of any one of embodiments 1-33, wherein the rAAV is administered at a volume that does not exceed 10% of the total cerebrospinal fluid volume of the subject.35. The method of any one of embodiments 1-34, wherein the rAAV is administered intrathecally to the subject.36. The method of any one of embodiments 1-35, wherein the rAAV is administered to the subject in a solution comprising:(a) sodium chloride at a concentration of about 8.77 g / L,(b) magnesium chloride, at a concentration of about 0.244 g / L,(c) potassium chloride at a concentration of about 0.224 g / L,(d) calcium chloride at a concentration of about 0.206 g / L,(e) dextrose at a concentration of about 0.793 g / L,(f) poloxamer 188 at a concentration of about 0.010 g / L,(g) sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and(h) sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L.37. The method of any one of embodiments 1-36, wherein the rAAV is administered at a dose of about 1.3 x 1010GC / g brain mass, about 6.5 x io10GC / g brain mass, or about 2 x 1011GC / g brain mass, wherein the brain mass is determined by MRI.38. The method of any one of embodiments 1-30, wherein the rAAV is administered to the at least one ear.39. The method of any one of embodiments 1-38, wherein the subject is 5 years old or older and less than 18 years old.40. The method of any one of embodiments 1-38, wherein the subject is 4 months old or older and less than 5 years old.41. The method of any one of embodiments 1-40, wherein the subject is receiving an enzyme replacement therapy (ERT) at the time of the administration of the rAAV.42. The method of embodiment 41, wherein the subject is not responsive to the ERT.43. The method of embodiment 41 or 42, wherein the ERT comprises a recombinant idursulfase.44. The method of any one of embodiments 41-43, wherein the ERT is discontinued about 6 months, about 24 weeks, about 9 months, about 12 months, about 52 weeks, about 15 months, about 18 months, about 21 months, or about 24 months after the administration of the rAAV.45. The method of any one of embodiments 1-44, wherein the rAAV is a recombinant adeno-associated virus serotype 9.46. The method of embodiment 45, wherein the rAAV comprises a human IDS expression cassette, wherein the expression of the human IDS is driven by a hybrid of a cytomegalovirus (CMV) enhancer and a chicken beta actin promoter (CB7).47. The method of embodiment 46, wherein the human IDS expression cassette comprises (i) an IDS transgene flanked by inverted terminal repeats (ITRs), (ii) the chicken beta actin intron, and (iii) a rabbit beta-globin polyadenylation (poly A) signal.48. The method of embodiment 47, wherein the ITRs are AAV2 ITRs.49. The method of any one of embodiments 46-48, wherein the human IDS expression cassette comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 45.50. The method of any one of embodiments 1-49, further comprising administering an immune suppression therapy to the subject before or concurrently with the rAAV, and optionally continuing the immune suppression therapy thereafter.51. The method of embodiment 50, wherein the immune suppression therapy comprises administering at least one corticosteroid, sirolimus, and / or tacrolimus.52. The method of embodiment 51, wherein the at least one corticosteroid is methylprednisolone and / or prednisone.53. The method of any one of embodiments 1-52, further comprising administering at least one antibiotics to the subject before or concurrently with the rAAV.54. The method of embodiment 53, wherein the at least one antibiotics is trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atovaquone.55. The method of any one of embodiments 1-54, further comprising administering at least one antifungal therapy to the subject before or concurrently with rAAV.56. A therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for use in preventing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear.57. A therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for use in stabilizing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof,wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents a further loss of the hearing in the at least one ear.58. A therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for use in improving mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves the hearing in the at least one ear.59. Use of a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) in preventing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear.60. Use of a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) in improving mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves the hearing in the at least one ear.61. Use of a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) in stabilizing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents a further loss of the hearing in the at least one ear.62. A formulation suitable for administration to the ear comprising a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS).63. The formulation of embodiment 62, wherein the rAAV comprises a human IDS expression cassette, wherein the expression of the human IDS is driven by a hybrid of a cytomegalovirus (CMV) enhancer and a chicken beta actin promoter (CB7).64. The formulation of embodiment 63, wherein the human IDS expression cassette comprises (i) an IDS transgene flanked by inverted terminal repeats (ITRs), (ii) the chicken beta actin intron, and (iii) a rabbit beta-globin polyadenylation (poly A) signal.65. The formulation of embodiment 64, wherein the ITRs are AAV2 ITRs.66. The formulation of any one of embodiments 63-65, wherein the human IDS expression cassette comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 45.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG. 1. The amino acid sequence of human IDS (SEQ ID NO: 1). A post- translational formylglycine modification of C84(shown in bold in FIG. 1) is required for enzyme activity. Eight N linked glycosylation sites (N31, N115, N144, N246, N280, N325, N513and N537) are bold and boxed. One tyrosine-O-sulfation site (Y) is bold and the full sulfation site sequence (PSSEKY165ENTKTCRGPD (SEQ ID NO: 47)) is boxed. The N-terminus of the mature 42 kDa and mature 14 kDa polypeptides are indicated by horizontal arrows. In the brain, the N-terminus of the mature 42 kDa form starts at positions 34 or 36 as follows: T34DALNVLLI (SEQ ID NO: 54); and A36LNVLLIIV (SEQ ID NO: 55) as indicated in FIG. 1. (See, Sleat, 2005, Proteomics 5: 1520-1532, Table S2). Two of the eight N-linked glycosylation sites, namely N280and N116, are mannose-6-phophorylated in IDS obtained from human brain. (Sleat et al., 2006, Mol & Cell Proeomics 5.4: 686-701, reported at Table V).

[0050] FIG. 2. Multiple sequence alignment of hIDS with known orthologs. The names of the species and protein IDs are as follows: SP|P22304|IDS_HUMAN [Homo sapiens]; TR|K6ZGI9_PANTR [Pan troglodytes (Chimpanzee)]; TR|K7BKV4_PANTR [Pan troglodytes (Chimpanzee)]; TR|H9FTX2_MACMU [Macaca mulatta (Rhesus macaque)]; TRF7EJG2 CALJA [Callithrix jacchus (White-tufted-ear marmoset)]; TR|U3DTL8_CALJA [Callithrix jacchus (White-tufted-ear marmoset)]; TR|G7NRX7_MACMU [Macaca mulatta (Rhesus macaque)]; TR|G7Q1V9_MACFA [Macaca fascicularis (Crab-eating macaque; Cynomologous monkey)]; TR|H2PX10_PONAB [Pongo abelii (Sumatran orangutan)];TR|A0A0D9R4Dl_CHLSB [Chlorocebus sabaeus (Green monkey)]; TR|G1RST8|G1RST8_NOMLE [Nomascus leucogenys (Northern white-cheeked gibbon)]; UPI0000D9F625 [Macaca mulatta (Rhesus macaque)]; UPI000274358B [Pan paniscus (Pygmy chimpanzee; Bonobo)]; UPI00027F6FC5 [Papio Anubis (Olive baboon)];UPI00027FAE03 [Saimiri boliviensis (Bolivian squirrel monkey)]; UPI0003ABBF28 [Macaca fascicularis (Crab-eating macaque; Cynomologous monkey)]; UPI000533297F [Rhinopithecus roxellana (Golden snub-nosed monkey; Pygathrix roxellana)];UPI0005F40BD2 [Colobus angolensis palliates (Peters’ Angolan colobus)] (SEQ ID NOs: 27-44).

[0051] FIG. 3. MPS II mutations in hIDS and corresponding disease phenotypes, mild, intermediate or severe, (from Uniprot).

[0052] FIG. 4. Human IDS processing as reported in Millat et al., 1997, Exp. Cell. Res. 230: 362-367, at Fig.7.

[0053] FIG. 5. Schematic Representation of Construct 1.

[0054] FIG. 6. Clustal Multiple Sequence Alignment of AAV capsids 1 - 9 (SEQ ID NOs: 16-26). Amino acid substitutions (shown in bold in the bottom rows, SEQ ID NOs: 48-53) can be made to AAV9 and AAV8 capsids by “recruiting” amino acid residues from the corresponding position of other aligned AAV capsids. Sequence regions designated by “HVR” = hypervariable regions.

[0055] FIG. 7. Heparan Sulfate (HS) digestion with Heparinase.

[0056] FIG. 8. Graph showing cerebral spinal fluid (CSF) heparan sulfate biomarker (ng / mL). Graph showed consistent HS decrease in the CSF after Construct 1 dosing. The median change from baseline at week 8 (N=6) was -30.3% (p-value = 0.03 as measured by Wilcoxon signed rank test). The median change from baseline at the last available timepoint (N=6) was -35.8% (p-value = 0.03 as measured by Wilcoxon signed rank test). The graph showed measurable CSF I2S enzyme concentration in cohort 2 after Construct 1 administration with a range of 1170-1940 pg / mL.

[0057] FIG. 9. Graph showing cerebral spinal fluid (CSF) D2S6 biomarker (ng / mL). The median change from baseline at week 8 (N=6) was -44.2% (p-value = 0.03 as measured by Wilcoxon signed rank test). The median change from baseline at the last available timepoint (N=6) was -39.2% (p-value = 0.03 as measured by Wilcoxon signed rank test).

[0058] FIGS. 10A-10C. Graphs showing neurodevelopment function comparing age equivalence (months) and age (months). The Graph in FIG. 10A showed continued cognitive development in 4 of 5 patients with > 6 months of follow-up. Patients 1, 3, and 5demonstrated continued cognitive development within a normal range. Patients 2 and 4 presented with significant cognitive delay at baseline. Patient 2 has continued cognitive development. Patient 4 acquired expressive and receptive language skills. FIGs. 10B and IOC show cognitive developmental quotient and age equivalence quotient, respectively, of patients in Construct 1 gene therapy clinical trial. The graphs in FIGs. 10B and IOC show that 3 out of 4 patients who entered the study with cognitive skill above the -2SD of the normal range continued to stay above -2SD as of > 6-month follow-up. One patient entered the study with significant delay in neurocognitive development at baseline but demonstrated relative stabilization following Construct 1 administration at an older age (59 months) and continued to acquire expressive and receptive language skills.

[0059] FIGS. 11A-11D. Graphs showing neurodevelopment function in terms of language and motor skills. FIG. 11A displays expressive communication; FIG. 11B displays receptive communication; FIG. 11C displays gross motor skills; and FIG. 11D displays fine motor skills. Graphs showed continued language and / or motor skills acquisition in patients with > 6 months of follow-up.

[0060] FIG. 12. Graph showing systemic efficacy by measuring plasma I2S protein concentration (pg / mL). The graph shows a general increase in plasma I2S enzyme levels in 5 out of 6 patients after Construct 1 administration (normal range (14,706 pg / mL ~ < 100,000 pg / mL)).

[0061] FIG. 13. Graph showing systemic efficacy by measuring urine total GAGs level (g / mol CK) in ERT -treated patients. Graph showed sustained decrease in urine GAG levels across all patients receiving ERT.

[0062] FIG. 14. Graph showing systemic efficacy by measuring urine total GAGs level (g / mol CK) in ERT naive and ERT discontinued patients. Graphs showed a rapid decrease in urine GAGs in ERT-naive patients after Construct 1 administration (absence of urine GAG rebound post ERT withdrawal).

[0063] FIG. 15. Ultrasound of liver or spleen in ERT naive patient. Liver and spleen dimensions in ERT-naive patient decreased 24 weeks after Construct 1 administration.

[0064] FIG. 16. Proof-of-concept Study Activities.

[0065] FIG. 17. Dose Rationale for the First-in-Human Clinical Trial.

[0066] FIG. 18. Total heparan sulfate in CSF samples from healthy subjects, MPS I (neuronopathic and non-neuronopathic) subjects, and MPS II (neuronopathic and non- neuronopathic) subjects.

[0067] FIGS. 19A-19C. Graphs showing the level of D0S0 disaccharide (FIG. 19A), D0A6 disaccharide (FIG. 19B), and D0A0 (FIG. 19C) in healthy subjects, MPS I (neuronopathic and non-neuronopathic) subjects, and MPS II (neuronopathic and non- neuronopathic) subjects.

[0068] FIG. 20. Graph showing the level of D2S6 disaccharide in healthy subjects, MPS I (neuronopathic and non-neuronopathic) subjects, and MPS II (neuronopathic and non- neuronopathic) subjects.

[0069] FIG. 21. Graph showing the percent composition of heparan sulfate disaccharides in healthy subjects, MPS I (neuronopathic and non-neuronopathic) subjects, and MPS II (neuronopathic and non-neuronopathic) subjects.

[0070] FIG. 22. Diagram showing MPS II phase 1 / 2 clinical study summary.

[0071] FIG. 23. Graphs showing widespread CNS and systemic biodistribution after Construct 1 IC administration in non-human. The term “LD” is low dose, “IS” is immunosuppression, and “HD” is high dose.

[0072] FIGS. 24A-24B. Graphs showing cerebral spinal fluid (CSF) Biomarker Heparan Sulfate (HS) in subjects in the Phase 1 / 2 study. CSF HS measurements showed dosedependent reductions in Cohorts 1-3 at Weeks 8 and 24 (medians show in FIG. 24A, individual participants data show in FIG. 24B).

[0073] FIGS. 25A-25B. Graphs showing concentration of D2S6 in patients in Phase 1 / 2 study. CSF D2S6 measurement showed dose-dependent reductions in Cohorts 1-3 at Week 8 and 24, with Cohort 3 participants approaching normal levels (medians show in FIG. 25A, individual participants data show in FIG. 25B).

[0074] FIGS. 26A-26C. Graphs showing cognition (FIG. 26A), expressive language (FIG. 26B), and fine motor (FIG. 26C) neurodevelopmental function for cohort 1 patients in Phase 1 / 2 study.

[0075] FIGS. 27A-27C. Graphs showing cognition (FIG. 27A), expressive language (FIG. 27B), and fine motor (FIG. 27C) neurodevelopmental function for cohort 2 patients in Phase 1 / 2 study.

[0076] FIGS. 28A-28B. Graphs showing maladaptive behavior index (FIG. 28A) and toileting skills (FIG. 28B) for patients in Phase 1 / 2 study.

[0077] FIGS. 29A-29B. Graphs showing plasma I2S protein levels (FIG. 29A) and urine GAG levels (FIG. 29B) in patients in Phase 1 / 2 study.

[0078] FIG. 30. Diagram showing MPS I phase 1 / 2 clinical study summary.

[0079] FIGS. 31A-31B. Graphs showing concentration of cerebral spinal fluid (CSF) biomarker and heparin sulfate in participants in the MPS I Phase 1 / 2 study (FIG. 31A) and in a single participant (FIG. 31B). Graphs show a decrease in CSF heparin sulfate in all participants through last time point available (e.g., week 24 for Phase 1 / 2 trial (FIG. 31 A) and week 59 for the single participant (FIG. 31B). Study showed a measurable CSF IDUA enzyme activity in the majority of participants in the Phase 1 / 2 trial and in the single participant.

[0080] FIGS. 32A-32F. Graphs showing neurodevelopment function BSID-III as cognition (FIG. 32A), expressive language (FIG. 32B), and fine motor (FIG. 32C) in MPS I Phase 1 / 2 study and the single participant study (FIGS. 32D-32F).

[0081] FIG. 33. Graph showing neurodevel opmental function BSID cognition in the MPS I single participant study.

[0082] FIG. 34. Tables showing the neurodevel opmental function (WASI-II and VABS-III) for a 13 year-old MPS I Phase 1 / 2 study participant.

[0083] FIGS. 35A-35B. Graphs showing level of I0S6 in MPS I Phase 1 / 2 participants (FIG. 35A) and in the single participant study (FIG. 35B).

[0084] FIGS. 36A-36B. Graphs showing level of total urine GAGs in MPS I Phase 1 / 2 participants (FIG. 36A) and in the single participant study (FIG. 36B).

[0085] FIGS. 37A and 37B. Composite audiograms showing average air conduction ABR thresholds for left ear (FIG. 37A) and right ear (FIG. 37B) at 500Hz, 1000Hz, 2000Hz and 4000 Hz in dBeHL.

[0086] FIG. 38. Graph showing change in ABR air conduction thresholds for each participant from baseline to last available timepoint showed majority of ears with improved or stable hearing thresholds.

[0087] FIG. 39 is a study flow chart of the presently disclosed Example 10, showing the schedule of events. AAV9 = Adeno-associated virus vector of serotype 9; ABR = auditory brainstem response; CSF = cerebrospinal fluid; CT = computed tomography; DNA = deoxyribonucleic acid; ECG = electrocardiogram; ELISPOT = enzyme-linked immunospot; EOS = end of study; ERT = enzyme replacement therapy; ET = early termination; GAG = glycosaminoglycan; Hep = hepatitis; HIV = human immunodeficiency virus; IC = intraci sternal; ICV = Intracerebroventricular; INR=International Normalized Ratio;IQ = intelligence quotient; IT = intrathecally; IV = intravenous; MRI = magnetic resonance imaging; PI = principal investigator; PO = orally; PT = prothrombin time; PTT = partial thromboplastin time; QD = once daily; qPCR = quantitative polymerase chain reaction;Construct 1 = Recombinant adeno-associated virus serotype 9 capsid containing human iduronate-2-sulfatase expression cassette; sAg = surface antigen; T cell = T lymphocyte. Visit 14 is deliberately omitted from the visit numbering scheme. Notations of the chart are listed as the following:•aSites can admit the subject between Day -2 and the morning of Day 1. Therefore, some of the pre-dose testing can be done on Day -2 or on Day -1, as per institution’s standards.•bFor an ET visit, lumbar puncture (and CSF assessments) is performed only if withdrawal occurs before Week 56 and cognitive function assessment is performed only if withdrawal occurs before Week 48.•cThe Investigator, in consultation with the Medical Monitor, should determine which assessments need to be performed at an unscheduled visit.•dVital signs and neurologic assessments are monitored frequently, including assessments every 45 ± 10 minutes for 3 hours starting in the post-anesthesia care unit, then hourly ± 15 minutes for the next 4 hours, and then every 4 hours ± 30 minutes for the remainder of the 24-hour period immediately post-dose.•eSubjects can be discharged home approximately 1-2 days after dosing if deemed appropriate by the PI. Subjects / car egivers are to receive instructions on neurologic function monitoring.•fWeek 64 visit is to be performed only for subjects who discontinue IV ERT.•gWeek 20 and 28 assessments, italicized in the table, will be limited to evaluation of adverse events and concomitant therapies by telephone contact.•hPre-dose neurologic assessment can be performed any time from Day -2 to Day 1.•1If clinically indicated.• J Performed according to each institution’s standards and may be performed prior to Day 1.•kResults should be assessed or reviewed by a neurologist.• Estimated glomerular filtration rate (eGFR) based on creatinine must be measured prior to screening MRI with gadolinium. If laboratory determines that creatinine is less than the lower limit of assay validation or detection then the lowest limit cutoff value will be used to estimate eGFR. The investigator must consult with the Medical Monitor before proceeding with MRI if the eGFR is < 30mL / min / 1.73 m2.•1Assessments to evaluate contraindication to IC or ICV injection is performed according to the appropriate Administration Manual and be completed prior to Day -2. Approval to proceed with the IC or ICV injection has to be documented before starting IS on Day -2.•mMRI with gadolinium is performed as a screening assessment only. All attempts should be made to perform MRI following confirmation of neurocognitive scores. If an MRI under sedation occurs prior to neurocognitive testing, then there must be at least 24 hours between the time of the MRI and when the neurocognitive testing can be performed.•nGenetic confirmation of MPS type 2. Genetic test done prior to the study is acceptable with adequate documentation. Subject with no prior genetic testing can have it done during screening.•nnAuditory capacity changes are measured by auditory brainstem response (ABR) and, if possible, behavioral audiometry (BAUD). Tympanometry is done at the time BAUD and / or ABR assessments are completed. Auditory capacity are assessed at visits specified in this Schedule of Events; an additional assessment is permitted at the Week 24 visit if deemed clinically necessary by the investigator.• Plasma biomarkers: samples are taken for GAGs and I2S. All attempts should be made to perform I2S assessments at trough in relation to IV ERT, defined as at least 96 hours after ERT infusion up until the start of the subsequent infusion.•pAbdominal ultrasound are done to assess the size of the liver and spleen. For subjects who have ERT withdrawn after Week 24, plasma and urine biomarkers are to be drawn. The abdominal ultrasound is performed at the same facility for every timepoint.•qAntibodies to AAV9 and I2S.•rUrine biomarkers: samples are taken for GAGs. All attempts should be made to collect urine GAGs at trough in relation to IV ERT, defined as at least 96 hours after ERT infusion up until the start of the subsequent infusion.•sIC or ICV injection are performed according to the appropriate Administration Manual.•1Tacrolimus: dose adjustments are made to maintain whole blood trough concentrations within 2-4 ng / mL Sirolimus: dose adjustments are made to maintain whole blood trough concentrations within 1-3 ng / mL. Subjects should continue the newmaintenance dose for at least 7-14 days before further dosage adjustment with concentration monitoring.• The methylprednisolone should be administered before the lumbar puncture and IC or ICV injection of the investigational product. Pre-medication with acetaminophen and an antihistamine is optional, at the discretion of the investigator.•uThe screening (Day -35 to Day -2) lumbar puncture is for assessment of CSF pressure and biomarkers only. Following the Day 1 pre-dose lumbar puncture, iodinated myelographic contrast are administered IT (see Administration Manual).•vCSF biomarkers: samples are taken for GAGs and I2S, and future use sample testing are only conducted from remnants of study samples, if available.•WCSF safety laboratory tests: CSF pressure, erythrocyte cell count, white blood cell count with differential, total protein, and glucose.•xCognition is assessed using BSID-III or KABC-II; adaptive function using VABS-II. If an MRI under sedation occurs prior to neurocognitive testing, then there must be at least 24 hours between the time of the MRI and when the neurocognitive testing can be performed.•zFor VZV : Ab titer at baseline. For EB V and CMV : Viral genome PCR at baseline and serial testing as described in able X. If positive at any timepoint then more frequent testing can be performed as per site standard of care.•yIf a subject has a relative with the same MPS II mutation, separate informed consent to review appropriate relative documentation is provided.•aaTo be completed by same caregiver throughout study if possible.•bbTo be completed by same clinician throughout study if possible.

[0088] FIG. 40 is a table providing a summary of audiometric data of Example 16. Audiometric data from 11 participants at Screening (baseline) and last timepoint post Construct 1 treatment. 'Average of estimated behavioral thresholds in dBeHL obtained from ABR for the frequencies measured. A difference in threshold of > 10 dB was used to determine change from baseline, indicates participants with last timepoints in long term study, indicates tube placement or tympanic membrane perforation.3Screening was Week 1 timepoint.

[0089] FIG. 41 depicts composite audiograms showing average air conduction ABR thresholds for each ear at 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz in dBeHL. Improvementsin hearing thresholds were observed at last timepoints post Construct 1 treatment compared to baseline.

[0090] FIG. 42 is a graph showing the average change in ABR air conduction thresholds for each participant from baseline to last timepoint available.

[0091] FIG. 43 is a table providing a summary of audiometric data of Example 17. Audiometric data from 11 participants at Screening (baseline) and last timepoint post Construct 1. 'Average of estimated behavioral thresholds in dBeHL obtained from ABR for the frequencies measured. A difference in threshold of > 10 dB was used to determine change from baseline, indicates tube placement or tympanic membrane perforation.3Conductive component at 500 Hz.

[0092] FIG. 44 depicts composite audiograms showing average air conduction ABR thresholds for each ear at 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz in dBeHL. Improvements in hearing thresholds were observed at last timepoints post Construct 1 treatment compared to baseline.

[0093] FIG. 45 is a graph showing the average change in ABR air conduction thresholds for each participant from baseline to last timepoint available.5. DETAILED DESCRIPTION OF THE INVENTION

[0094] The invention involves the delivery of recombinant human iduronate-2-sulfatase (rhIDS) produced by human neuronal or glial cells to the cerebrospinal fluid (CSF) of the central nervous system (CNS) of a human subject diagnosed with mucopolysaccharidosis II (MPS II), including, but not limited to patients diagnosed with Hunter syndrome. See, also, International Patent Application No. PCT / US2017 / 027770, filed April 14, 2017 (published as WO / 2017 / 181113 on October 19, 2017), which is incorporated by reference herein in its entirety, for compositions and methods that can be used according to the invention described herein.

[0095] In a preferred embodiment, the treatment is accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding human IDS (hIDS), or a derivative of hIDS, to the CSF of a patient (human subject) diagnosed with MPS II, so that a permanent depot of transduced neuronal and / or glial cells is generated that continuously supplies the transgene product to the CNS. The rhIDS secreted from the neuronal / glial cell depot into the CSF will be endocytosed by cells in the CNS, resulting in “cross-correction” of the enzymatic defect in the recipient cells. Moreover, it has been found, unexpectedly, that the depot of transduced neural and glial cells in the CNS can deliver therecombinant enzyme to both the CNS and systemically, which may reduce or eliminate the need for systemic treatment, e.g., weekly i.v. injections of the enzyme. Also provided herein is the treatment of MPS I by administering a viral vector or other DNA expression construct encoding human IDUA to a subject (e.g., non-replicating recombinant AAV of serotype 9 capsid containing an hIDUA expression cassette; Construct 2; refer to PCT / US2021 / 014129; PCT / US2018 / 015910; and PCT / US2019 / 042205, each of which is incorporated by reference herein in its entirety).

[0096] In an alternative embodiment, the hIDS can be produced by human neuronal or glial cells in cell culture (e.g., bioreactors) and administered as an enzyme replacement therapy (“ERT”), e.g., by injecting the enzyme - into the CSF, directly into the CNS, and / or systemically. However, the gene therapy approach offers several advantages over ERT since systemic delivery of the enzyme will not result in treating the CNS because the enzyme cannot cross the blood brain barrier; and, unlike the gene therapy approach of the invention, direct delivery of the enzyme to the CSF and / or CNS would require repeat injections which are not only burdensome, but pose a risk of infection.

[0097] The hIDS encoded by the transgene can include, but is not limited to human IDS (hIDS) having the amino acid sequence of SEQ ID NO. 1 (as shown in FIG. 1), and derivatives of hIDS having amino acid substitutions, deletions, or additions, e.g., including but not limited to amino acid substitutions selected from corresponding non-conserved residues in orthologs of IDS shown in FIG. 2, with the proviso that such mutations do not include replacement of the cysteine residue at position 84 (C84) which is required for enzyme activity (Millat et al., 1997, Biochem J 326: 243-247); or a mutation that has been identified in severe, severe-intermediate, intermediate, or attenuated MPS II phenotypes e.g., as shown in FIG. 3, or as reported by Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-761 (reporting “attenuated” mutants R48P, A85T, W337R, and the truncated mutant Q531X; and “severe” mutants P86L, S333L, S349I, R468Q, R468L); Millat et al., 1998, BBA 1406: 214- 218 (reporting “attenuated” mutants P480L and P480Q; and “severe” mutant P86L); and Bonucelli et al., 2001, BBA 1537:233-238, each of which is incorporated by reference herein in its entirety.

[0098] For example, amino acid substitutions at a particular position of hIDS can be selected from among corresponding non-conserved amino acid residues found at that position in the IDS orthologs aligned in FIG. 2, with the proviso that such substitutions do not include any of the deleterious mutations shown in FIG. 3 or as reported by Sukegawa-Hayasaka et al., 2006, supra, Millat et al., 1998, supra, or Bonucelli et al., 2001, supra, each of which isincorporated by reference herein in its entirety. The resulting transgene product can be tested using conventional assays in vitro, in cell culture or test animals to ensure that the mutation does not disrupt IDS function. Preferred amino acid substitutions, deletions or additions selected should be those that maintain or increase enzyme activity, stability or half-life of IDS, as tested by conventional assays in vitro, in cell culture or animal models for MPS II. For example, the enzyme activity of the transgene product can be assessed using a conventional enzyme assay with, for example, 4-Methylumbelliferyl a-L-idopyranosiduronic acid 2-sulfate or 4-methylumbelliferyl sulfate as the substrate (see, e.g., Lee et al., 2015, Clin. Biochem. 48(18): 1350-1353, Dean et al., 2006, Clin. Chem. 52(4):643-649 for exemplary IDS enzyme assays that can be used, each of which is incorporated by reference herein in its entirety). The ability of the transgene product to correct MPS II phenotype can be assessed in cell culture; e.g., by transducing MPS II cells in culture with a viral vector or other DNA expression construct encoding hIDS or a derivative; by adding the transgene product or a derivative to MPS II cells in culture; or by co-culturing MPS II cells with human neuronal / glial host cells engineered to express and secrete rhIDS or a derivative, and determining correction of the defect in the MPS II cultured cells, e.g., by detecting IDS enzyme activity and / or reduction in GAG storage in the MPS II cells in culture (see, e.g., Stroncek et al., 1999, Transfusion 39(4):343-350, which is incorporated by reference herein in its entirety).

[0099] Animal models for MPS II have been described that can be used to assess the therapeutics described herein. For example, a knockout mouse model (IDS -knockout) of MPS II was engineered by replacing exons 4 and 5 of the IDS gene with the neomycin resistance gene. (Garcia et al., 2007, J Inherit Metab Dis 30: 924-34). This IDS-knockout mouse exhibits many of the characteristics of MPS II, including skeletal abnormalities, hepatosplenomegaly, elevated urinary and tissue GAG, and brain storage lesions (Muenzer et al., 2001, Acta Paediatr Suppl 91 : 98-99) and was used to assess the effect of enzyme replacement therapy in MPS II in support of clinical trials for ERT. This mouse model, therefore, is a relevant model for studying the effects of gene therapy delivering rIDS produced by neuronal or glial cells as a treatment for MPS II (see, e.g., Polito and Cosma, 2009, Am. J. Hum. Genet. 85(2):296-301, which is incorporated by reference herein in its entirety).

[0100] Preferably, the hIDS transgene produced by the human neuronal / glial cells should be controlled by expression control elements that function in neurons and / or glial cells, e.g., the CB7 promoter (a chicken [B-actin promoter and CMV enhancer), and can include otherexpression control elements that enhance expression of the transgene driven by the vector (e.g., chicken [3-actin intron and rabbit P-globin poly A signal). The cDNA construct for the hIDS transgene should include a coding sequence for a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced CNS cells. Such signal peptides used by CNS cells may include but are not limited to:- Oligodendrocyte-myelin glycoprotein (hOMG) signal peptide having the amino acid sequence of MEYQILKMSLCLFILLFLTPGILC (SEQ ID NO:2)- Cellular repressor of ElA-stimulated genes 2 (hCREG2) signal peptide having the amino acid sequence of MSVRRGRRPARPGTRLSWLLCCSALLSPAAG (SEQ ID NO:3)- V-set and transmembrane domain containing 2B (hVSTM2B) signal peptide having the amino acid sequence of MEQRNRLGALGYLPPLLLHALLLFVADA (SEQ ID NO:4)- Protocadherin alpha-1 (hPCADHAl) signal peptide having the amino acid sequence of MVFSRRGGLGARDLLLWLLLLAAWEVGSG (SEQ ID NO:5)- FAM19A1 (TAFA1) signal peptide having the amino acid sequence of MAMVSAMSWVLYLWISACA (SEQ ID NO: 6)- Interleukin-2 signal peptide having the amino acid sequence of MYRMQLLSCIALILALVTNS (SEQ ID NO: 14)

[0101] Signal peptides may also be referred to herein as leader sequences or leader peptides.

[0102] The recombinant vector used for delivering the transgene should have a tropism for cells in the CNS, including but limited to neurons and / or glial cells. Such vectors can include non-replicating recombinant adeno-associated virus vectors (“rAAV”), particularly those bearing an AAV9 or AAVrhlO capsid are preferred. AAV variant capsids can be used, including but not limited to those described by Wilson in US Patent No. 7,906,111 which is incorporated by reference herein in its entirety, with AAV / hu.31 and AAV / hu.32 being particularly preferred; as well as AAV variant capsids described by Chatterjee in US Patent No. 8,628,966, US Patent No. 8,927,514 and Smith et al., 2014, Mol Ther 22: 1625-1634, each of which is incorporated by reference herein in its entirety. However, other viral vectors may be used, including but not limited to lentiviral vectors, vaccinia viral vectors, or non- viral expression vectors referred to as “naked DNA” constructs.

[0103] Pharmaceutical compositions suitable for administration to the CSF comprise a suspension of the rhIDS vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients. In certain embodiments, thepharmaceutical compositions are suitable for intrathecal administration. In certain embodiments, the pharmaceutical compositions are suitable for intraci sternal administration (injection into the cisterna magna). In certain embodiments, the pharmaceutical compositions are suitable for injection into the subarachnoid space via a Cl -2 puncture. In certain embodiments, the pharmaceutical compositions are suitable for intracerebroventricular administration. In certain embodiments, the pharmaceutical compositions are suitable for administration via lumbar puncture. In some embodiments, the pharmaceutical composition comprising the rAAV of the present disclosure comprises sodium chloride at a concentration of about 8.77 g / L, magnesium chloride 6-hydrate, at a concentration of about 0.244 g / L, potassium chloride at a concentration of about 0.224 g / L, calcium chloride dihydrate at a concentration of about 0.206 g / L, dextrose anhydrous at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.010 g / L, sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L.

[0104] Therapeutically effective doses of the recombinant vector should be administered to the CSF via intrathecal administration (i.e., injection into the subarachnoid space so that the recombinant vectors distribute through the CSF and transduce cells in the CNS). In some embodiments, the recombinant vector is administered in a solution comprising sodium chloride at a concentration of about 8.77 g / L, magnesium chloride 6-hydrate, at a concentration of about 0.244 g / L, potassium chloride at a concentration of about 0.224 g / L, calcium chloride dihydrate at a concentration of about 0.206 g / L, dextrose anhydrous at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.010 g / L, sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L. This can be accomplished in a number of ways - e.g., by intracranial (cisternal or ventricular) injection , or injection into the lumbar cistern. For example, intracistemal (IC) injection (into the cistema magna) can be performed by CT-guided suboccipital puncture; or injection into the subarachnoid space can be performed via a Cl -2 puncture when feasible for the patient; or lumbar puncture (typically diagnostic procedures performed in order to collect a sample of CSF) can be used to access the CSF. Alternatively, intracerebroventricular (ICV) administration (a more invasive technique used for the introduction of anti-infective or anticancer drugs that do not penetrate the blood-brain barrier) can be used to instill the recombinant vectors directly into the ventricles of the brain. Alternatively, intranasal administration may be used to deliver the recombinant vector to the CNS.

[0105] Because of the relatively rapid brain growth that occurs early in a developing child, the total dose of AAV9.hIDS administered IC depends on the assumed brain mass across different age strata, see, e.g, Table 2 below. For brain mass by age for the study subjects see, e.g., AS Dekaban, Ann Neurol, 1978 Oct; 4(4): 345-56.Table 2. Total dose administered by age*GC was determined using a Poly-A-specific PCR assay

[0106] CSF concentrations can be monitored by directly measuring the concentration of rhIDS in the CSF fluid obtained from occipital or lumbar punctures or estimated by extrapolation from concentrations of the rhIDS detected in the patient’s serum.

[0107] By way of background, human IDS is translated as a 550 amino acid polypeptide that contains eight potential N-glycosylation sites (N31, N115, N144, N246, N280, N325, N513and N537) depicted in FIG. 1 and includes a 25 amino acid signal sequence which is cleaved during processing. An initial 76 kDa intracellular precursor is converted into a phosphorylated 90 kDa precursor after modification of its oligosaccharide chains in the Golgi apparatus. This precursor is processed by glycosylation modifications and proteolytic cleavage through various intracellular intermediates to a major 55 kDa form. To summarize, after removal of the 25 aa signal sequence, proteolytic processing involves N-terminal proteolytic cleavage downstream of N31removing a propeptide of eight amino acids (residues 26-33), and C-terminal proteolytic cleavage upstream of N513which releases an 18 kDa polypeptide and produces a 62 kDa intermediate that is converted to a 55 kDa mature form. Further proteolytic cleavage yields a 45 kDa mature form located in the lysosomal compartment. (See FIG. 4 for diagram reproduced from Millat et al., 1997, Exp Cell Res 230: 362-367 (“Millat 1997”); Millat et al. 1997, Biochem J. 326: 243-247 (“Millat 1997a”); and Froissart et al., 1995, Biochem J. 309:425-430, each of which is incorporated by reference herein in its entirety).

[0108] A formylglycine modification of C84(shown in bold in FIG. 1) required for enzyme activity probably occurs as an early post-translational or co-translational event, most probably in the endoplasmic reticulum. (See, Millat 1997a, citing Schmidt et al., 1995, Cell 82: 271- 278). Post-translational processing continues in the Golgi to include addition of complexsialic acid-containing glycans and acquisition of mannose-6-phosphate residues which tag the enzyme for delivery to the lysosomal compartment. (See, Clarke, 2008, Expert Opin Pharmacother 9: 311-317 for a concise review which is incorporated by reference herein in its entirety). While no single glycosylation site is essential for IDS stability, glycosylation at position N280is important for cellular internalization and lysosomal targeting via the mannose-6-phosphate (M6P) receptor. (Chung et al., 2014, Glycoconj J 31 :309-315 at p. 310, first column). In the normal physiologic state, IDS is produced at very low levels and very little, if any, enzyme is secreted from the cell. (Clarke, 2008, supra).

[0109] The invention is based, in part, on the following principles:(i) Neuronal and glial cells in the CNS are secretory cells that possess the cellular machinery for post-translational processing of secreted proteins - including glycosylation, mannose-6-phosphorylation, and tyrosine-O-sulfation - robust processes in the CNS. See, e.g., Sleat et al., 2005, Proteomics 5: 1520-1532, and Sleat 1996, J Biol Chem 271 : 19191-98 which describes the human brain mannose-6-phosphate glycoproteome and notes that the brain contains more proteins with a much greater number of individual isoforms and mannose-6- phosphorylated proteins than found in other tissues; and Kanan et al., 2009, Exp. Eye Res. 89: 559-567 and Kanan & Al-Ubaidi, 2015, Exp. Eye Res. 133: 126-131 reporting the production of tyrosine-sulfated glycoproteins secreted by neuronal cells, each of which is incorporated by reference in its entirety for post- translational modifications made by human CNS cells.(ii) The human brain produces multiple isoforms of natural / native IDS. In particular, N-terminal sequencing of human brain mannose-6-phosphorylated glycoproteins revealed that the N-terminal sequence of the mature 42 kDa chain of hIDS varies in the brain, starting at positions 34 or 36 as follows: T34DALNVLLI (SEQ ID NO: 54); and A36LNVLLIIV (SEQ ID NO: 55). (Sleat, 2005, Proteomics 5: 1520- 1532, Table S2). Two of the eight N-linked glycosylation sites, namely N280and N116, were found to be mannose-6-phophorylated in IDS obtained from human brain. (Sleat et al., 2006, Mol & Cell Proeomics 5.4: 686-701, reported at Table V).(iii) During processing of hIDS, two polypeptides, 76 kDa and 90 kDa, are secreted by neural and glial cells, but only the 90 kDa polypeptide is mannose-6- phosphorylated, which is necessary for secreted forms of the enzyme to achieve cross correction. (See, Millat, 1997, Fig. 1 results for transduced lymphoblastoidcells, and Froissart 1995, Fig. 4 showing similar results for transduced fibroblasts - in culture medium, only the 90 kDa form is phosphorylated). Interestingly, it has been demonstrated that recombinant IDS produced by neuronal and glial cells may be endocytosed by recipient CNS cells more avidly than recombinant IDS produced by other cells such as kidney. Daniele 2002 demonstrated M6P-receptor mediated endocytosis of recombinant IDS from conditioned media of transduced neuronal and glial cell cultures by a recipient population of non-transduced neuronal and glial cells which properly processed the precursor to the 45 kDa mature active form. Uptake of the recombinant IDS produced by the neuronal and glial cell lines (74% endocytosis) far exceeded uptake of the enzyme produced by a kidney cell line (5.6% endocytosis). In each case, uptake was inhibited by M6P, indicating that recombinant IDS uptake was M6P -receptor mediated. (See Daniele 2002, Tables 2 and 4 and accompanying description in Results at pp. 205- 206 summarized in Table 3 below).Table 3. Summary of Results Reported in Daniele 2002(iv) The gene therapy approach described herein should result in the continuous secretion of an hIDS glycoprotein precursor of about 90 kDa as measured by polyacrylamide gel electrophoresis (depending on the assay used) that is enzymatically active. First, the enzyme responsible for the formylglycine modification of C84which is required for IDS activity — the FGly-Generating Enzyme (FGE, aka SUMF1) — is expressed in the cerebral cortex of the human brain (gene expression data for SUMF1 may be found, for example, at GeneCards, accessible at http: / / www.genecards.org). Second, the secreted glycosylated / phosphorylated rIDS produced by transduced neurons and glial cells in situ should be taken up and correctly processed by untransduced neural and glial cells in the CNS. Without being bound to any theory, it appears that the secreted rhIDS precursor produced in situ by gene therapy may be more avidly endocytosed by recipient cells in the CNS than would traditional recombinantenzymes used for ERT if administered to the CNS. For example, Elaprase® (made in HT1080, a fibrosarcoma cell line) is a purified protein reported to have a molecular weight of about 76 kDa - not the 90 kDa species secreted by neuronal and glial cells that appears to be more heavily phosphorylated. While the eight N- linked glycosylation sites are reported to be fully occupied in Elaprase® and contain two bis-mannose-6-phosphate terminated glycans as well as complex highly sialylated glycans, the post-translational modification of C84to FGly, which is an absolute requirement for enzyme activity, is only about 50%. (Clarke, 2008, Expert Opin Pharmacother 9:311-317; Elaprase® Full Prescribing Information and EMA filing). Another recombinant product, Hunterase® is made in CHO cells. While reported to have higher FGly and activity than Elaprase®, mannose-6-phosphorylation and uptake did not differ. (Chung, 2014, Glycoconj J 31 :309-315).(v) The extracellular IDS efficacy in vivo depends on uptake (cell and lysosome internalization) through mannose-6-phosphate (M6P) and its active site formylglycine (FGly), which is converted from C84through post-translational modification by formylglycine-generating enzyme. As shown above in Table 3, brain cells (neuronal and glial cells) show higher enzyme activities when incubated with IDS precursor media secreted by transduced neuronal and glial cells than with IDS precursor media secreted by genetically engineered kidney cells. The resultant five-fold increase in activity can likely be attributed to the efficient uptake of IDS (See Daniele 2002, Tables 2 and 4). Commercial forms of IDS, which are generated by CHO cells or HT-1080 cells, have a FGly content of about 50% to 70%, which determines the enzyme activity. However, neuronal and glial cells may improve upon this activity, due to improvement of IDS uptake.(vi) The cellular and subcellular trafficking / uptake of lysosomal proteins, including IDS, is through M6P. IDS from brain cells may contain higher M6P content, as reported in Daniele 2002, and in Sleat, Proteomics, 2005 (indicating that the human brain contains more (in both a quantitative and qualitative sense) Man6-P glycoproteins than other tissues.). It is possible to measure the M6P content of an IDS precursor, as done in Daniele 2002. In the presence of inhibitory M6P (e.g., 5 mM), the uptake of IDS precursor generated by non-neuronal or non-glial cells, such as the genetically engineered kidney cells of Daniele 2002, is predicted to decrease to levels close to that of the control cells, as was shown in Daniele 2002.While in the presence of inhibitory M6P, the uptake of IDS precursor generated by brain cells, such as neuronal and glial cells, is predicted to remain at a high level, as was shown in Daniele 2002, where the uptake was four times higher than control cells and comparable to the level of IDS activity (or uptake) of IDS precursor generated by genetically engineered kidney cells without the presence of inhibitory M6P. This assay allows for a way to predict the M6P content in IDS precursor generated by brain cells, and, in particular, to compare the M6P content in IDS precursors generated by different types of cells. The gene therapy approach described herein should result in the continuous secretion of an hIDS precursor that may be taken up into neuronal and glial cells at a high level in the presence of inhibitory M6P in such an assay.(vii) The M6P content and uptake of IDS precursor may also be demonstrated by 90 kDa and 76 kDa gel bands (e.g., SDS-PAGE gel bands). The 90 kDa is reported to be highly glycosylated / phosphorylated and contains M6P, while 76 kDa is not. A very broad gel band with a range from 76 kDa to 95 kDa and with an average MW of 80-85 kDa, similar to the IDS precursor gel band generated from genetically engineered kidney cells (Daniele 2002, Figure 1), may be contrasted with a gel band of IDS precursor generated from brain cells. In Daniele 2002, the gel band cannot be obtained due to unsuccessful immunoprecipitation of the IDS precursor. The gene therapy approach described herein should result in the continuous secretion of an hIDS precursor that differs from the IDS precursor gel band generated from genetically engineered kidney cells.(viii) The M6P content of commercial IDS precursor is 2 to 2.5 mol / mol, majority of which is present in a form of di-phosphorylated glycans. Although in average, every IDS precursor is phosphorylated, a normal distribution of glycans will have some IDS precursor with 2, 1 and 0 of di-phosphorylated M6P glycans assuming multiple phosphorylation sites. Uptake rate should be significant higher with multiple phosphorylation.(ix) The glycosylation of hIDS by human cells of the CNS will result in the addition of glycans that can improve stability, half-life and reduce unwanted aggregation of the transgene product. Significantly, the glycans that are added to hIDS of the invention include 2,6-sialic acid, incorporating Neu5Ac (“NANA”) but not its hydroxylated derivative, NeuGc (N-Glycolylneuraminic acid, i.e., “NGNA” or “Neu5Gc”). Such glycans are not present in recombinant IDS products, such asHunterase®, made in CHO cells because CHO cells do not have the 2,6- sialyltransferase required to make this post-translational modification; nor do CHO cells produce bisecting GlcNAc, although they do add Neu5Gc (NGNA) as sialic acid not typical (and potentially immunogenic) to humans instead of Neu5Ac (NANA). See, e.g., Dumont et al., 2016, Critical Rev in Biotech 36(6): 1110-1122 (Early Online pp. 1-13 at p. 5); and Hague et al., 1998 Electrophor 19:2612-2630 (“[t]he CHO cell line is considered ‘phenotypically restricted,’ in terms of glycosylation, due to the lack of an a2,6-sialyl- transferase”). Moreover, CHO cells can also produce an immunogenic glycan, the a-Gal antigen, which reacts with anti-a-Gal antibodies present in most individuals, and at high concentrations can trigger anaphylaxis. See, e.g., Bosques, 2010, Nat Biotech 28: 1153-1156. The human glycosylation pattern of the rhIDS of the invention should reduce immunogenicity of the transgene product and improve efficacy.(x) Immunogenicity of a transgene product could be induced by various factors, including the immune condition of the patient, the structure and characteristics of the infused protein drug, the administration route, and the duration of treatment. Process-related impurities, such as host cell protein (HCP), host cell DNA, and chemical residuals, and product-related impurities, such as protein degradants and structural characteristics, such as glycosylation, oxidation and aggregation (sub- visible particles), may also increase immunogenicity by serving as an adjuvant that enhances the immune response. The amounts of process-related and product- related impurities can be affected by the manufacturing process: cell culture, purification, formulation, storage and handling, which can affect commercially manufactured IDS products. In gene therapy, proteins are produced in vivo, such that process-related impurities are not present and protein products are not likely to contain product-related impurities / degradants associated with proteins produced by recombinant technologies, such as protein aggregation and protein oxidation. Aggregation, for example, is associated with protein production and storage due to high protein concentration, surface interaction with manufacturing equipment and containers, and the purification process with certain buffer systems. But these conditions that promote aggregation are not present when a transgene is expressed in vivo. Oxidation, such as methionine, tryptophan and histidine oxidation, is also associated with protein production and storage, caused, for example, by stressedcell culture conditions, metal and air contact, and impurities in buffers and excipients. The proteins expressed in vivo may also oxidize in a stressed condition, but humans, like many organisms, are equipped with an antioxidation defense system, which not only reduces the oxidation stress, but can also repairs and / or reverses the oxidation. Thus, proteins produced in vivo are not likely to be in an oxidized form. Both aggregation and oxidation could affect the potency, pharmacokinetics (clearance) and can increase immunogenicity concerns. The gene therapy approach described herein should result in the continuous secretion of an hIDS precursor with a reduced immunogenicity compared to commercially manufactured products.(xi) In addition to the N-linked glycosylation sites, hIDS contains a tyrosine (“Y”) sulfation site (PSSEKY165ENTKTCRGPD (SEQ ID NO: 47)). (See, e.g., Yang et al., 2015, Molecules 20:2138-2164, esp. at p. 2154 which is incorporated by reference in its entirety for the analysis of amino acids surrounding tyrosine residues subjected to protein tyrosine sulfation. The “rules” can be summarized as follows: Y residues with E or D within +5 to -5 position of Y, and where position -1 of Y is a neutral or acidic charged amino acid - but not a basic amino acid, e.g., R, K, or H that abolishes sulfation). While not intending to be bound by any theory, sulfation of this site in hIDS may improve stability of the enzyme and binding affinity for substrate. Tyrosine-sulfation of hIDS - a robust post- translational process in human CNS cells - should result in improved processing and activity of transgene products. The significance of tyrosine-sulfation of lysosomal proteins has not been elucidated; but in other proteins it has been shown to increase avidity of protein-protein interactions (antibodies and receptors), and to promote proteolytic processing (peptide hormone). (See, Moore, 2003, J Biol. Chem. 278: 24243-46; and Bundegaard et al., 1995, The EMBO J 14: 3073-79). The tyrosylprotein sulfotransferase (TPST1) responsible for tyrosine-sulfation (which may occur as a final step in IDS processing) is apparently expressed at higher levels (based on mRNA) in the brain (gene expression data for TPST1 may be found, for example, at the EMBL-EBI Expression Atlas, accessible at http: / / www.ebi.ac.uk / gxa / home). Such post- translational modification, at best, is under-represented in CHO cell products. Unlike human CNS cells, CHO cells are not secretory cells and have a limitedcapacity for post-translational tyrosine-sulfation. (See, e.g., Mikkelsen & Ezban, 1991, Biochemistry 30: 1533-1537, esp. discussion at p. 1537).

[0110] For the foregoing reasons, the production of rhIDS by human neuronal and / or glial cells should result in a “biobetter” molecule for the treatment of MPS II accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding rhIDS to the CSF of a patient (human subject) diagnosed with an MPS II disease (including but not limited to Hunter) to create a permanent depot in the CNS that continuously supplies a fully human-glycosylated, mannose-6-phosphorylated, sulfated transgene product secreted by the transduced CNS cells. The hIDS transgene product secreted from the depot into the CSF will be endocytosed by cells in the CNS, resulting in “cross-correction” of the enzymatic defect in the MPS II recipient cells.

[0111] It is not essential that every rhIDS molecule produced either in the gene therapy or protein therapy approach be fully glycosylated, phosphorylated, and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation (including 2,6- sialylation and mannose-6-phosphorylation) and sulfation to demonstrate efficacy. The goal of gene therapy treatment of the invention is to slow or arrest the progression of disease. Efficacy may be monitored by measuring cognitive function (e.g., prevention or decrease in neurocognitive decline); reductions in biomarkers of disease (such as GAG) in CSF and or serum; and / or increase in IDS enzyme activity in CSF and / or serum. Signs of inflammation and other safety events may also be monitored.

[0112] As an alternative, or an additional treatment to gene therapy, the rhIDS glycoprotein can be produced in human neural or glial cell lines by recombinant DNA technology and the glycoprotein can be administered to patients diagnosed with MPS II systemically and / or into the CSF for ERT). Human cell lines that can be used for such recombinant glycoprotein production include but are not limited to HT-22, SK-N-MC, HCN-1 A, HCN-2, NT2, SH- SY5y, hNSCl 1, or ReNcell VM (see, e.g., Dumont et al., 2016, Critical Rev in Biotech 36(6): 1110-1122 “Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives” which is incorporated by reference in its entirety for a review of the human cell lines that could be used for the recombinant production of the rHuGlylDS glycoprotein). To ensure complete glycosylation, especially sialylation, and tyrosinesulfation, the cell line used for production can be enhanced by engineering the host cells to co-express a-2,6-sialyltransferase (or both a-2,3- and a-2,6-sialyltransferases) and / or TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation.

[0113] While the delivery of rhIDS should minimize immune reactions, the clearest potential source of toxicity related to CNS-directed gene therapy is generating immunity against the expressed rhIDS protein in human subjects who are genetically deficient for IDS and, therefore, potentially not tolerant of the protein and / or the vector used to deliver the transgene.

[0114] Thus, in a preferred embodiment, it is advisable to co-treat the patient with immune suppression therapy — especially when treating patients with severe disease who have close to zero levels of IDS. Immune suppression therapies involving a regimen of tacrolimus or rapamycin (sirolimus) in combination with mycophenolic acid, or other immune suppression regimens used in tissue transplantation procedures can be employed. Such immune suppression treatment may be administered during the course of gene therapy, and in certain embodiments, pre-treatment with immune suppression therapy may be preferred. Immune suppression therapy can be continued subsequent to the gene therapy treatment, based on the judgment of the treating physician, and may thereafter be withdrawn when immune tolerance is induced; e.g., after 180 days.

[0115] Combinations of delivery of the rhIDS to the CSF accompanied by delivery of other available treatments are encompassed by the methods of the invention. The additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment. Available treatments for MPS II that could be combined with the gene therapy of the invention include but are not limited to enzyme replacement therapy using Elaprase® administered systemically or to the CSF; and / or HSCT therapy.

[0116] In certain embodiments, described herein is a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising delivering to the cerebrospinal fluid (CSF) of said human subject a therapeutically effective amount of a recombinant human iduronate-2-sulfatase (IDS) precursor produced by human neuronal or human glial cells.

[0117] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising delivering to the cerebrospinal fluid (CSF) of said human subject, a therapeutically effective amount of a recombinant human iduronate-2-sulfatase (IDS) glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, has a formylglycine residue at C84(Fig. 1), is a2,6-sialylated, does not contain detectable NeuGc, and is mannose-6- phosphorylated.

[0118] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising delivering to the cerebrospinal fluid (CSF) of said human subject, a therapeutically effective amount of a recombinant human iduronate-2-sulfatase (IDS) glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, has a formylglycine residue at C84(Fig. 1), is a2,6-sialylated, does not contain detectable NeuGc and / or a-Gal antigen, and is mannose-6-phosphorylated.

[0119] In certain embodiments, the human IDS precursor is delivered to the CSF from a depot of cells in the central nervous system genetically engineered to secrete said IDS precursor into the CSF. In certain embodiments, the depot is formed in the subject’s brain. In certain embodiments, the human subject is deficient in IDS activity. In certain embodiments, the human IDS comprises the amino acid sequence of SEQ ID NO. 1.

[0120] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering to the cerebrospinal fluid (CSF) of said human subject a recombinant nucleotide expression vector encoding human iduronate-2-sulfatase (IDS), wherein said expression vector when used to transduce a primary human neuronal cell in culture directs the expression of a secreted human IDS glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, has a formylglycine residue at C84(Fig. 1), is a2,6-sialylated and mannose-6-phosphorylated.

[0121] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering to the cerebrospinal fluid of the brain of said human subject, a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS, so that a depot is formed in the subject’s central nervous system that secretes a recombinant human IDS glycoprotein precursor that is a2,6-sialylated and mannose-6-phosphorylated.

[0122] In certain embodiments, secretion of said recombinant human IDS glycoprotein precursor that is a2,6-sialylated is confirmed by transducing a human neuronal cell line with said recombinant nucleotide expression vector in cell culture. In certain embodiments, secretion of said recombinant human IDS glycoprotein precursor that is mannose-6- phosphorylated is confirmed by transducing a human neuronal cell line with said recombinantnucleotide expression vector in cell culture. In certain embodiments, the secretion is confirmed in the presence and absence of mannose-6-phosphate.

[0123] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering to the cerebrospinal fluid of the brain of said human subject, a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS, so that a depot is formed that secretes a glycosylated IDS precursor containing a a2,6-sialylated glycan; wherein said recombinant vector, when used to transduce human neuronal cells in culture results in secretion of said glycosylated IDS precursor containing a a2,6-sialylated glycan in said cell culture.

[0124] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering to the cerebrospinal fluid of the brain of said human subject, a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS, so that a depot is formed that secretes a glycosylated IDS precursor that contains a mannose-6-phosphate; wherein said recombinant vector, when used to transduce human neuronal cells in culture results in secretion of said glycosylated IDS precursor that is mannose-6-phosphorylated in said cell culture.

[0125] In certain embodiments, described herein is a method of treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering to the cerebrospinal fluid of the brain of said human subject, a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS, so that a depot is formed that secretes a glycosylated IDS precursor that contains a formylglycine; wherein said recombinant vector, when used to transduce human neuronal cells in culture results in secretion of said glycosylated IDS precursor that contains a formylglycine in said cell culture.

[0126] In certain embodiments, the human IDS comprises the amino acid sequence of SEQ ID NO. 1. In certain embodiments, the IDS transgene encodes a leader peptide. In certain embodiments, the expression vector is a replication defective AAV vector. In certain embodiments, the expression vector is delivered to the CSF of the subject by intrathecal (e.g., intracisternal, Cl -2 puncture if feasible for the patient, or lumbar puncture), intracerebroventricular, or intranasal administration. In certain embodiments, the human subject is deficient in IDS activity. In some embodiments, the expression vector is delivered to the CSF of the subject by intrathecal administration in a solution comprising sodium chloride at a concentration of about 8.77 g / L, magnesium chloride 6-hydrate, at aconcentration of about 0.244 g / L, potassium chloride at a concentration of about 0.224 g / L, calcium chloride dihydrate at a concentration of about 0.206 g / L, dextrose anhydrous at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.010 g / L, sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L.

[0127] In preferred embodiments, the glycosylated IDS does not contain detectable NeuGc and / or a-Gal. The phrase “detectable NeuGc and / or a-Gal” used herein means NeuGc and / or a-Gal moieties detectable by standard assay methods known in the art. For example, NeuGc may be detected by HPLC according to Hara et al., 1989, “Highly Sensitive Determination of N-Acetyl-and N-Glycolylneuraminic Acids in Human Serum and Urine and Rat Serum by Reversed-Phase Liquid Chromatography with Fluorescence Detection.” J. Chromatogr., B: Biomed. 377: 111-119, which is hereby incorporated by reference for the method of detecting NeuGc. Alternatively, NeuGc may be detected by mass spectrometry. The a-Gal may be detected using an ELISA, see, for example, Galili et al., 1998, “A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody.” Transplantation. 65(8): 1129-32, or by mass spectrometry, see, for example, Ayoub et al., 2013, “Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques.” Landes Bioscience. 5(5): 699-710. See also the references cited in Platts-Mills et al., 2015, “Anaphylaxis to the Carbohydrate Side-Chain Alpha-gal” Immunol Allergy Clin North Am. 35(2): 247-260.

[0128] In one aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising delivering to the CSF of the human subject a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or human glial cells, wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain MRI of the human subject’s brain.

[0129] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising determining the human subject’s brain mass from the human subject’s brain MRI, and subsequently delivering to the CSF of the human subject a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal cells or human glial cells, wherein the glycosylatedrecombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, and wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass.

[0130] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising (a) determining the human subject’s brain mass from the human subject’s brain MRI, (b) calculating the dose based on the human subject’s brain mass, and (c) subsequently administrating to the CSF of the subject the dose of recombinant nucleotide expression vector encoding human IDS.

[0131] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising, in the following order: (a) delivering to the CSF of the human subject a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or human glial cells; (b) measuring level of heparan sulfate in the CSF of the human subject; and (c) comparing the level of heparan sulfate in the CSF of the human subject with level of heapran sulfate in a reference population; wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain magnetic resonance imaging (MRI) of the human subject’s brain. In certain embodiments, the reference population consists of: (a) at least 1, 2, 3, 4, 5, 10, 25, 50, 75, 100, 200, 250, 300, 400, 500, or 1000 individual healthy people without MPS II, preferably of similar age, weight, and / or of the same gender as the human subject.

[0132] In another aspect, provided herein is a method for treating a human subject diagnosed with MPS II, comprising, in the following order: (a) taking a first measurement of the level of heparan sulfate in the CSF of the human subject; (b) delivering to the CSF of the human subject a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal or human glial cells; and (c) after a period of time, taking a second measurement of the level of heparan sulfate; wherein the glycosylated recombinant human IDS precursor is delivered by administration of a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain magnetic resonance imaging (MRI) of the human subject’s brain. In certain embodiments, the period of time is about 1 day, 2 days, 3days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 moths, 4 months, 5 months, 6 months, 7 months, 8 months, 11 months, or 1 year.

[0133] In a preferred embodiment, the glycosylated recombinant human IDS precursor will be endocytosed by cells in the CNS. In a preferred embodiment, the glycosylated recombinant human IDS precursor is delivered to lysosomes of cells in the CNS of the human subject.

[0134] In certain embodiments of the method for treating described herein, the human subject’s brain mass is converted from the human subject’s brain volume by multiplying the human subject’s brain volume in cm3by a factor of 1.046 g / cm3, wherein the human subject’s brain volume is determined by brain MRI of the subject’s brain.

[0135] In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x 1010GC / g brain mass as determined by MRI, or about 6.5 x 1O10GC / g brain mass as determined by MRI. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x io10GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x io10GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x 1O10GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x io10GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x io11GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x io11GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay).

[0136] In various embodiments of the method for treating described herein, the human subject is 5 years old or older and less than 18 years old. In specific embodiments, the human subject is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 years old. In specific embodiments, the human subject is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 years old. In specific embodiments, the human subject is 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13- 14, 14-15, 15-16, 16-17, 17-18 or 18-19 years old. In specific embodiments, the human subject is about 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17- 18 or 18-19 years old. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x io10GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose according to Table 7.

[0137] In various embodiments of the method for treating described herein, the human subject is 4 months old or older and less than 5 years old. In specific embodiments, the human subject is 4, 5, 6, 7, 8, 9, 10, or 11 months old. In specific embodiments, the human subject is about 4, 5, 6, 7, 8, 9, 10, or 11 months old. In specific embodiments, the human subject is 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months old. In specific embodiments, the human subject is about 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months old. In specific embodiments, the human subject is 1, 2, 3, 4, or 5 years old. In specific embodiments, the human subject is about 1, 2, 3, 4, or 5 years old. In specific embodiments, the human subject is 1-2, 2-3, 3-4, 4-5, or 5-6 years old. In specific embodiments, the human subject is about 1-2, 2-3, 3-4, 4-5, or 5-6 years old. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x io10GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x io10GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x io11GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.9 x io11GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x io10GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x io10GC / g brain mass as determined by MRI. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 2.0 x 1011GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments, therecombinant nucleotide expression vector is administered at a dose of about 2.9 x io11GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.3 x io10GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 1.9 x io10GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 6.5 x io10GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a Poly-A-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of about 9.6 x io10GC / g brain mass (e.g., the brain mass is determined by MRI and the genome count is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose chosen from Dose 1 or Dose 2 according to Table 5. In certain embodiments, the recombinant nucleotide expression vector is administered at a dose according to Table 6.

[0138] In some embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered via intraci sternal (IC) administration. In other embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered via intracerebroventricular (ICV) administration.

[0139] In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is administered at a volume that does not exceed 10% of the total cerebrospinal fluid volume of the human subject.

[0140] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is secreted at a detectable level.

[0141] In certain embodiments of the method for treating described herein, the human neuronal or human glial cells carry at least one mutation in the endogenous gene encoding human IDS precursor.

[0142] In certain embodiments of the method for treating described herein, the human neuronal or human glial cells are transduced with a recombinant adeno-associated virus vector (rAAV).

[0143] In a preferred embodiment, the recombinant nucleotide expression vector is an AAV9 or AAVrhlO vector.

[0144] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is expressed under the control of a CB7 promoter.

[0145] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is expressed from a cDNA encoding human IDS precursor.

[0146] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor is about 90 kDa as measured by polyacrylamide gel electrophoresis.

[0147] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor contains a formylglycine.

[0148] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor (a) is a2,6-sialylated; (b) does not contain detectable NeuGc; (c) does not contain detectable a-Gal antigen; (d) contains tyrosine-sulfation; and / or (e) is mannose-6-phosphorylated.

[0149] In certain embodiments of the method for treating described herein, the glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO. 1.

[0150] In certain embodiments provided herein, the method further comprising administering an immune suppression therapy to the human subject before or concurrently with the human IDS precursor treatment and optionally continuing immune suppression therapy thereafter.

[0151] In some embodiments, the immune suppression therapy comprises administering one or more corticosteroids, sirolimus, and / or tacrolimus. In a specific embodiment, the one or more corticosteroids are methylprednisolone and / or prednisone.

[0152] In a specific embodiment, the immune suppression therapy comprises administering prednisone at a dose of about 0.10 mg / kg, 0.11 mg / kg, 0.12 mg / kg, 0.13 mg / kg, 0.14 mg / kg, 0.15 mg / kg, 0.16 mg / kg, 0.17 mg / kg, 0.18 mg / kg, 0.19 mg / kg, 0.20 mg / kg, 0.21 mg / kg, 0.22 mg / kg, 0.23 mg / kg, 0.24 mg / kg, 0.25 mg / kg, 0.26 mg / kg, 0.27 mg / kg, 0.28 mg / kg, 0.29 mg / kg, 0.30 mg / kg, 0.31 mg / kg, 0.32 mg / kg, 0.33 mg / kg, 0.34 mg / kg, 0.35 mg / kg, 0.36 mg / kg, 0.37 mg / kg, 0.38 mg / kg, 0.39 mg / kg, 0.40 mg / kg, 0.41 mg / kg, 0.42 mg / kg, 0.43 mg / kg, 0.44 mg / kg, 0.45 mg / kg, 0.46 mg / kg, 0.47 mg / kg, 0.48 mg / kg, 0.49 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or 1 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering prednisone at a dose ranging from about 0.10 mg / kg to about 0.20 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering prednisone at a dose ranging from about 0.20 mg / kg toabout 0.30 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering prednisone at a dose ranging from about 0.30 mg / kg to about 0.40 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering prednisone at a dose ranging from about 0.40 mg / kg to about 0.50 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering prednisone at a dose ranging from about 0.50 mg / kg to about 1 mg / kg. In a particular embodiment, the dose is administered daily. In a particular embodiment, the immune suppression therapy comprises administering prednisone at a dose of 0.5 mg / kg daily. In another particular embodiment, the immune suppression therapy comprises administering prednisone at a dose of 0.5 mg / kg daily with gradual tapering and discontinuation.

[0153] In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose of about 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 0.50 mg / kg to about 1.0 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 1.0 mg / kg to about 2.0 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 2.0 mg / kg to about 3.0 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 3.0 mg / kg to about 5.0 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 5.0 mg / kg to about 10.0 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 10.0 mg / kg to about 15.0 mg / kg. In a specific embodiment, the immune suppression therapy comprises administering methylprednisolone at a dose ranging from about 15.0 mg / kg to about 20.0 mg / kg. In a particular embodiment, the methylprednisolone is administered once. In a particular embodiment, the methylprednisolone is administered intravenously. In a particular embodiment, the methylprednisolone is administered for a maximum of 500 mg, In a particular embodiment, the methylprednisolone s administered over at least 30 minutes. In a particular embodiment, the immune suppression therapycomprises administering methylprednisolone at a dose of 10 mg / kg IV for maximum of 500 mg over at least 30 minutes.

[0154] In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose to maintain a target blood level of 1-3 ng / mL. In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose of about 0.25 mg / m2 / day, 0.3 mg / m2 / day, 0.4 mg / m2 / day, 0.5 mg / m2 / day, 0.6 mg / m2 / day, 0.7 mg / m2 / day, 0.8 mg / m2 / day, 0.9 mg / m2 / day, 1 mg / m2 / day, 1.25 mg / m2 / day, 1.5 mg / m2 / day, 1.75 mg / m2 / day, 2 mg / m2 / day, 2.25 mg / m2 / day, 2.5 mg / m2 / day, 2.75 mg / m2 / day, 3 mg / m2 / day, 3.25 mg / m2 / day, 3.5 mg / m2 / day, 3.75 mg / m2 / day, 4 mg / m2 / day, 4.25 mg / m2 / day, 4.5 mg / m2 / day, 4.75 mg / m2 / day, or 5 mg / m2 / day. In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose ranging from about 0.25 mg / m2 / day to about 0.5 mg / m2 / day. In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose ranging from about 0.50 mg / m2 / day to about 1.0 mg / m2 / day. In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose ranging from about 1.0 mg / m2 / day to about 1.5 mg / m2 / day. In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose ranging from about 1.5 mg / m2 / day to about 2 mg / m2 / day. In a specific embodiment, the immune suppression therapy comprises administering sirolimus at a dose ranging from about 2 mg / m2 / day to about 5 mg / m2 / day. In a particular embodiment, the dose is divided in BID dosing. In a particular embodiment, the immune suppression therapy comprises administering sirolimus at a dose of about 1 mg / m2 / day every 4 hours. In a particular embodiment, the immune suppression therapy comprises administering sirolimus at a dose of about 0.5 mg / m2 / day divided in BID dosing.

[0155] In a specific embodiment, the immune suppression therapy comprises administering tacrolimus at a dose to maintain a target blood level of 2-4 ng / mL. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose of about 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg 0.04 mg / kg 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, or 0.10 mg / kg. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose ranging from 0.01 mg / kg to 0.02 mg / kg. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose ranging from 0.02 mg / kg to 0.03 mg / kg. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose ranging from 0.03 mg / kg to 0.05 mg / kg. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose ranging from 0.05 mg / kg to 0.07mg / kg. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose ranging from 0.07 mg / kg to 0.10 mg / kg. In a particular embodiment, the dose is administered twice daily. In a particular embodiment, the immune suppression therapy comprises administering tacrolimus at a dose of about 0.05 mg / kg twice daily.

[0156] In some embodiments, the method further comprises administering one or more antibiotics to the human subject before or concurrently with the immune suppression therapy. In a specific embodiment, the one or more antibiotics are trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atovaquone. In another specific embodiment, the one or more antibiotics are trimethoprim and / or sulfamethoxazole. In another specific embodiment, the one or more antibiotics are pentamidine, dapsone, and / or atovaquone. In specific embodiments, the one or more antibiotics are administered at a dose of about 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg. In specific embodiments, the one or more antibiotics are administered at a dose ranging from about 1 mg / kg to 2 mg / kg. In specific embodiments, the one or more antibiotics are administered at a dose ranging from about 2 mg / kg to 3 mg / kg. In specific embodiments, the one or more antibiotics are administered at a dose ranging from about 3 mg / kg to 5 mg / kg. In specific embodiments, the one or more antibiotics are administered at a dose ranging from about 5 mg / kg to 7 mg / kg. In specific embodiments, the one or more antibiotics are administered at a dose ranging from about 7 mg / kg to 10 mg / kg. In a specific embodiment, the one or more antibiotics are administered at a dose of about three times a week. In certain embodiments, the one or more antibiotics are administered to prevent Pneumocystis carinii pneumonia.

[0157] In some embodiments, the method further comprises administering one or more antifungal therapies to the human subject before or concurrently with the immune suppression therapy. In certain embodiments, the one or more antifungal therapies are initiated if the absolute neutrophil count is < 500 mm3.

[0158] In some embodiments, the method further comprises a step of measuring one or more of the following biomarkers after administration of the recombinant nucleotide expression vector: (a) level of glycosaminoglycans (GAGs) in CSF; (b) level of iduronate-2- sulfatase (I2S) in CSF; (c) level of GAGs in plasma; (d) level of I2S in plasma; (e) level of leukocyte I2S enzyme activity; and (f) level of GAGs in urine. In a specific embodiment, the GAGs in CSF comprise heparin sulfate in CSF. In another specific embodiment, the GAGs in CSF are heparin sulfate in CSF. In another specific embodiment, the GAGs in plasma comprise heparin sulfate in plasma. In another specific embodiment, the GAGs in plasma areheparin sulfate in plasma. In another specific embodiment, the GAGs in urine comprise heparin sulfate in urine. In another specific embodiment, the GAGs in urine are heparin sulfate in urine. In a specific embodiment, the step of measuring comprises mearing level of heparin sulfate in CSF. In another specific embodiment, the step of measuring comprises measuring level of leukocyte I2S enzyme activity.

[0159] In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is a liquid composition. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is a frozen composition. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector is a lyophilized composition or a reconstituted lyophilized composition. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector provided herein may be formulated in various dosage forms for IC or ICV administration. In certain embodiments of the method for treating described herein, the recombinant nucleotide expression vector provided herein may be provided in a unit-dosage form or multiple-dosage form. A unit-dosage form, as used herein, refers to a physically discrete unit suitable for administration to human and animal subjects, and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the said recombinant nucleotide expression vector and / or other ingredient(s) sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carriers or excipients. Examples of a unit-dosage form include an ampoule, a vial, a prefilled syringe, or a cartridge.

[0160] In certain embodiments of the method for treating described herein, a unit-dosage form may be administered in fractions or multiples thereof. In certain embodiments of the method for treating described herein, a multiple-dosage form is a plurality of identical unitdosage forms packaged in a single container to be administered in segregated unit-dosage form. Examples of a multiple-dosage form include a vial, a prefilled syringe, or a cartridge. In certain embodiments, the prefilled syringe comprises 8.5* 1012GC of the recombinant nucleotide expression vector In certain embodiments, the prefilled syringe comprises 9 8* 1012GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.1 x 1013GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.3x 1013GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.5x 1013GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.7x 1013GC of the recombinant nucleotide expression vector. Incertain embodiments, the prefilled syringe comprises 4.2* 1013GC of the recombinant nucleotide expression vector In certain embodiments, the prefilled syringe comprises 4.9x 1013GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 5.5 1013GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 6.3 x 1013GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 7.3 x 1013GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 8.5 1013GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 9. Ox 1013GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises l.Ox 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.1 x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.2x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.3x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.4x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.5x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.6x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.7x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.8x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1 9x io14of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2. Ox 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2. l x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.2x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.3 x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.4x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.5x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.6x 1014GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 1.3x 1010GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises1.9x IO10GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 6.5* IO10GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 9.6* IO10GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2. Ox 1011GC of the recombinant nucleotide expression vector. In certain embodiments, the prefilled syringe comprises 2.9x 1011GC of the recombinant nucleotide expression vector.

[0161] As used herein, the term “about” means within plus or minus 10% of a given value or range. In certain embodiments, the term “about” means within plus or minus 1% of a given value or range, wherein the value is a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain MRI of the human subject’s brain. In certain embodiments, the term “about” means within plus or minus 2% of a given value or range, wherein the value is a dose that is determined by brain MRI of the subject’s brain, and wherein the brain mass is determined by brain MRI of the human subject’s brain. In certain embodiments, the term “about” means within plus or minus 5% of a given value or range, wherein the value is a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain MRI of the human subject’s brain. In certain embodiments, the term “about” means within plus or minus 7% of a given value or range, wherein the value is a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain MRI of the human subject’s brain. In certain embodiments, the term “about” means within plus or minus 10% of a given value or range, wherein the value is a dose that is dependent on the human subject’s brain mass, and wherein the brain mass is determined by brain MRI of the human subject’s brain. However, it is to be understood that in this specification, the term “about” also affords support for recitation of the exact value with which the term is connected. For example, “about 10” also provides support for the number “10” exactly.5.1 PROCESSING, N-GLYCOSYLATION AND TYROSINE SULFATION5.1.1. Processing

[0162] Human IDS includes a 25 amino acid signal sequence which is cleaved during processing. An initial 76 kDa intracellular IDS precursor is converted into a phosphorylated 90 kDa IDS precursor after modification of its oligosaccharide chains in the Golgi apparatus. This precursor is processed by glycosylation modifications and proteolytic cleavage through various intracellular intermediates to a major 55 kDa form. To summarize, after removal ofthe 25 aa signal sequence, proteolytic processing involves N-terminal proteolytic cleavage downstream of N31removing a propeptide of eight amino acids (residues 26-33), and C- terminal proteolytic cleavage upstream of N513which releases an 18 kDa polypeptide and produces a 62 kDa intermediate that is converted to a 55 kDa mature form. Further proteolytic cleavage yields a 45 kDa mature form located in the lysosomal compartment. (See FIG. 4 for diagram reproduced from Millat et al., 1997, Exp Cell Res 230: 362-367 (“Millat 1997”); Millat et al. 1997, Biochem J. 326: 243-247 (“Millat 1997a”); and Froissart et al., 1995, Biochem J. 309:425-430, each of which is incorporated by reference herein in its entirety).

[0163] A formylglycine modification of C84(shown in bold in FIG. 1) required for enzyme activity probably occurs as an early post-translational or co-translational event, most probably in the endoplasmic reticulum. See, Millat 1997a, citing Schmidt et al., 1995, Cell 82: 271- 278). Post-translational processing continues in the Golgi to include addition of complex sialic acid-containing glycans and acquisition of mannose-6-phosphate residues which tag the enzyme for delivery to the lysosomal compartment. (See, Clarke, 2008, Expert Opin Pharmacother 9: 311-317 for a concise review which is incorporated by reference herein in its entirety).

[0164] In a specific embodiment, HuGlylDS used in accordance with the methods described herein, when expressed in a neuronal or glial cell, in vivo or in vitro, can be the 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) mannose-6-phosphorylated form of the enzyme. IDS produced from neuronal and glial cells may contain higher M6P content, as reported in Daniele 2002, and in Sleat, Proteomics, 2005 (indicating that the human brain contains more (in both a quantitative and qualitative sense) M6P glycoproteins than other tissues.). It is possible to measure the M6P content of an IDS precursor, as done in Daniele 2002.

[0165] Accordingly, in certain embodiments, HuGlylDS used in accordance with the methods described herein, when expressed in a neuronal or glial cell, in vivo or in vitro, is mannose-6-phosphorylated at a higher level than IDS expressed in a non-neuronal or glial cell. In particular, HuGlylDS used in accordance with the methods described herein, when expressed in a neuronal or glial cell, in vivo or in vitro, is mannose-6-phosphorylated at a higher level than IDS expressed in a HT1080 or CHO cell. In certain embodiments, the mannose-6-phosphorylation level of the expressed IDS is measured by uptake of the IDS by a human neuronal cell in the presence of M6P (e.g., 5 mM M6P). In certain embodiments, when expressed in a neuronal or glial cell, in vivo or in vitro, 10% - 20%, 20% - 30%, 30% -40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% of HuGlylDS molecules used in accordance with the methods described herein are mannose-6- phosphorylated.5.1.2. N-Glycosylation

[0166] Neuronal and glial cells in the CNS are secretory cells that possess the cellular machinery for post-translational processing of secreted proteins - including glycosylation and tyrosine-O-sulfation. hIDS has eight asparaginal (“N”) glycosylation sites identified in FIG. 1 (N31ST; N115FS; N144HT; N246IT; N280IS; N325ST; N513FS; N537DS). TWO of the eight N- linked glycosylation sites, namely N280and N116, are mannose-6-phophorylated in IDS obtained from human brain. (Sleat et al., 2006, Mol & Cell Proeomics 5.4: 686-701, reported at Table V).

[0167] While no single glycosylation site is essential for IDS stability, glycosylation at position N280is important for cellular internalization and lysosomal targeting via the mannose-6-phosphate (M6P) receptor. (Chung et al., 2014, Glycoconj J 31 :309-315 at p. 310, first column). In the normal physiologic state, IDS is produced at very low levels and very little, if any, enzyme is secreted from the cell. (Clarke, 2008, supra).

[0168] It is not essential that every molecule produced either in the gene therapy or protein therapy approach be fully glycosylated and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation and sulfation to demonstrate efficacy.

[0169] In a specific embodiment, HuGlylDS used in accordance with the methods described herein, when expressed in a neuronal or glial cell, in vivo or in vitro, could be glycosylated at 100% of its N-glycosylation sites. However, one of skill in the art will appreciate that not every N-glycosylation site of HuGlylDS need be N-glycosylated in order for benefits of glycosylation to be attained. Rather, benefits of glycosylation can be realized when only a percentage of N-glycosylation sites are glycosylated, and / or when only a percentage of expressed IDS molecules are glycosylated. Accordingly, in certain embodiments, HuGlylDS used in accordance with the methods described herein, when expressed in a neuronal or glial cell, in vivo or in vitro, is glycosylated at 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% of its available N-glycosylation sites. In certain embodiments, when expressed in a neuronal or glial cell, in vivo or in vitro, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% of HuGlylDS moleculesused in accordance with the methods described herein are glycosylated at least one of their available N-glycosylation sites.

[0170] In a specific embodiment, at least 10%, 20% 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites present in HuGlylDS used in accordance with the methods described herein are glycosylated at an Asn residue (or other relevant residue) present in an N-glycosylation site, when the HuGlylDS is expressed in a neuronal or glial cell, in vivo or in vitro. That is, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites of the resultant HuGlylDS are glycosylated.

[0171] In another specific embodiment, at least 10%, 20% 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites present in a HuGlylDS molecule used in accordance with the methods described herein are glycosylated with an identical attached glycan linked to the Asn residue (or other relevant residue) present in an N- glycosylation site, when the HuGlylDS is expressed in a neuronal or glial cell, in vivo or in vitro. That is, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N- glycosylation sites of the resultant HuGlylDS have an identical attached glycan.

[0172] Importantly, when the IDS proteins used in accordance with the methods described herein are expressed in neuronal or glial cells, the need for in vitro production in prokaryotic host cells (e.g., E. coli) or eukaryotic host cells (e.g., CHO cells) is circumvented. Instead, as a result of the methods described herein (e.g., use of neuronal or glial cells to express IDS), N-glycosylation sites of the IDS proteins are advantageously decorated with glycans relevant to and beneficial to treatment of humans, and, in particular, at the target location of treatment. Such an advantage is unattainable when CHO cells or E. coli are utilized in protein production, because e.g., CHO cells (1) do not express 2,6 sialyltransferase and thus cannot add 2,6 sialic acid during N-glycosylation and (2) can add Neu5Gc as sialic acid instead of Neu5 Ac; and because E. coli does not naturally contain components needed for N- glycosylation. Furthermore, such an advantage may be unattainable when human cells that are not neuronal or glial cells are utilized in protein production. Accordingly, in one embodiment, an IDS protein expressed in a neuronal or glial cell to give rise to a HuGlylDS used in the methods of treatment described herein is glycosylated in the manner in which a protein is N-glycosylated in human neuronal or glial cells, but is not glycosylated in the manner in which proteins are glycosylated in CHO cells. In another embodiment, an IDS protein expressed in a neuronal or glial cell to give rise to a HuGlylDS used in the methods of treatment described herein is glycosylated in the manner in which a protein is N-glycosylated in a neuronal or glial cells, wherein such glycosylation is not naturally possible using aprokaryotic host cell, e.g., using E. colt. In one embodiment, an IDS protein expressed in a human neuronal or glial cell to give rise to a HuGlylDS used in the methods of treatment described herein is glycosylated in the manner in which a protein is N-glycosylated in human neuronal or glial cells, but is not glycosylated in the manner in which proteins are glycosylated in human cells which are not neuronal or glial cells.

[0173] Assays for determining the glycosylation pattern of proteins are known in the art. For example, hydrazinolysis can be used to analyze glycans. First, polysaccharides are released from their associated protein by incubation with hydrazine (the Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK can be used). The nucleophile hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein and allows release of the attached glycans. N-acetyl groups are lost during this treatment and have to be reconstituted by re-N-acetylation. The free glycans can be purified on carbon columns and subsequently labeled at the reducing end with the fluorophore 2-amino benzamide. The labeled polysaccharides can be separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al, Anal Biochem 2002, 304(l):70-90. The resulting fluorescence chromatogram indicates the polysaccharide length and number of repeating units. Structural information can be gathered by collecting individual peaks and subsequently performing MS / MS analysis. Thereby the monosaccharide composition and sequence of the repeating unit can be confirmed and additionally in homogeneity of the polysaccharide composition can be identified. Specific peaks of low molecular weight can be analyzed by MALDI-MS / MS and the result used to confirm the glycan sequence. Each peak corresponds to a polymer consisting of a certain number of repeat units and fragments thereof. The chromatogram thus allows measurement of the polymer length distribution. The elution time is an indication for polymer length, while fluorescence intensity correlates with molar abundance for the respective polymer.

[0174] Homogeneity of the glycan patterns associated with proteins, as it relates to both glycan length and numbers glycans present across glycosylation sites, can be assessed using methods known in the art, e.g., methods that measure glycan length and hydrodynamic radius. Size exclusion-HPLC allows the measurement of the hydrodynamic radius. Higher numbers of glycosylation sites in a protein lead to higher variation in hydrodynamic radius compared to a carrier with less glycosylation sites. However, when single glycan chains are analyzed, they may be more homogenous due to the more controlled length. Glycan length can measured by hydrazinolysis, SDS PAGE, and capillary gel electrophoresis. In addition,homogeneity can also mean that certain glycosylation site usage patterns change to a broader / narrower range. These factors can be measured by Glycopeptide LC-MS / MS.

[0175] N-glycosylation confers numerous benefits on the HuGlylDS used in the methods described herein. Such benefits are unattainable by production of proteins in E. coli, because E. coli does not naturally possess components needed for N-glycosylation. Further, some benefits are unattainable through protein production in, e.g, CHO cells, because CHO cells lack components needed for addition of certain glycans (e.g., 2,6 sialic acid) and because CHO cells can add glycans, e.g., Neu5Gc not typical to humans, and the a-Gal antigen which is immunogenic in most individuals and at high concentrations can trigger anaphylaxis. Even further, some benefits are unattainable through protein production in human cells that are not neuronal or glial cells. Thus, the expression of IDS in human neuronal or glial cells results in the production of HuGlylDS comprising beneficial glycans that otherwise would not be associated with the protein if produced in CHO cells, in E. coli, or in human cells which are not neuronal or glial cells.5.1.3. Tyrosine Sulfation

[0176] In addition to the N-linked glycosylation sites, hIDS contains a tyrosine (“Y”) sulfation site (PSSEKY165ENTKTCRGPD (SEQ ID NO: 47)). (See, e.g, Yang et al., 2015, Molecules 20:2138-2164, esp. at p. 2154 which is incorporated by reference in its entirety for the analysis of amino acids surrounding tyrosine residues subjected to protein tyrosine sulfation. The “rules” can be summarized as follows: Y residues with E or D within +5 to -5 position of Y, and where position -1 of Y is a neutral or acidic charged amino acid - but not a basic amino acid, e.g, R, K, or H that abolishes sulfation).

[0177] Importantly, tyrosine-sulfated proteins cannot be produced in E. coli, which naturally does not possess the enzymes required for tyrosine-sulfation. Further, CHO cells are deficient for tyrosine sulfation-they are not secretory cells and have a limited capacity for post-translational tyrosine-sulfation. See, e.g., Mikkelsen & Ezban, 1991, Biochemistry 30: 1533-1537. Advantageously, the methods provided herein call for expression of IDS, e.g., HuGlylDS, in neurons or glial cells, which are secretory and do have capacity for tyrosine sulfation. Assays for detection tyrosine sulfation are known in the art. See, e.g., Yang et al., 2015, Molecules 20:2138-2164.

[0178] Tyrosine-sulfation of hIDS - a robust post-translational process in human CNS cells - should result in improved processing and activity of transgene products. The significance of tyrosine-sulfation of lysosomal proteins has not been elucidated; but in other proteins it hasbeen shown to increase avidity of protein-protein interactions (antibodies and receptors), and to promote proteolytic processing (peptide hormone). (See, Moore, 2003, J Biol. Chem. 278:24243-46; and Bundegaard et al., 1995, The EMBO J 14: 3073-79). The tyrosylprotein sulfotransferase (TPST1) responsible for tyrosine-sulfation (which may occur as a final step in IDS processing) is apparently expressed at higher levels (based on mRNA) in the brain (gene expression data for TPST1 may be found, for example, at the EMBL-EBI Expression Atlas, accessible at http: / / www.ebi.ac.uk / gxa / home).5.2 CONSTRUCTS AND FORMULATIONS

[0179] For use in the methods provided herein are viral vectors or other DNA expression constructs encoding iduronate-2-sulfatase (IDS), e.g., human IDS (hIDS). For use in the methods provided herein are viral vectors or other DNA expression constructs encoding glycosylated (HuGly) a-L-iduronidase (IDUA), e.g., human IDUA (hIDUA). The viral vectors and other DNA expression constructs provided herein include any suitable method for delivery of a transgene to the cerebrospinal fluid (CSF). The means of delivery of a transgene include viral vectors, liposomes, other lipid-containing complexes, other macromolecular complexes, synthetic modified mRNA, unmodified mRNA, small molecules, non-biologically active molecules (e.g., gold particles), polymerized molecules (e.g., dendrimers), naked DNA, plasmids, phages, transposons, cosmids, or episomes. In some embodiments, the vector is a targeted vector, e.g., a vector targeted to neuronal cells.

[0180] In some aspects, the disclosure provides for a nucleic acid for use, wherein the nucleic acid encodes an IDS, e.g., hIDS, operatively linked to a promoter selected from the group consisting of: cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65 promoter and opsin promoter.

[0181] In certain embodiments, provided herein are recombinant vectors that comprise one or more nucleic acids (e.g. polynucleotides). The nucleic acids may comprise DNA, RNA, or a combination of DNA and RNA. In certain embodiments, the DNA comprises one or more of the sequences selected from the group consisting of promoter sequences, the sequence of the gene of interest (the transgene, e.g., IDS), untranslated regions, and termination sequences. In certain embodiments, viral vectors provided herein comprise a promoter operably linked to the gene of interest.

[0182] In certain embodiments, nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149-161).

[0183] In another aspect, the disclosure provides for a formulation comprising a recombinant nucleotide expression vector encoding human IDS, wherein the formulation is suitable for administration to the cerebrospinal fluid of human brain, so that a depot is formed in the human central nervous system that secretes a recombinant human IDS glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains a formylglycine, is a2,6-sialylated, does not contain detectable NeuGc, does not contain a-Gal antigen, and / or is mannose-6-phosphorylated. For example, the formulation may contain buffer (such as, a buffer having a particular pH, or a buffer containing a particular ingredient) that makes it suitable for administration to the cerebrospinal fluid of human brain, so that a depot is formed in the human central nervous system that secretes a recombinant human IDS glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains a formylglycine, is a2,6-sialylated, does not contain detectable NeuGc, does not contain a-Gal antigen, and / or is mannose-6-phosphorylated. In a specific embodiment, the buffer comprises a physiologically compatible aqueous buffer, a surfactant and optional excipients.

[0184] In another aspect, the disclosure provides for a kit comprising a recombinant nucleotide expression vector encoding human IDS and a pharmaceutically acceptable carrier, wherein the recombinant nucleotide expression vector is suitable for administration to the cerebrospinal fluid (CSF) of human brain, so that a depot is formed in the human central nervous system that secretes a recombinant human IDS glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains a formylglycine, is a2,6-sialylated, does not contain detectable NeuGc, does not contain detectable a-Gal antigen, and / or is mannose-6-phosphorylated. In another aspect, the disclosure provides for a kit comprising a formulation comprising a recombinant nucleotide expression vector encoding human IDS, wherein the formulation is suitable for administration to the CSF of human brain, so that a depot is formed in the human central nervous system that secretes a recombinant human IDS glycoprotein precursor that is about 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains aformylglycine, is a2,6-sialylated, does not contain detectable NeuGc, does not contain detectable a-Gal antigen, and / or is mannose-6-phosphorylated. A kit described herein comprises the recombinant nucleotide expression vector or the formulation in one or more containers. Optionally associated with such one or more containers can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0185] The formulations and kits encompassed herein can be used in accordance with the methods for treating a human patient as provided in this disclosure.5.2.1. mRNA

[0186] In certain embodiments, the vectors provided herein are modified mRNA encoding for the gene of interest (e.g., the transgene, for example, IDS). The synthesis of modified and unmodified mRNA for delivery of a transgene to the CSF is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is incorporated by reference herein in its entirety. In certain embodiments, provided herein is a modified mRNA encoding for IDS, e.g., hIDS.5.2.2. Viral Vectors

[0187] Viral vectors include adenovirus, adeno-associated virus (AAV, e.g., AAV9, AAVrhlO), lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus, hemagglutinin virus of Japan (HVJ), alphavirus, vaccinia virus, and retrovirus vectors. Retroviral vectors include murine leukemia virus (MLV)- and human immunodeficiency virus (HlV)-based vectors. Alphavirus vectors include semliki forest virus (SFV) and sindbis virus (SIN). In certain embodiments, the viral vectors provided herein are recombinant viral vectors. In certain embodiments, the viral vectors provided herein are altered such that they are replication-deficient in humans. In certain embodiments, the viral vectors are hybrid vectors, e.g., an AAV vector placed into a “helpless” adenoviral vector. In certain embodiments, provided herein are viral vectors comprising a viral capsid from a first virus and viral envelope proteins from a second virus. In specific embodiments, the second virus is vesicular stomatitus virus (VSV). In more specific embodiments, the envelope protein is VSV-G protein.

[0188] In certain embodiments, the viral vectors provided herein are HIV based viral vectors. In certain embodiments, HIV-based vectors provided herein comprise at least twopolynucleotides, wherein the gag and pol genes are from an HIV genome and the env gene is from another virus.

[0189] In certain embodiments, the viral vectors provided herein are herpes simplex virusbased viral vectors. In certain embodiments, herpes simplex virus-based vectors provided herein are modified such that they do not comprise one or more immediately early (IE) genes, rendering them non-cytotoxic.

[0190] In certain embodiments, the viral vectors provided herein are MLV based viral vectors. In certain embodiments, MLV-based vectors provided herein comprise up to 8 kb of heterologous DNA in place of the viral genes.

[0191] In certain embodiments, the viral vectors provided herein are lentivirus-based viral vectors. In certain embodiments, lentiviral vectors provided herein are derived from human lentiviruses. In certain embodiments, lentiviral vectors provided herein are derived from nonhuman lentiviruses. In certain embodiments, lentiviral vectors provided herein are packaged into a lentiviral capsid. In certain embodiments, lentiviral vectors provided herein comprise one or more of the following elements: long terminal repeats, a primer binding site, a polypurine tract, att sites, and an encapsidation site.

[0192] In certain embodiments, the viral vectors provided herein are alphavirus-based viral vectors. In certain embodiments, alphavirus vectors provided herein are recombinant, replication-defective alphaviruses. In certain embodiments, alphavirus replicons in the alphavirus vectors provided herein are targeted to specific cell types by displaying a functional heterologous ligand on their virion surface.

[0193] In certain embodiments, the viral vectors provided herein are AAV based viral vectors. In preferred embodiments, the viral vectors provided herein are AAV9 or AAVrhlO based viral vectors. In certain embodiments, the AAV9 or AAVrhlO based viral vectors provided herein retain tropism for CNS cells. Multiple AAV serotypes have been identified. In certain embodiments, AAV-based vectors provided herein comprise components from one or more serotypes of AAV. In certain embodiments, AAV based vectors provided herein comprise components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAV10 or AAV11. In preferred embodiments, AAV based vectors provided herein comprise components from one or more of AAV8, AAV9, AAVrhlO, AAV10, or AAV11 serotypes. AAV9-based viral vectors are used in the methods described herein. Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCTZEP2014 / 076466, each of which is incorporated herein by reference in its entirety. In one aspect, provided herein are AAV (e.g., AAV9 or AAVrhlO)-based viral vectors encoding a transgene e.g., IDS). In specific embodiments, provided herein are AAV9-based viral vectors encoding IDS. In more specific embodiments, provided herein are AAV9-based viral vectors encoding hIDS.

[0194] Provided in particular embodiments are AAV9 vectors comprising an artificial genome comprising (i) an expression cassette containing the transgene under the control of regulatory elements and flanked by ITRs; and (ii) a viral capsid that has the amino acid sequence of the AAV9 capsid protein or is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAV9 capsid protein (SEQ ID NO: 26) while retaining the biological function of the AAV9 capsid. In certain embodiments, the encoded AAV9 capsid has the sequence of SEQ ID NO: 26 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions and retaining the biological function of the AAV9 capsid. FIG. 6 provides a comparative alignment of the amino acid sequences of the capsid proteins of different AAV serotypes with potential amino acids that may be substituted at certain positions in the aligned sequences based upon the comparison in the row labeled SUBS. Accordingly, in specific embodiments, the AAV9 vector comprises an AAV9 capsid variant that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions identified in the SUBS row of FIG. 6 that are not present at that position in the native AAV9 sequence.

[0195] In certain embodiments, the AAV that is used in the methods described herein is Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety. In certain embodiments, the AAV that is used in the methods described herein comprises one of the following amino acid insertions: LGETTRP (SEQ ID NO: 56) or LALGETTRP (SEQ ID NO: 57), as described in United States Patent Nos. 9,193,956; 9458517; and 9,587,282 and US patent application publication no. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV that is used in the methods described herein is AAV.7m8, as described in United States Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and US patent application publication no. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV that is used in the methods described herein is any AAV disclosed in United States Patent No. 9,585,971, such as AAV-PHP.B. Incertain embodiments, the AAV that is used in the methods described herein is an AAV disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282 US patent application publication nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335.

[0196] In certain embodiments, a single-stranded AAV (ssAAV) may be used supra. In certain embodiments, a self-complementary vector, e.g., scAAV, may be used see, e.g., Wu, 2007, Human Gene Therapy, 18(2): 171-82, McCarty et al, 2001, Gene Therapy, Vol 8, Number 16, Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).

[0197] In certain embodiments, the viral vectors used in the methods described herein are adenovirus based viral vectors. A recombinant adenovirus vector may be used to transfer in the IDS. The recombinant adenovirus can be a first generation vector, with an El deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region. The recombinant adenovirus can be a second generation vector, which contains full or partial deletions of the E2 and E4 regions. A helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi). The transgene is inserted between the packaging signal and the 3’ITR, with or without stuffer sequences to keep the artificial genome close to wild-type size of approx. 36 kb. An exemplary protocol for production of adenoviral vectors may be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,” Gene Therapy 12:S18-S27, which is incorporated by reference herein in its entirety.

[0198] In certain embodiments, the viral vectors used in the methods described herein are lentivirus based viral vectors. A recombinant lentivirus vector may be used to transfer in the IDS. Four plasmids are used to make the construct: Gag / pol sequence containing plasmid, Rev sequence containing plasmids, Envelope protein containing plasmid (i.e. VSV-G), and Cis plasmid with the packaging elements and the IDS gene.

[0199] For lentiviral vector production, the four plasmids are co-transfected into cells (i.e., HEK293 based cells), whereby polyethylenimine or calcium phosphate can be used as transfection agents, among others. The lentivirus is then harvested in the supernatant (lentiviruses need to bud from the cells to be active, so no cell harvest needs / should be done). The supernatant is filtered (0.45 pm) and then magnesium chloride and benzonase added.Further downstream processes can vary widely, with using TFF and column chromatography being the most GMP compatible ones. Others use ultracentrifugation with / without column chromatography. Exemplary protocols for production of lentiviral vectors may be found in Lesch et al., 2011, “Production and purification of lentiviral vector generated in 293T suspension cells with baculoviral vectors,” Gene Therapy 18:531-538, and Ausubel et al., 2012, “Production of CGMP-Grade Lentiviral Vectors,” Bioprocess Int. 10(2):32-43, both of which are incorporated by reference herein in their entireties.

[0200] In a specific embodiment, a vector for use in the methods described herein is one that encodes an IDS (e.g., hIDS) such that, upon transduction of cells in the CNS, or a relevant cell (e.g., a neuronal cell in vivo or in vitro), a glycosylated variant of IDS is expressed by the transduced cell. In a specific embodiment, a vector for use in the methods described herein is one that encodes an IDS (e.g., hIDS) such that, upon transduction of a cell in the CNS, or a relevant cell (e.g., a neuronal cell in vivo or in vitro), a sulfated variant of IDS is expressed by the cell.5.2.3. Promoters and Modifiers of Gene Expression

[0201] In certain embodiments, the vectors provided herein comprise components that modulate gene delivery or gene expression (e.g., “expression control elements”). In certain embodiments, the vectors provided herein comprise components that modulate gene expression. In certain embodiments, the vectors provided herein comprise components that influence binding or targeting to cells. In certain embodiments, the vectors provided herein comprise components that influence the localization of the polynucleotide (e.g., the transgene) within the cell after uptake. In certain embodiments, the vectors provided herein comprise components that can be used as detectable or selectable markers, e.g., to detect or select for cells that have taken up the polynucleotide.

[0202] In certain embodiments, the viral vectors provided herein comprise one or more promoters. In certain embodiments, the promoter is a constitutive promoter. In alternate embodiments, the promoter is an inducible promoter. The native IDS gene, like most housekeeping genes, primarily uses a GC-rich promoter. In a preferred embodiment, strong constitutive promoters that provide for sustained expression of hIDS are used. Such promoters include “CAG” synthetic promoters that contain: “C” - the cytomegalovirus (CMV) early enhancer element; “A” - the promoter as well as the first exon and intron of the chicken beta-actin gene; and “G” - the splice acceptor of the rabbit beta-globin gene (see, Miyazaki et al., 1989, Gene 79: 269-277; and Niwa et al., Gene 108: 193-199).

[0203] In certain embodiments, the promoter is a CB7 promoter (see Dinculescu et al., 2005, Hum Gene Ther 16: 649-663, incorporated by reference herein in its entirety). In some embodiments, the CB7 promoter includes other expression control elements that enhance expression of the transgene driven by the vector. In certain embodiments, the other expression control elements include chicken [3-actin intron and / or rabbit P-globin polA signal. In certain embodiments, the promoter comprises a TATA box. In certain embodiments, the promoter comprises one or more elements. In certain embodiments, the one or more promoter elements may be inverted or moved relative to one another. In certain embodiments, the elements of the promoter are positioned to function cooperatively. In certain embodiments, the elements of the promoter are positioned to function independently. In certain embodiments, the viral vectors provided herein comprise one or more promoters selected from the group consisting of the human CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus (RS) long terminal repeat, and rat insulin promoter. In certain embodiments, the vectors provided herein comprise one or more long terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2 LTRs. In certain embodiments, the vectors provided herein comprise one or more tissue specific promoters (e.g., a neuronal cell-specific promoter).

[0204] In certain embodiments, the viral vectors provided herein comprise one or more regulatory elements other than a promoter. In certain embodiments, the viral vectors provided herein comprise an enhancer. In certain embodiments, the viral vectors provided herein comprise a repressor. In certain embodiments, the viral vectors provided herein comprise an intron or a chimeric intron. In certain embodiments, the viral vectors provided herein comprise a polyadenylation sequence.5.2.4. Signal Peptides

[0205] In certain embodiments, the vectors provided herein comprise components that modulate protein delivery. In certain embodiments, the viral vectors provided herein comprise one or more signal peptides. In certain embodiments, the signal peptides allow for the transgene product (e.g., IDS) to achieve the proper packaging (e.g. glycosylation) in the cell. In certain embodiments, the signal peptides allow for the transgene product (e.g., IDS) to achieve the proper localization in the cell. In certain embodiments, the signal peptides allow for the transgene product (e.g., IDS) to achieve secretion from the cell. Examples of signal peptides to be used in connection with the vectors and transgenes provided herein maybe found in Table 4. Signal peptides may also be referred to herein as leader sequences or leader peptides.Table 4. Signal peptides for use with the vectors provided herein.5.2.5. Untranslated Regions

[0206] In certain embodiments, the viral vectors provided herein comprise one or more untranslated regions (UTRs), e.g., 3’ and / or 5’ UTRs. In certain embodiments, the UTRs are optimized for the desired level of protein expression. In certain embodiments, the UTRs are optimized for the mRNA half life of the transgene. In certain embodiments, the UTRs are optimized for the stability of the mRNA of the transgene. In certain embodiments, the UTRs are optimized for the secondary structure of the mRNA of the transgene.5.2.6. Inverted Terminal Repeats

[0207] In certain embodiments, the viral vectors provided herein comprise one or more inverted terminal repeat (ITR) sequences. ITR sequences may be used for packaging therecombinant gene expression cassette into the virion of the viral vector. In certain embodiments, the ITR is from an AAV, e.g., AAV9 (see, e.g., Yan et al., 2005, J. Virol., 79(l):364-379; United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety).5.2.7. Transgenes

[0208] In certain embodiments, the vectors provided herein encode an IDS transgene. In specific embodiments, the IDS is controlled by appropriate expression control elements for expression in neuronal cells: In certain embodiments, the IDS (e.g., hIDS) transgene comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the IDS (e.g, hIDS) transgene comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 1.

[0209] The HuGlylDS encoded by the transgene can include, but is not limited to human IDS (hIDS) having the amino acid sequence of SEQ ID NO. 1 (as shown in FIG. 1), and derivatives of hIDS having amino acid substitutions, deletions, or additions, e.g., including but not limited to amino acid substitutions selected from corresponding non-conserved residues in orthologs of IDS shown in FIG. 2, with the proviso with the proviso that such mutations do not include replacement of the cysteine residue at position 84 (C84) which is required for enzyme activity (Millat et al., 1997, Biochem J 326: 243-247); or a mutation that has been identified in severe, severe-intermediate, intermediate, or attenuated MPS II phenotypes e.g., as shown in FIG. 3, or as reported by Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-761 (reporting “attenuated” mutants R48P, A85T, W337R, and the truncated mutant Q531X; and “severe” mutants P86L, S333L, S349I, R468Q, R468L); Millat et al., 1998, BBA 1406: 214-218 (reporting “attenuated” mutants P480L and P480Q; and “severe” mutant P86L); and Bonucelli et al., 2001, BBA 1537:233-238, each of which is incorporated by reference herein in its entirety.

[0210] For example, amino acid substitutions at a particular position of hIDS can be selected from among corresponding non-conserved amino acid residues found at that position in the IDS orthologs aligned in FIG. 2, with the proviso that such substitutions do not include any of the deleterious mutations shown in FIG. 3 or as reported by Sukegawa-Hayasaka et al., 2006, supra, Millat et al., 1998, supra, or Bonucelli et al., 2001, supra, each of which isincorporated by reference herein in its entirety. The resulting transgene product can be tested using conventional assays in vitro, in cell culture or test animals to ensure that the mutation does not disrupt IDS function. Preferred amino acid substitutions, deletions or additions selected should be those that maintain or increase enzyme activity, stability or half-life of IDS, as tested by conventional assays in vitro, in cell culture or animal models for MPS II. For example, the enzyme activity of the transgene product can be assessed using a conventional enzyme assay with, for example, 4-Methylumbelliferyl a-L-idopyranosiduronic acid 2-sulfate or 4-methylumbelliferyl sulfate as the substrate (see, e.g., Lee et al., 2015, Clin. Biochem. 48(18): 1350-1353, Dean et al., 2006, Clin. Chem. 52(4):643-649 for exemplary IDS enzyme assays that can be used, each of which is incorporated by reference herein in its entirety). The ability of the transgene product to correct MPS II phenotype can be assessed in cell culture; e.g., by transducing MPS II cells in culture with a viral vector or other DNA expression construct encoding hIDS or a derivative; by adding the transgene product or a derivative to MPS II cells in culture; or by co-culturing MPS II cells with human neuronal / glial host cells engineered to express and secrete rhIDS or a derivative, and determining correction of the defect in the MPS II cultured cells, e.g., by detecting IDS enzyme activity and / or reduction in GAG storage in the MPS II cells in culture (see, e.g., Stroncek et al., 1999, Transfusion 39(4):343-350, which is incorporated by reference herein in its entirety).

[0211] In some embodiments, a dose of a recombinant AAV of the disclosure is determined using a PCR assay. In some embodiments, the PCR assay is a Poly-A PCR assay. In some embodiments, the PCR assay is a transgene-specific PCR assay. In some embodiments, the dose determined in one assay is different (e.g., higher or lower) than a dose determined in another assay. For example, a dose determined using a transgene-specific PCR assay is higher (e.g., about 50% higher) than a dose determined using a Poly-A-specific PCR assay. In some embodiments, a dose determined by a transgene-specific PCR assay is higher by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or higher than about 75% higher than a dose determined by a Poly-A-specific PCR assay. In some embodiments, a dose of 2.0 x io11GC / g brain mass, where the number of genome copies is determined using a Poly-A-specific PCR assay is equivalent to a dose of 2.9 x io11GC / g brain mass, where the number of genome copies is determined using a transgene- specific PCR assay. In some embodiments, the total dose administered to a subject accounts for the estimated brain mass of the subject, which can be determined using a magnetic resonance imaging (MRI) screening.5.2.8. Constructs

[0212] In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a sequence encoding the transgene (e.g., IDS), h) a fourth linker sequence, i) a poly A sequence, j) a fifth linker sequence, and k) a second ITR sequence.

[0213] In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a promoter sequence, and b) a sequence encoding the transgene (e.g., IDS). In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a promoter sequence, and b) a sequence encoding the transgene (e.g., IDS), wherein the transgene comprises a signal peptide.

[0214] In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding the transgene (e.g., IDS), i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, 1) a fifth linker sequence, and m) a second ITR sequence.

[0215] In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding the transgene (e.g., IDS), i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, 1) a fifth linker sequence, and m) a second ITR sequence, wherein the transgene comprises a signal peptide, and wherein the transgene encodes hIDS.

[0216] In a specific embodiment, the viral vector described herein comprises the elements and in the order as illustrated in FIG. 5.5.2.9. Manufacture and Testing of Vectors

[0217] The viral vectors provided herein may be manufactured using host cells. The viral vectors provided herein may be manufactured using mammalian host cells, for example, A549 , WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, andmyoblast cells. The viral vectors provided herein may be manufactured using host cells from human, monkey, mouse, rat, rabbit, or hamster.

[0218] The host cells are stably transformed with the sequences encoding the transgene and associated elements (i.e., the vector genome), and the means of producing viruses in the host cells, for example, the replication and capsid genes (e.g., the rep and cap genes of AAV). For a method of producing recombinant AAV vectors with AAV8 capsids, see Section IV of the Detailed Description of U.S. Patent No. 7,282,199 B2, which is incorporated herein by reference in its entirety. Genome copy titers of said vectors may be determined, for example, by TAQMAN® analysis. Virions may be recovered, for example, by CsCh sedimentation.

[0219] In vitro assays, e.g., cell culture assays, can be used to measure transgene expression from a vector described herein, thus indicating, e.g., potency of the vector. For example, the HT-22, SK-N-MC, HCN-1 A, HCN-2, NT2, SH-SY5y, hNSCl 1, or ReNcell VM cell lines, or other cell lines that are derived from neuronal or glial cells or progenitors of neuronal or glial cells can be used to assess transgene expression. Once expressed, characteristics of the expressed product (i.e., HuGlylDS) can be determined, including determination of the glycosylation and tyrosine sulfation patterns associated with the HuGlylDS.5.2.10. Compositions

[0220] Compositions are described comprising a vector encoding a transgene described herein and a suitable carrier. A suitable carrier (e.g., for administration to the CSF, and, for example, to neuronal cells) would be readily selected by one of skill in the art.5.3 GENE THERAPY

[0221] Methods are described for the administration of a therapeutically effective amount of a transgene construct to human subjects having MPS II in order to prevent, improve or stabilize MPS Il-associated hearing loss. More particularly, methods for administration of a therapeutically effective amount of a transgene construct to patients having MPS II, in particular, for administration to the CSF are described. In particular embodiments, such methods for administration to the CSF of a therapeutically effective amount of a transgene construct can be used to treat to patients having Hunter’s syndrome in order to prevent, improve or stabilize MPS Il-associated hearing loss.5.3.1. Target Patient Populations

[0222] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to a patient diagnosed with MPS II. In certain embodiments, the patient has MPS Il-associated hearing loss. In specific embodiments, the patients have been diagnosed with mild MPS II. In specific embodiments, the patients have been diagnosed with severe MPS II. In specific embodiments, the patients have been diagnosed with Hunter’s syndrome. In specific embodiments, the patients have been diagnosed with neuronopathic MPS II. In some embodiments, a patient has been diagnosed with hepatosplenomegaly, has a symptom associated with hepatosplenomegaly, is suspected of having hepatosplenomegaly, and / or has a predisposition to suffer from hepatosplenomegaly. Examples of symptoms associated with hepatosplenomegaly include, but are not limited to, brown urine, clay-colored bowel movements, enlarged or swollen abdomen, fever, itching, jaundice or yellowing of the eyes and skin, nausea, pain (e.g., I the upper right portion of the stomach), fatigue, and / or vomiting. In some embodiments, a patient diagnosed with MPS II has hepatosplenomegaly. In some embodiments, a patient is suffering from hepatosplenomegaly associated with MPS II. In some embodiments, a patient is being treated or has been treated with ERT.

[0223] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients diagnosed with MPS II who have been identified as responsive to treatment with IDS, e.g., hIDS.

[0224] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to pediatric patients. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are less than three years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are aged 2 to 4 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are4 months old or older and less than 5 years old. In a specific embodiment, therapeutically effective doses of the recombinant vector are administered to patients that have severe MPS II and are 4 months old or older and less than 5 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are5 years old or older and less than 18 years old. In a specific embodiment, therapeutically effective doses of the recombinant vector are administered to patients that have neuronopathic MPS II and are 5 years old or older and less than 18 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered topatients that are 18 months old or older and 8 years old or younger. In a specific embodiment, therapeutically effective doses of the recombinant vector are administered to patients that are pediatric male patients and are 18 months old or older and 8 years old or younger. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are aged 3 to 8 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are aged 8 to 16 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are aged 5 to 18 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 10 years old or younger. In a specific embodiment, therapeutically effective doses of the recombinant vector are administered to patients that have severe MPS II and are 10 years old or younger. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 18 years old or younger. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are more than 5 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are more than 10 years old.

[0225] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 4, 5, 6, 7, 8, 9, 10, or 11 months old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are about 4, 5, 6, 7, 8, 9, 10, or 11 months old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are about 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 1, 2, 3, 4, or 5 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are about 1, 2, 3, 4, or 5 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 1-2, 2-3, 3-4, 4-5, or 5-6 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are about 1-2, 2-3, 3-4, 4-5, or 5-6 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients that are 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, or 18-19 years old. In certain embodiments, therapeutically effective doses of the recombinant vector are administered topatients that are about 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16- 17, 17-18, or 18-19 years old.

[0226] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to adolescent patients. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to adult patients. In some embodiments, therapeutically effective doses of the recombinant vector are administered to male patients. In other embodiments, therapeutically effective doses of the recombinant vector are administered to female patients.

[0227] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to patients diagnosed with MPS II who have been identified as responsive to treatment with IDS, e.g., hIDS, injected into the CSF prior to treatment with gene therapy.5.3.2. Dosage and Mode of Administration

[0228] In certain embodiments, therapeutically effective doses of the recombinant vector are administered to the CSF via intrathecal administration (z.e., injection into the subarachnoid space so that the recombinant vectors distribute through the CSF and transduce cells in the CNS). This can be accomplished in a number of ways - e.g., by intracranial (cisternal or ventricular) injection , or injection into the lumbar cistern. In certain embodiments, intrathecal administration is performed via intraci sternal (IC) injection (e.g., into the cistema magna). In specific embodiments, intracisternal injection is performed by CT-guided suboccipital puncture. In specific embodiments, intrathecal injection is performed by lumbar puncture. In specific embodiments, injection into the subarachnoid space is performed by Cl -2 puncture if feasible for the patient. Alternatively, intracerebroventricular (ICV) administration (a more invasive technique used for the introduction of anti-infective or anticancer drugs that do not penetrate the blood-brain barrier), for example, image-assisted ICV injection, can be used to instill the recombinant vectors directly into the ventricles of the brain. In a specific embodiment, the recombinant vector is administered via a single image-assisted ICV injection. In a further specific embodiment, the recombinant vector is administered via a single image-assisted ICV injection with immediate removal of the administration catheter. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to the CNS via intranasal administration. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to the CNS via intraparenchymal injection. In certain embodiments, intraparenchymal injection is targeted to the striatum. In certain embodiments,intraparenchymal injection is targeted to the white matter. In certain embodiments, therapeutically effective doses of the recombinant vector are administered to the CSF by any means known to the art, for example, by any means disclosed in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is hereby incorporated by reference in its entirety.

[0229] In preferred embodiments, for intrathecal administration (including IC and ICV administration), therapeutically effective doses of the recombinant vector are administered to the CSF in an injection volume that does not exceed 10% of the total CSF volume, which total CSF volume is about 50 mL in infants and about 150 mL in adults. A carrier suitable for intrathecal injection, such as Elliott’s B Solution or a modified Elliott’s B Solution, should be used as a vehicle for the recombinant vectors. Elliott’s B Solution (generic name: sodium chloride, sodium bicarbonate, anhydrous dextrose, magnesium sulfate, potassium chloride, calcium chloride and sodium phosphate) is a sterile, nonpyrogenic, isotonic solution containing no bacteriostatic preservatives and is used as a diluent for intrathecal administration of chemotherapeutics. The modified Elliott’s B solution includes 8.77 g / L sodium chloride, 0.244 g / L magnesium chloride, 0.0278 g / L sodium phosphate monobasic monohydrate, 0.114 g / L sodium phosphate dibasic anhydrous, 0.224 g / L potassium chloride, 0.206 g / L calcium chloride, 0.793 g / L dextrose, 0.010 g / L pol oxamer 188, pH 7.26. In some embodiments, the AAV or a composition comprising the AAV of the present disclosure is provided in a modified Elliott’s B solution for intrathecal administration.

[0230] In one embodiment, a non-replicating recombinant AAV9 vector expressing human iduronate-2-sulfatase (IDS) is used for treatment. In certain embodiments, the IDS expression cassette is flanked by inverted terminal repeats (ITRs) and expression is driven by a hybrid of the cytomegalovirus (CMV) enhancer and the chicken beta actin promoter (CB7). In certain embodiments, the transgene includes the chicken beta actin intron and a rabbit beta-globin polyadenylation (poly A) signal.

[0231] In certain embodiments, the recombinant nucleotide expression vector is administered at a dose that is dependent on the human subject’s brain mass. In preferred embodiments, the brain mass is determined by brain magnetic resonance imaging (MRI) of the human subject’s brain. In certain embodiments, the human subject’s brain mass is converted from the human subject’s brain volume by multiplying the human subject’s brain volume in cm3by a factor of 1.046 g / cm3, wherein the human subject’s brain volume is obtained from the human subject’s brain MRI. In some embodiments, a dose is the number of genome copies per weight of brain mass. In some embodiments, the number of genomecopies in a dose (e.g., the dose of the recombinant nucleotide expression vector) is determined by a Poly-A-specific PCR assay. In some embodiments, the number genome copies in a dose (e.g., the dose of the recombinant nucleotide expression vector) is determined by a transgene-specific PCR assay. In some embodiments, the weight of brain mass is determined by MRI.

[0232] In certain embodiments, the rAAV9.hIDS is administered IC (by suboccipital injection) as a single flat dose ranging from 1.4 * 1013GC (1.1 x IO10GC / g brain mass) to 7.0 x 1013GC (5.6 x 1O10GC / g brain mass) in a volume of about 5 to 20 ml. In the event the patient has neutralizing antibodies to AAV, doses at the high range may be used. In some embodiments, a single dose of an rAAV encoding hIDS is administered to the subject in the central nervous system (e.g., in the cerebrospinal fluid) and, surprisingly, treatment effects are observed outside of the CNS. For example, changes in organ sizes are observed outside of the CNS (e.g., spleen or liver) after the rAAV of the disclosure is administered to a subject in the CNS. In some embodiments, changes in biomarker levels (e.g., D2S6, HS, total GAG, and / or anti-IDS antibody) are detected outside of the CNS after administration of the rAAV of the disclosure in the cerebrospinal fluid (e.g., changes in biomarker levels detected in the liver, spleen, urine, plasma, or blood). In some embodiments, no additional therapy for MPS II is administered to the subject outside the CNS.

[0233] In certain embodiments, the recombinant vector described herein may be administered intrathecally as a single flat dose ranging from about 1.3 x io10GC / g brain mass to about 6.5 x 1O10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 1.3 x io10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 1.9 x io10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 6.5 x io10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 9.6 x io10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 2.0 x io11GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 2.9 * 1011GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at Dose 1 or Dose 2 as listed in and according to Table 5 below (for example, when the human patient is 4 months old or older and less than 5 years old).

[0234] In certain embodiments, the recombinant vector described herein may be administered intrathecally as a single flat dose ranging from about 1.3 x IO10GC / g brain mass to about 2.0 x 1011GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In certain embodiments, the recombinant vector described herein may be administered intrathecally as a single flat dose ranging from about 1.3 x io10GC / g brain mass to about 2.9 x io11GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 2.0 x io11GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 2.9 x io11GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at Dose 3 as listed in and according to Table 6 below (for example, when the human patient is 4 months old or older and less than 5 years old).

[0235] In certain embodiments, the recombinant vector described herein may be administered by IC administration as a single flat dose ranging from about 1.3 x 1O10GC / g brain mass to about 6.5 x 1O10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 1.3 x 1O10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 1.9 x io10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein maybe administered by IC administration as a single flat dose at about 6.5 * IO10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 9.6 x io10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at Dose 1 or Dose 2 as listed in and according to Table 5 below (for example, when the human patient is 4 months old or older and less than 5 years old).

[0236] In certain embodiments, the recombinant vector described herein may be administered by IC administration as a single flat dose ranging from about 1.3 x 1O10GC / g brain mass to about 2.0 x 1011GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In certain embodiments, the recombinant vector described herein may be administered by IC administration as a single flat dose ranging from about 1.3 x 1O10GC / g brain mass to about 2.9 x 1011GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 2.0 x io11GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 2.9 x io11GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at Dose 3 as listed in and according to Table 6 below (for example, when the human patient is 4 months old or older and less than 5 years old).

[0237] In certain embodiments, the recombinant vector described herein may be administered by ICV administration as a single flat dose ranging from about 1.3 x 1O10GC / g brain mass to about 6.5 x 1O10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 1.3 x 1O10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 1.9 x io10GC / g brain mass (for example, when the human patient is 4 months old or older and less than5 years old). In another specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 6.5 * IO10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 9.6 x io10GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at Dose 1 or Dose 2 as listed in and according to Table 5 below (for example, when the human patient is 4 months old or older and less than 5 years old).

[0238] In certain embodiments, the recombinant vector described herein may be administered by ICV administration as a single flat dose ranging from about 1.3 x 1O10GC / g brain mass to about 2.0 x 1011GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In certain embodiments, the recombinant vector described herein may be administered by ICV administration as a single flat dose ranging from about 1.3 x 1O10GC / g brain mass to about 2.9 x 1011GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 2.0 x io11GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In a specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 2.9 x io11GC / g brain mass (for example, when the human patient is 4 months old or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at Dose 3 as listed in and according to Table 6 below (for example, when the human patient is 4 months old or older and less than 5 years old).Table 5. Total Dose Administered by Brain Mass (Dose 1 or Dose 2)*GC determined based on a Poly-A-specific PCR assayTable 6. Total Dose Administered by Brain Mass (Dose 3)

[0239] In a specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose at about 6.5 x io10GC / g brain mass (for example, when the human patient is 5 years old or older and less than 18 years old). In another specific embodiment, the recombinant vector described herein may be administered intrathecally as a single flat dose as listed in and according to Table 7 below (for example, when the human patient is 5 years old or older and less than 18 years old).

[0240] In a specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose at about 6.5 x io10GC / g brain mass (for example, when the human patient is 5 years old or older and less than 18 years old). In another specific embodiment, the recombinant vector described herein may be administered by IC administration as a single flat dose as listed in and according to Table 7 below (for example, when the human patient is 5 years old or older and less than 18 years old).

[0241] In a specific embodiment, the recombinant vector described herein may be administered by ICV administration as a single flat dose at about 6.5 x io10GC / g brain mass (for example, when the human patient is 5 years old or older and less than 18 years old). In another specific embodiment, the recombinant vector described herein may be administeredby ICV administration as a single flat dose as listed in and according to Table 7 below (for example, when the human patient is 5 years old or older and less than 18 years old).Table 7. Total Dose Administered by Brain Mass5.3.3. Preventing and Treating MPS Il-Associated Hearing Loss

[0242] The present disclosure further provides methods and formulations for preventing or treating MPS Il-associated hearing loss in a subject in need thereof using recombinant human iduronate-2-sulfatase (IDS). In certain embodiments, the recombinant human IDS is delivered to the subject by a presently disclosed recombinant vectors (see Section 5.2). In certain embodiments, the subject is 4 months old or older and less than 5 years old. In certain embodiments, the subject is 5 years old or older and less than 18 years old.

[0243] The present disclosure is based, in part, on the surprising findings that a therapeutically effective amount of the presently disclosed recombinant vectors was able to stabilize and improve hearing in subjects suffering from MPS Il-associated hearing loss. In addition, in subjects who have not developed hearing loss (having normal hearing), a therapeutically effective amount of the presently disclosed recombinant vectors was able to prevent hearing loss for at least 3 or 4 years after the administration.

[0244] In one aspect, the present disclosure provides a method for preventing MPS II- associated hearing loss in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a recombinant adeno- associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) disclosed herein (see Section 5.2), wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear. In certain embodiments, the at least one ear comprises the left ear or the right ear. In certain embodiments, the at least one ear comprises the left ear and the right ear.

[0245] In certain embodiments, the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear (e.g., unilateral or bilateral) for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 24 weeks, at least 48 weeks, at least about 1 year, at least about 52 weeks, at least about 1.5 years, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after the administration of the rAAV. In certain embodiments, the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear for at least about 3 years. In certain embodiments, the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear for at least about 4 years. In certain embodiments, the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear for at least about 5 years.

[0246] In certain embodiments, preventing the onset of hearing loss is indicated by no change or a small change in a hearing threshold as measured by an auditory brain response (ABR) testing in the at least one ear after the administration of the rAAV as compared to a hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV. In certain embodiments, the hearing threshold is an estimate of behavioral threshold (eHL) derived from an ABR threshold (nHL) determined by the ABR testing. In certain embodiments, preventing the onset of hearing loss is indicated by an about 25 dB or less, an about 20 dB or less, about 15 dB or less, about 10 dB or less, about 5 dB or less, or about 1 dB or less of change in the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV. In certain embodiments, preventing the onset of hearing loss is indicated by an about 25 dB or less of change in the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV. In certain embodiments, preventing the onset of hearing loss is indicated by an about 10 dB or less of change in the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV.

[0247] In certain embodiments, the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural as determined by a tympanometric testing.

[0248] In another aspect, the present disclosure provides a method for treating mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), wherein the subject has developed hearing loss in at least one ear e.g., unilateral or bilateral) prior tothe administration of the rAAV. In certain embodiments, the therapeutically effective amount of the rAAV stabilizes the hearing loss or improves hearing in the at least one ear. In certain embodiments, the at least one ear comprises the left ear or the right ear. In certain embodiments, the at least one ear comprises the left ear and the right ear.

[0249] In another aspect, the present disclosure provides a method for stabilizing mucopolysaccharidosis II (MPS II)-associated hearing loss or preventing the further loss of hearing associated with MPS II in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), wherein the subject has developed hearing loss in at least one ear (e.g., unilateral or bilateral) prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents a further loss of the hearing (e.g., the hearing impairment is getting worse or deteriorating, the hearing is further decreasing) in the at least one ear. In certain embodiments, the at least one ear comprises the left ear or the right ear. In certain embodiments, the at least one ear comprises the left ear and the right ear. As used herein, stabilizing MPS Il-associated hearing loss refers to preventing the hearing impairment from getting worse or deteriorating.

[0250] In certain embodiments, the therapeutically effective amount of the rAAV stabilizes the hearing loss or prevents the further loss of the hearing in the at least one ear for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 24 weeks, at least 48 weeks, at least about 1 year, at least about 52 weeks, at least about 1.5 years, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after the administration of the rAAV. In certain embodiments, the therapeutically effective amount of the rAAV prevents the further loss of the hearing in the at least one ear for at least about 16 weeks after the administration of the rAAV. In certain embodiments, the therapeutically effective amount of the rAAV prevents the further loss of the hearing in the at least one ear for at least about 24 weeks after the administration of the rAAV. In certain embodiments, the therapeutically effective amount of the rAAV prevents the further loss of the hearing in the at least one ear for at least about 48 week after the administration of the rAAV.

[0251] In certain embodiments, stabilizing the hearing loss or preventing the further loss of the hearing is indicated by an increase in a hearing threshold as measured by an ABR testing in the at least one ear after the administration of the rAAV as compared to a hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV. In certain embodiments, the hearing threshold is an estimate of behavioral threshold(eHL) derived from an ABR threshold (nHL). In certain embodiments, stabilizing the hearing loss or preventing the further loss of the hearing is indicated by an about 25 dB or less, about 20 dB or less, about 15 dB or less, about 10 dB or less, about 5 dB or less, or about 1 dB or less of change in the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV. In certain embodiments, stabilizing the hearing loss or preventing the further loss of the hearing is indicated by an about 10 dB or less of change in the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV.

[0252] In certain embodiments, the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural as determined by a tympanometric testing.

[0253] In another aspect, the present disclosure provides a method for improving mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), wherein the subject has developed hearing loss in at least one ear (e.g., unilateral or bilateral) prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves the hearing in the at least one ear. In certain embodiments, the at least one ear comprises the left ear or the right ear. In certain embodiments, the at least one ear comprises the left ear and the right ear.

[0254] In certain embodiments, the therapeutically effective amount of the rAAV improves the hearing in the at least one ear in less than about 1 week, less than about 2 weeks, less than about 3 weeks, less than about 1 month, less than about 2 months, less than about 3 months, less than about 4 months, less than about 5 months, less than about 24 weeks, less than about 6 months, less than about 48 weeks, less than about 52 weeks, or less than about 1 year after the administration of the rAAV. In certain embodiments, the therapeutically effective amount of the rAAV improves the hearing in the at least one ear in about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 24 weeks, about 6 months, about 48 weeks, about 52 weeks, or about 1 year after the administration of the rAAV. In certain embodiments, the therapeutically effective amount of the rAAV improves the hearing in the at least one ear in about 16 weeks after the administration of the rAAV. In certain embodiments, the therapeutically effective amount of the rAAV improves the hearing in the at least one ear in about 24 weeks after the administration of the rAAV. In certain embodiments, the therapeutically effective amount ofthe rAAV improves the hearing in the at least one ear in about 48 weeks after the administration of the rAAV.

[0255] In certain embodiments, improving the hearing is indicated by a decrease in a hearing threshold as measured by an ABR testing in the at least one ear after the administration of the rAAV as compared to a hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV. In certain embodiments, the therapeutically effective amount of the rAAV decreases the hearing threshold as measured by the ABR testing in the at least one ear for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV. In certain embodiments, the hearing threshold is an estimate of behavioral threshold (eHL) derived from an ABR threshold (nHL). In certain embodiments, improving the hearing is indicated by a more than about 5 dB, more than about 10 dB, more than about 15 dB, more than about 20 dB, more than about 25 dB, or more than about 30 dB decrease of the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV. In certain embodiments, improving the hearing is indicated by a more than about 10 dB decrease of the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV.

[0256] In certain embodiments, the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural as determined by a tympanometric testing.

[0257] In certain embodiments, the methods disclosed herein further comprise performing an ABR testing in the at least one ear prior to and after the administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV. ABR testing is a physiologic procedure which can be used to estimate behavioral hearing thresholds in individuals who are unable to reliably perform behavioral audiometry. The ABR measures electrical activity in response to the presentation of various sound stimuli.

[0258] One skilled in the art would know how to perform the ABR testing in a subject. In certain embodiments, the ABR testing is performed by delivering tone burst stimuli through insert earphones to measure ABR thresholds in nHL for each ear at the following frequencies: 500Hz, 1000Hz, 2000Hz, and 4000Hz. Once nHL thresholds are obtained, appropriate correction factors are applied by frequency to obtain estimates of behavioral thresholds (eHL). The average degree of hearing loss is then classified as mild, moderate, severe, or profound for each ear using the average of the thresholds measured. ABR testing is alsocompleted using a high intensity (80-90 dBnHL) click stimuli at a rate of 17-30 clicks / sec. Absolute waveform latency values are then recorded along with a comment regarding the quality of the waveform morphology. These measures are conducted to evaluate the integrity of the auditory brainstem pathway.

[0259] In certain embodiments, the method disclosed herein further comprises performing a tympanometric testing in the at least one ear prior to and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural. Tympanometry tests are done to assess middle ear function.

[0260] One skilled in the art would know how to perform the tympanometric testing in a subject. In certain embodiments, a tympanometric testing is performed for both ears in conjunction with ABR testing to help differentiate between conductive and sensorineural hearing loss. The following parameters are recorded: Equivalent Canal Volume (cc), Static Admittance (mmho), Tympanometric Width (daPa) and Tympanometric Peak Pressure (daPa). Tympanometric results are then classified using the following key: A=normal, Bl=flat tympanogram with normal equivalent volume (suggesting middle ear dysfunction), B2=T-tube or tympanic membrane perforation, C=negative middle ear pressure.

[0261] In certain embodiments, the present disclosure provides a method for preventing MPS Il-associated hearing loss in a subject in need thereof, wherein the method comprises: (a) administering to the subject a therapeutically effective amount of a recombinant adeno- associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) disclosed herein (see Section 5.2), and (b) performing an ABR testing in the at least one ear prior to and after the administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV; wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear. In certain embodiments, the method further comprises (c) performing a tympanometric testing in the at least one ear prior to and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.

[0262] In certain embodiments, the present disclosure provides a method for treating mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the method comprises: (a) administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2- sulfatase (hIDS) disclosed herein (see Section 5.2), and (b) performing an ABR testing in theat least one ear prior to and after the administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV; wherein the subject has developed hearing loss in at least one ear (e.g., unilateral or bilateral) prior to the administration of the rAAV. In certain embodiments, the therapeutically effective amount of the rAAV stabilizes the hearing loss or improves hearing in the at least one ear. In certain embodiments, the method further comprises (c) performing a tympanometric testing in the at least one ear prior to and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.

[0263] In certain embodiments, the present disclosure provides a method for stabilizing MPS Il-associated hearing loss in a subject in need thereof, wherein the method comprises: (a) administering to the subject a therapeutically effective amount of a recombinant adeno- associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) disclosed herein (see Section 5.2), and (b) performing an ABR testing in the at least one ear prior to and after the administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV; wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents the further loss of hearing in the at least one ear In certain embodiments, the method further comprises (c) performing a tympanometric testing in the at least one ear prior to and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.

[0264] In certain embodiments, the present disclosure provides a method for improving mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the method comprises: (a) administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2- sulfatase (hIDS) disclosed herein (see Section 5.2), and (b) performing an ABR testing in the at least one ear prior to and after the administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV; wherein the subject has developed hearing loss in at least one ear (e.g., unilateral or bilateral) prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves the hearing in the at least one ear. In certain embodiments, the method further comprises (c) performing a tympanometric testing in the at least one ear prior to and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.

[0265] In another aspect, the present disclosure provides a formulation suitable for administration to the ear (e.g., inner ear) comprising a therapeutically effective amount of a presently disclosed recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) (see Section 5.2). In certain embodiments, the formulation is suitable for intra-tympanic delivery, intra-cochlear delivery, or intra-labyrinthine delivery (see e.g., Liu et al., Front Neurosci. 2022; 16: 867453, the content of which is incorporated by reference in its entirety). In certain embodiments, the formulation is suitable for targeted delivery of the rAAV to the cochlea. In certain embodiments, the formulation is suitable for intra-cochlear delivery or intra-labyrinthine delivery. In certain embodiments, the formulation comprises adjuvants (e.g., histamine, benzyl alcohol, saponin, and N-methyl-2- pyrrolidone (NMP)) to increase drug permeation across round window membrane (RWM) (see e.g., Saber et al, Round Window Membrane and Delivery of Biologically Active Agents into the Cochlea. Soina: Karolinska Institutet.; Creber et al., Eur. J. Pharm. Sci. 126 69-81.; Zhang et al., Adv. Funct. Mater. 31 :2008701., the content of each of which is incorporated by reference in its entirety). In certain embodiments, the formulation comprises nanocarriers, such as liposome, micelles, polymeric nanoparticle, and dendrimers to deliver the rAAV. In certain embodiments, the formulation comprises hydrogel, nanocarriers, permeation enhancers, or microfluidic systems to improve drug permeation and sustained release.5.4 COMBINATION THERAPIES

[0266] Combinations of administration of the HuGlylDS to the patients accompanied by administration of other available treatments are encompassed by the methods of the invention. The additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment. Available treatments for MPS II that could be combined with the gene therapy of the invention include but are not limited to enzyme replacement therapy (ERT) using idursulfase administered systemically or to the CSF; and / or HSCT therapy. In another embodiment, ERT can be administered using the rHuGlylDS glycoprotein produced in human neuronal and glial cell lines by recombinant DNA technology. Human neuronal and glial cell lines that can be used for such recombinant glycoprotein production include but are not limited to HT-22, SK-N-MC, HCN-1 A, HCN-2, NT2, SH-SY5y, hNSCl 1, or ReNcell VM to name a few. To ensure complete glycosylation, especially sialylation, and tyrosine-sulfation, the cell line used for production can be enhanced by engineering the host cells to co-express a-2,6-sialyltransferase (or both a-2,3- and a-2,6-sialyltransferases) and / or TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation.5.5 BIOMARKERS / SAMPLING / MONITORING EFFICACY

[0267] Efficacy may be monitored by measuring cognitive function (e.g., prevention or decrease in neurocognitive decline); reductions in biomarkers of disease (such as GAG, including heparan sulfate and dermatan sulfate) in CSF and or serum; and / or increase in IDS enzyme activity in CSF and / or serum. Signs of inflammation and other safety events may also be monitored.

[0268] In one aspect, provided herein is a method of monitoring efficacy by determining if a subject undergoing ERT treatment or a subject who has received ERT treatment can discontinue ERT treatment after administration of a gene therapy (e.g., rAAV encoding hIDS) of the disclosure. For example, provided herein is a method of treating and / or identifying a subject diagnosed with MPS II (e.g., a subject who is likely to be responsive to discontinuing treatment with ERT), comprising: (a) administering a therapeutically effective amount of a gene therapy (e.g., an rAAV encoding hIDS) of the disclosure to the subject, wherein the subject was treated with ERT or is being treated with ERT; (b) identifying the subject as being likely to be responsive to discontinuing ERT treatment, comprising: i. obtaining or having obtained a biological sample from the subject; ii. determining the level of at least one biomarker in the biological sample; and iii. identifying the subject as being likely to be responsive to discontinuing ERT treatment if the level of the biomarker in the biological sample is different (e.g., higher or lower) than a reference (e.g., reference level of the at least one biomarker); and (c) discontinuing ERT treatment in the subject. In some embodiments, a biomarker is D2S6, HS, total GAG, and / or anti-IDS antibody. In some embodiments, ERT is recombinant idursulfase. In some embodiments, the subject diagnosed with MPS II has hepatosplenomegaly.

[0269] In another aspect, provided herein is a method of selectively treating a human subject with MPS II, comprising administering to the subject a therapeutically effective amount of an rAAV encoding hIDS, wherein the subject was treated with ERT or is being treated with ERT, and wherein the subject has been determined likely to be responsive to discontinuing treatment with ERT according to a method comprising: (a) obtaining a biological sample from the subject; and (b) determining the level of at least one biomarker in the biological sample; wherein the subject is determined likely to be responsive to discontinuing treatment with ERT when the level of the at least one biomarker is different (e.g., higher or lower) than a reference. In some embodiments, a biomarker is D2S6, HS, totalGAG, and / or anti-IDS antibody. In some embodiments, ERT is recombinant idursulfase. In some embodiments, the subject diagnosed with MPS II has hepatosplenomegaly.

[0270] In another aspect provided herein is a method of identifying or diagnosing a subject as having neuronopathic or non-neuronopathic MPS II or MPS I. In some embodiments, the method comprises determining the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, D2S6) in a biological sample from a subject. In some embodiments, the method comprises determining the level of undegraded glycosaminoglycans (GAGs) in a biological sample from a subject. In some embodiments, the subject is identified or diagnosed as having neuronopathic MPS II or MPS I if the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) is elevated as compared to a reference level. In some embodiments, the subject is identified or diagnosed as having neuronopathic MPS II or MPS I if the level of GAG heparan sulfate (HS) is elevated (e.g., in the brain) as compared to a reference level. In some embodiments, the total amount of heparin sulfate (t-HS) is the sum of four disaccharides (D2S6, D0A0, D0S0, D0A6) in cerebrospinal fluid (CSF) following enzymatic digestion (e.g., as determined based on a bioanalytical mass spectrometry method). In some embodiments, an elevated level of D2S6 in e.g., CSF of a subject (e.g., presymptomatic subject) is indicative of neuronopathic MPS II or MPS I. In some embodiments, the level of D2S6 is indicative of iduronate-2-sulfatase enzyme activity and can be used for therapy monitoring. In some embodiments, the one or more disaccharides comprises one or more of D0A0, D0S0, D0A6, D2S6, or a combination thereof. In another aspect provided herein is a method of identifying or diagnosing a subject as having neuronopathic or non-neuronopathic MPS II or MPS I, wherein the subject is identified or diagnosed as having neuronopathic MPS II if the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample from the subject is elevated as compared to a reference level. In some embodiments, the subject is presymptomatic or has no visible or detectable MPS II or MPS I symptom. In some embodiments, the subject has or has been diagnosed with MPS I or MPS II. In some embodiments, the reference level is the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample from one or more healthy subjects and / or from one or more non-neuronopathic subjects. In some embodiments, the reference level is the level of D2S6 in a biological sample (e.g., CSF sample) from one or more healthy subjects and / or from one or more non-neuronopathic subjects. In some embodiments, the reference level is a pre-determined level. In some embodiments, the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, D2S6) is about or at least about 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, or higher than 40% of the totalheparan sulfate disaccharides (HS) in a biological sample from a subject (e.g., a subject with MPS I or MPS II). In some embodiments, the level of one or more disaccharides (e.g., DOAO, DOSO, D0A6, and / or D2S6) is about or at least about 20% the total heparan sulfate disaccharides (HS) in a biological sample from a subject (e.g., a subject with MPS I or MPS II). In some embodiments, the level of one or more disaccharides (e.g., DOAO, DOSO, D0A6, and / or D2S6) in a biological sample from a subject (e.g., a subject with MPS I or MPS II) is about or at least about 5%, 10%, 115, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more than 90% higher than the level of one or more disaccharides (e.g., DOAO, DOSO, D0A6, and / or D2S6) in a biological sample from a reference (e.g., a healthy subject). In some embodiments, the level of total heparin sulfate (e.g., DOAO, DOSO, D0A6, D2S6) is about or at least about 250 ng / mL, 275 ng / mL, 300 ng / mL, 325 ng / mL, 350 ng / mL, 375 ng / mL, 400 ng / mL, 425 ng / mL, 450 ng / mL, 475 ng / mL, 500 ng / mL, 525 ng / mL, 550 ng / mL, 575 ng / mL, 600 ng / mL, 625 ng / mL, 650 ng / mL, 675 ng / mL, 700 ng / mL, 725 ng / mL, 750 ng / mL, 775 ng / mL, 800 ng / mL, 825 ng / mL, 850 ng / mL, 875 ng / mL, 900 ng / mL, 925 ng / mL, 950 ng / mL, 975 ng / mL, 1000 ng / mL, or more than 1000 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., DOAO, DOSO, D0A6, and / or D2S6) is about or at least about 500 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., DOAO, DOSO, D0A6, and / or D2S6) is about or at least about 600 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., DOAO, DOSO, D0A6, and / or D2S6) is about or at least about 700 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., DOAO, DOSO, D0A6, and / or D2S6) is about or at least about 800 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., DOAO, DOSO, D0A6, and / or D2S6) is between about 500 ng / mL to about 1000 ng / mL, about 300 ng / mL to about 1000 ng / mL, about 400 ng / mL to about 1000 ng / mL, about 550 ng / mL to about 1000 ng / mL about 600 ng / mL to about 1000 ng / mL, about 700 ng / mL to about 1000 ng / mL 500 ng / mL to about 900 ng / mL, about 300 ng / mL to about 900 ng / mL, about 400 ng / mL to about 900 ng / mL, about 550 ng / mL to about 900 ng / mL about 600 ng / mL to about 900 ng / mL, or about 700 ng / mL to about 900 ng / mL. In some embodiments, the level of at least one heparin sulfate (e.g., DOAO, DOSO, D0A6, and / or D2S6) is about or at least about 50 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 270 ng / mL, 280 ng / mL, 290 ng / mL, 300 ng / mL, 310ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, or more than 400 ng / mL. In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, or more than 200 ng / mL. In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 80 ng / mL (e.g., for DOSO or D0A6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 100 ng / mL (e.g., for DOSO, D0A6, or D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 90 ng / mL (e.g., for DOSO, D0A6, or D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 110 ng / mL (e.g., for DOSO, D0A6, or D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 120 ng / mL (e.g., for DOSO, D0A6, or D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 160 ng / mL (e.g., for D2S6 or D0A6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 250 ng / mL (e.g., for D2S6 or D0A0). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 300 ng / mL (e.g., for D2S6 or D0A0). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 350 ng / mL (e.g., for D2S6 or D0A0). ). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 150 ng / mL (e.g., for D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 170 ng / mL (e.g., for D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 180 ng / mL (e.g., for D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, DOSO, D0A6, and / or D2S6) is about or at least about 200 ng / mL (e.g., for D2S6). In some embodiments, the level of one or more heparan sulfate disaccharide(s) (e.g., D0A0, DOSO, D0A6, and / or D2S6) or the level of D2S6 in a biological sample from a subject is elevated by about or at least about 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL,250 ng / mL, 260 ng / mL, 270 ng / mL, 280 ng / mL, 290 ng / mL, 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, 410 ng / mL, 420 ng / mL, 430 ng / mL, 440 ng / mL, 450 ng / mL, 460 ng / mL, 470 ng / mL, 480 ng / mL, 490 ng / mL, 500 ng / mL, or more than 500 ng / mL as compared to a reference level (e.g., level of one or more heparan sulfate disaccharide(s) (e.g., D0A0, D0S0, D0A6, and / or D2S6) or the level of D2S6 in a biological sample from one or more healthy subjects and / or from one or more non-neuronopathic subjects, or a pre-determined value). In some embodiments, the total level of heparan sulfate disaccharide(s) (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample from a subject is elevated by about or at least about 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 270 ng / mL, 280 ng / mL, 290 ng / mL, 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, 410 ng / mL, 420 ng / mL, 430 ng / mL, 440 ng / mL, 450 ng / mL, 460 ng / mL, 470 ng / mL, 480 ng / mL, 490 ng / mL, 500 ng / mL, 510 ng / mL, 520 ng / mL, 530 ng / mL, 540 ng / mL, 550 ng / mL, 560 ng / mL, 570 ng / mL, 580 ng / mL, 590 ng / mL, 600 ng / mL, 610 ng / mL, 620 ng / mL, 630 ng / mL, 640 ng / mL, 650 ng / mL, 660 ng / mL, 670 ng / mL, 680 ng / mL, 690 ng / mL, 700 ng / mL, 710 ng / mL, 720 ng / mL, 730 ng / mL, 740 ng / mL, 750 ng / mL, 760 ng / mL, 770 ng / mL, 780 ng / mL, 790 ng / mL, 800 ng / mL, 850 ng / mL, 900 ng / mL, 950 ng / mL, 1000 ng / mL, or more than 1000 ng / mL as compared to a reference level (e.g., the total level of heparan sulfate disaccharides (e.g., D0A0, D0S0, D0A6, and D2S6) in a biological sample from one or more healthy subjects and / or from one or more non-neuronopathic subjects, or a pre-determined value).

[0271] In some embodiments, a subject is determined to be responsive to a treatment of the disclosure (e.g., rAAV9 encoding hIDUA for treating MPS I; Construct 2) based on the level of I0S6 in a biological sample from the subject. In some embodiments, an elevated level of I0S6 in a biological sample from the subject (e.g., subject with MPS I) as compared to a reference, is indicative that the subject is responsive to a treatment of the disclosure (or treatment with rAAV9 encoding hIDUA for MPS I). In some embodiments, the reference is a level of I0S6 in a biological sample from a healthy subject or a population of healthy subjects. In some embodiments, the reference is a level of I0S6 in a biological sample from a subject with MPS I or a population of subjects with MPS I. In some embodiments, the reference is a level of I0S6 in a biological sample from a subject who does not have MPS I or is not diagnosed with MPS I or a population of subjects not diagnosed with MPS I. In someembodiments, the reference is a level of IS06 in a biological sample from the same subject but taken at a different time point (e.g., obtained at an earlier time point). In some embodiments, the reference is a predetermined value.5.5.1. Disease Markers

[0272] In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring the level of a disease biomarker in the patient. In certain embodiments, the level of the disease biomarker is measured in the CSF of the patient. In certain embodiments, the level of the disease biomarker is measured in the serum of the patient. In certain embodiments, the level of the disease biomarker is measured in the plasma of the patient. In certain embodiments, the level of the disease biomarker is measured in the urine of the patient. In certain embodiments, the disease biomarker is GAG. In some embodiments, the disease biomarker is I0S6. In preferred embodiments, the disease biomarker is heparan sulfate. In certain embodiments, the disease biomarker is D2S6. 12S enzyme cleaves sulfates from HS in the lysosome and absence of I2S causes long chains of fully sulfated D2S6 to accumulate. In some embodiments, quantitative measurement of D2S6 is reflective of I2S enzyme activity level and elevated levels of HS and D2S6 correlate closely with the neuronopathic phenotype of MPS II. In some embodiments, levels of D2S6 inversely correlate with neurocognitive development. In some embodiments, the disease biomarker is an anti -AAV antibody (e.g., anti-AAV9 antibody). In certain embodiments, the disease biomarker is dermatan sulfate. In certain embodiments, the disease biomarker is IDS enzyme activity. In certain embodiments, the disease biomarker is inflammation. In certain embodiments, the disease biomarker is a safety event.

[0273] In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring one or more of the following biomarkers in a sample from the patient: (a) level of GAGs in CSF; (b) level of I2S in CSF; (c) level of GAGs in plasma; (d) level of I2S in plasma; (e) level of leukocyte I2S enzyme activity; (f) level of GAGs in urine, (g) level of heparan sulfate in CSF, and (h) level of dermatan sulfate in CSF. In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring I2S and / or GAGs in CSF, urine, and / or plasma. In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring heparan sulfate in CSF, plasma, and / or urine. In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring non-reducing heparan sulfate. In some embodiments, heparan sulfatemeasured in CSF is the primary endpoint for determining efficacy of treatment. In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring D2S6 in CSF. In some embodiments, D2S6 is measured using any detectable / available assay or biological sample used for detecting D2S6 (e.g., CSF, urine, and / or plasma). In certain embodiments, efficacy of treatment with the recombinant nucleotide expression vector is monitored by measuring total urine GAGs, urine HS and / or plasma I2S enzyme activity. In some embodiments, urine GAG is indicative of systemic effect and / or is independent of ERT treatment. In some embodiments, efficacy of a treatment of the disclosure is determined based on the level of I2S protein concentration in a sample from a subject (e.g., an increase in the level of I2S protein concentration is indicative of efficacy). In some embodiments, Heparan sulfate (HS) and D2S6 (glycosaminoglycans (GAGs) are measured in the cerebrospinal fluid (CSF) at baseline and / or after administration of the recombinant vector of the present disclosure. In some embodiments, determining or monitoring efficacy of MPS II treatment in a subject is determined by detecting a level of at least one biomarker (e.g., D2S6) in a biological sample from a subject obtained at about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 days, or 1, 2, 3, 4, 5, 6, 7, 8, 10, 16, 20, 24, 30, 35, 40, 45, 48, 50, 52, 56, 104 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years after the rAAV of the present disclosure is administered to the subject (e.g., and comparing the level with a reference). In some embodiments, determining or monitoring efficacy of MPS I treatment in a subject is determined by detecting a level of at least one biomarker (e.g, I0S6) in a biological sample from a subject obtained at about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,470, 480, 490 days, or 1, 2, 3, 4, 5, 6, 7, 8, 10, 16, 20, 24, 30, 35, 40, 45, 48, 50, 52, 56, 104 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years after the rAAV of the present disclosure is administered to the subject (e.g, and comparing the level with a reference).

[0274] In some embodiments, the level of HS and / or D2S6 is decreased in a subject after administration of a recombinant vector of the present disclosure as compared to a reference (e.g., compared to the level of HS and / or D2S6 in the subject before administration of arecombinant vector of the present disclosure, or as compared to baseline, or a predetermined value). In some embodiments, the level of HS is decreased by about or at least about 3%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or more than 100% after administration of a recombinant vector of the present disclosure as compared to a reference, e.g., before ERT is discontinued, (e.g., compared to the level of HS in the subject before administration of a recombinant vector of the present disclosure). In some embodiments, the level of D2S6 is decreased by about or at least about 3%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or more than 100% after administration of a recombinant vector of the present disclosure as compared to a reference, e.g., before ERT is discontinued, (e.g., compared to the level of D2S6 in the subject before administration of a recombinant vector of the present disclosure). In some embodiments, the level of I0S6 is decreased in a subject after administration of a recombinant vector of the present disclosure as compared to a reference (e.g., compared to the level of I0S6 in the subject before administration of a recombinant vector of the present disclosure, or as compared to baseline, or a predetermined value). In some embodiments, the level of IS06 is decreased by about or at least about 3%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or more than 100% after administration of a recombinant vector of the present disclosure as compared to a reference, e.g., before ERT is discontinued, (e.g., compared to the level of I0S6 in the subject before administration of a recombinant vector of the present disclosure). In some embodiments, a biomarker (e.g., HS, D2S6, 10S6, total GAG, and / or I2S enzyme) is measured before administration of the recombinant vector of the present disclosure, on the same day as the administration of the recombinant vector of the present disclosure, on one day after the administration of the recombinant vector of the present disclosure, and / or after about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 30 weeks, 35 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, 1 year, 2 years, or more than 2 years after the administrationof the recombinant vector of the present disclosure. In some embodiments, a biomarker (e.g., HS, D2S6, 10S6, total GAG, and / or I2S enzyme) is measured before administration of the recombinant vector of the present disclosure, on the same day as the administration of the recombinant vector of the present disclosure, 8 weeks after, 16 weeks after, 24 weeks after, 32 weeks after, 40 weeks after, 48 weeks after, 56 weeks after, 72 weeks after, 104 weeks after, 1 year after, 2 years after, or more than 2 years after administration of the recombinant vector of the present disclosure. In some embodiments, HS and / or D2S6 is measured in CSF in ng / ml. In some embodiments, a biomarker (e.g., HS, I0S6, and / or D2S6) is decreased by about or at least about 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 mg / ml, 40 mg / ml, 45 ng / ml, 50 ng / ml, 55 ng / ml, 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml, 100 ng / ml, 110 ng / ml, 120 ng / ml, 130 ng / ml, 140 ng / ml, 150 ng / ml, 160 ng / ml, 170 ng / ml, 180 ng / ml, 190 ng / ml, 200 ng / ml, 220 ng / ml, 250 ng / ml, 270 ng / ml, 300 ng / ml, or more than 300 ng / ml after the administration of the recombinant vector of the present disclosure as compared to a reference (e.g., as compared to the amount from baseline or the amount before treatment with the recombinant vector of the present disclosure). In some embodiments, total GAG (e.g., in urine) is measured in g / mol, CK. In some embodiments, a biomarker (e.g., total GAG) is decreased by about or at least about 5 g / mol, 10 g / mol, 15 g / mol, 20 g / mol, 25 g / mol, 30 g / mol, 35 mg / ml, 40 mg / ml, 45 g / mol, 50 g / mol, 55 g / mol, 60 g / mol, 65 g / mol, 70 g / mol, 75 g / mol, 80 g / mol, 85 g / mol, 90 g / mol, 95 g / mol, 100 g / mol, 110 g / mol, 120 g / mol, 130 g / mol, 140 g / mol, 150 g / mol, 160 g / mol, 170 g / mol, 200 g / mol, or more than 200 g / mol after the administration of the recombinant vector of the present disclosure as compared to a reference (e.g., as compared to the amount from baseline or the amount before treatment with the recombinant vector of the present disclosure). In some embodiments, a biomarker (e.g., HS, D2S6, 10S6, and / or total GAG) is decreased and / or hepatosplenomegaly is decreased by about or at least about 3%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or more than 100% after the administration of the recombinant vector of the present disclosure as compared to a reference, e.g., before ERT is discontinued, (e.g., as compared to the amount from baseline or the amount before treatment with the recombinant vector of the present disclosure). In some embodiments, determining that a biomarker is increased or decreased in a subject is determined before ERT is discontinued in the subject. In some embodiments, a biomarker (e.g., HS, I0S6, D2S6, and / or total GAG) is decreased and / or hepatosplenomegaly is decreased for about, after about, atleast about, or up to about 2 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 30 weeks, 35 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, more than 104 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years after the administration of the recombinant vector of the present disclosure as compared to a reference (e.g., compared to the level of a biomarker in the subject before administration of a recombinant vector of the present disclosure, or compared to a biomarker level from a previously obtained biological sample from the subject, or compared to baseline, or compared to the level of a biomarker in a healthy subject). For example, in some embodiments, a biomarker (e.g., HS, D2S6, 10S6, and / or total GAG) is decreased and / or hepatosplenomegaly is decreased by about or at least about 5%, 20%, 25%, 30%, 35%, 40% 45%, 50%, or more than 50% for about, at least about, or up to about 2 weeks, 4 weeks, 6 weeks, 8 weeks, 16 weeks, 24 weeks, 48 weeks, 56 weeks, 104 weeks, 2 years, or more than 2 years after the administration of the recombinant vector of the present disclosure (e.g., as compared to the level of the biomarker or hepatosplenomegaly from baseline or the level of the biomarker or hepatosplenomegaly before treatment with the recombinant vector of the present disclosure, or as compared to the level of a biomarker or hepatosplenomegaly obtained from the subject at an earlier time point). In some embodiments, a decrease in a biomarker (e.g., HS, D2S6, 10S6, and / or total GAG), a decrease in hepatosplenomegaly, and / or an increase in a biomarker (e.g., I2S) is sustained for about or at least about 2 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 30 weeks, 35 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, more than 104 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years after the administration of the recombinant vector of the present disclosure.

[0275] In some embodiments, the level of a biomarker (e.g., I2S enzyme) is increased by about or at least about 3%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or more than 100% after the administration of the recombinant vector of the present disclosure as compared to a reference (e.g., compared to the level of the biomarker in the subject before administration of the recombinant vector of the present disclosure). In some embodiments, a biomarker (e.g., I2S enzyme) is increased for about, after about, at least about, or up to about 2 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 30 weeks, 35 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, more than 104 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years after the administration of the recombinant vector of the present disclosure as compared to a reference (e.g., compared to the level of the biomarker in the subject before administration of the recombinant vector of the present disclosure). In some embodiments, a biomarker (e.g., I2S enzyme) is increased by about or at least about 5%, 20%, 25%, 30%, 35%, 40% 45%, 50%, or more than 50% at about, for about, at least about, or up to about 2 weeks, 4 weeks, 6 weeks, 8 weeks, 16 weeks, 24 weeks, 48 weeks, 56 weeks, 104 weeks, 2 years, or more than 2 years after the administration of the recombinant vector of the present disclosure (e.g., as compared to the amount from baseline or the amount before treatment with the recombinant vector of the present disclosure). In some embodiments, abiomarker (e.g., I2S enzyme) is increased by about or at least about 500 pg / ml, 1000 pg / ml, 1500 pg / ml, 10000 pg / ml, 15000 pg / ml, 20000 pg / ml, 25000 mg / ml, 30000 mg / ml, 35000 pg / ml, 40000 pg / ml, 45000 pg / ml, 50000 pg / ml, or more than 50000 pg / ml after the administration of the recombinant vector of the present disclosure (e.g., as compared to the amount from baseline or the amount before treatment with the recombinant vector of the present disclosure).

[0276] In some embodiments, heparan sulfate (HS) glycosaminoglycan (CAGs) in CSF is measured using a bioanalytical LC / MS. In some embodiments, heparan sulfate non-reducing ends and total heparan sulfate in CNS is measured using a bioanalytical LC / MS. In some embodiments, heparan sulfate non-reducing ends and total heparan sulfate CAGs in plasma is measured using a bioanalytical LC / MS. In some embodiments, HS CAGs in urine is measured using a bioanalytical LC / MS. In some embodiments, urine total CAGs concentration is measured using a colorimetric assay. In some embodiments, more details about the assays that can be used is provided in Section 6 of the disclosure.

[0277] In some embodiments, if a biomarker (e.g., D2S6, 10S6, HS, GAG (e.g., total GAG in urine)) and / or hepatosplenomegaly is increased and / or a biomarker (e.g., I2S) is decreased after a recombinant vector of the present disclosure is administered to the subject, another dose (e.g., same dose, lower dose, or higher dose as compared to previous dose) of the recombinant vector of the present disclosure is administered to the subject. In some embodiments, if a biomarker (e.g., D2S6, HS, GAG (e.g., total GAG in urine)) and / or hepatosplenomegaly is increased and / or a biomarker (e.g., I2S) is decreased after a recombinant vector of the present disclosure is administered to the subject, another therapy or treatment is administered to the subject (e.g., a therapy or treatment that is used to treat MPS II, a therapy or treatment used to treat a symptom that the subject is suffering from, a therapy or treatment for hepatosplenomegaly, and / or a therapy or treatment used to treat or prevent a symptom from MPS II). In some embodiments, if a biomarker (e.g., I0S6)) and / or hepatosplenomegaly is increased and / or a biomarker (e.g., I2S) is decreased after a recombinant vector of the present disclosure is administered to the subject, another therapy or treatment is administered to the subject (e.g., a therapy or treatment that is used to treat MPS I, a therapy or treatment used to treat a symptom that the subject is suffering from, a therapy or treatment for hepatosplenomegaly, and / or a therapy or treatment used to treat or prevent a symptom from MPS I). For example, if a biomarker (e.g., D2S6, 10S6, HS, GAG (e.g., total GAG in urine)) and / or hepatosplenomegaly is increased and / or if a biomarker (e.g., I2S) is decreased by about or by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100% as compared to a reference, another dose (e.g., same dose, lower dose, or higher dose as compared to previous dose) of the recombinant vector of the present disclosure is administered to the subject and / or another therapy or treatment is administered to the subject (e.g., ERT treatment). In some embodiments, a reference is a control. In some embodiments, a reference is the biomarker level and / or hepatosplenomegaly in a previous biological sample or from an image (e.g., ultrasound) obtained from the subject. In some embodiments, a reference is the biomarker level and / or hepatosplenomegaly from the subject at baseline (e.g., before the recombinant vector of the present disclosure is administered to the subject). In some embodiments, a reference is the biomarker level and / or hepatosplenomegaly in a healthy subject or in a subject who does not have or has not been diagnosed with MPS II. In some embodiments, a reference is the level of the at least one biomarker and / or hepatosplenomegaly in a biological sample obtained from a subject diagnosed with MPS II who never received ERT or is not receiving ERT. In some embodiments, a reference is the biomarker level and / or hepatosplenomegaly in a subject or group of subjects with MPS II. In some embodiments, if an increase in a biomarker (e.g., D2S6, 10S6, HS, GAG (e.g., total GAG in urine)) and / or an increase in hepatosplenomegaly and / or a decrease in a biomarker (e.g., I2S) is observed over a period of about, at least about, or up to about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 30 weeks, 35 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, 53 weeks, 54 weeks, 55 weeks, 56 weeks, 57 weeks, 58 weeks, 59 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, more than 104 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years after the administration of the recombinant vector of the present disclosure, another dose (e.g., same dose, lower dose, or higher dose as compared to previous dose) of the recombinant vector of the present disclosureis administered to the subject and / or another therapy or treatment is administered to the subject.

[0278] In some embodiments, a subject is receiving or has received enzyme replacement therapy (ERT) prior to and / or continuously with the administration of a recombinant vector of the present disclosure. In some embodiments, ERT treatment is discontinued in a subject prior to the administration of a recombinant vector of the present disclosure. In some embodiments, ERT treatment is discontinued in a subject at about 52 weeks after a recombinant vector of the present disclosure is administered to a subject. In some embodiments, ERT treatment is discontinued in a subject at about 56 weeks after a recombinant vector of the present disclosure is administered to a subject. In some embodiments, ERT treatment is discontinued in a subject at about, before about, or after about 52 weeks after a recombinant vector of the present disclosure is administered to a subject (e.g., at about, before about, or after about one day, two days, three days, four days, five days, six days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, or more than 100 weeks after a recombinant vector of the present disclosure is administered to a subject). In some embodiments, determining the level of a biomarker and / or hepatosplenomegaly is used to determine if a patient can discontinue enzyme replacement therapy (ERT) treatment after a recombinant vector of the present disclosure is administered to a subject. In some embodiments, an increase in a biomarker (e.g., I2S) after a recombinant vector of the present disclosure is administered to a subject (e.g., as compared to baseline) is indicative that a subject can stop treatment with ERT. In some embodiments, an increase in a biomarker (e.g., I2S) over a period of time after a recombinant vector of the present disclosure is administered to a subject is indicative that a subject can stop treatment with ERT. In some embodiments, if a subject is negative for an anti-AAV antibody (e.g., a biological sample from the subject does not show a detectable level of anti-AAV9 antibodies), ERT treatment is discontinued before a recombinant vector of the present disclosure is administered to a subject. In some embodiments, ERT treatment is discontinued by about or at least about 1 year, 10 months, 8 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 3 weeks, 2 weeks, 1 week, 10 days, 5 days, 4 days, 3 days, 2 days, or 1 day before a recombinant vector of the present disclosure is administered to a subject. In some embodiments, a decrease in abiomarker (e.g., D2S6, HS, GAG (e.g., total GAG in urine)) and / or a decrease in hepatosplenomegaly after a recombinant vector of the present disclosure is administered to a subject (e.g., as compared to baseline) is indicative that a subject can stop treatment with ERT. In some embodiments, a decrease in a biomarker (e.g., D2S6, 10S6, HS, GAG (e.g., total GAG in urine)) and / or decrease in hepatosplenomegaly over a period of time after a recombinant vector of the present disclosure is administered to a subject is indicative that a subject can stop treatment with ERT. In some embodiments, a decrease in a biomarker (e.g., D2S6, 10S6, HS, GAG (e.g., total GAG in urine)) and / or a decrease in hepatosplenomegaly and / or an increase in a biomarker (e.g., I2S) over a period of about, at least about, or up to about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 30 weeks, 35 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, 53 weeks, 54 weeks, 55 weeks, 56 weeks, 57 weeks, 58 weeks, 59 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, more than 104 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years after the administration of the recombinant vector of the present disclosure is indicative that the subject can discontinue or stop treatment with ERT. In some embodiments, if a biomarker level (e.g., D2S6, 10S6, HS, GAG (e.g., total GAG in urine)) decreased and / or hepatosplenomegaly is decreased and / or if a biomarker (e.g., I2S) is increased by about or by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100% as compared to a reference level, ERT treatment is stopped or discontinued. In some embodiments, if a biomarker level (e.g., D2S6, 10S6, HS, GAG (e.g., total GAG in urine)) is decreased and / or hepatosplenomegaly is decreased and / or if a biomarker (e.g., I2S) is increased by about or by at least about 1-fold, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90- fold, 95-fold, 100-fold, or more than 100-fold (e.g., after a recombinant vector of the presentdisclosure is administered) as compared to a reference level, ERT treatment is stopped or discontinued. In some embodiments, ERT is discontinued indefinitely. In some embodiments, ERT is discontinued or stopped for about, at least about, or at most about 1 day, 5 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 30 weeks, 35 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks,51 weeks, 52 weeks, 53 weeks, 54 weeks, 55 weeks, 56 weeks, 57 weeks, 58 weeks, 59 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years. In some embodiments, ERT is stopped at about or at least about52 weeks after the administration of the recombinant vector of the present disclosure.

[0279] In some embodiments, a biomarker (e.g., I2S, I0S6, D2S6, HS, GAG (e.g., total GAG in urine)) and / or hepatosplenomegaly is monitored after ERT treatment is discontinued. For example, in some embodiments, a biomarker (e.g., I2S, I0S6, D2S6, HS, GAG (e.g., total GAG in urine)) and / or hepatosplenomegaly (e.g., size of the liver or spleen) is measured at about, at least about, or every 2 days, 5 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 30 weeks, 35 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, 53 weeks, 54 weeks, 55 weeks, 56 weeks, 57 weeks, 58 weeks, 59 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years after ERT treatment isstopped or discontinued and / or after the recombinant vector of the present disclosure is administered to the subject.

[0280] In some embodiments, if a biomarker (e.g., D2S6, 10S6, HS, GAG (e.g., total GAG in urine)) is increased and / or hepatosplenomegaly is increased and / or a biomarker (e.g., I2S) is decreased after ERT treatment is stopped or discontinued, ERT treatment is readministered to the subject. For example, if a biomarker (e.g., D2S6, 10S6, HS, GAG (e.g., total GAG in urine)) is increased and / or hepatosplenomegaly is increased and / or if a biomarker (e.g., I2S) is decreased by about or by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100% (e.g., after a recombinant vector of the present disclosure is administered to the subject) as compared to a reference, ERT treatment is re-administered to the subject (e.g., for a period of time until a biomarker (e.g., D2S6, 10S6, HS, GAG (e.g., total GAG in urine)) is reduced or controlled and / or hepatosplenomegaly is reduced or controlled and / or a biomarker (e.g., I2S) is increased or controlled. In some embodiments, a reference is a control. In some embodiments, a reference is the biomarker level and / or hepatosplenomegaly in a previous biological sample or from an image (e.g., ultrasound) obtained from the subject. In some embodiments, a reference is the biomarker level and / or hepatosplenomegaly from the subject at baseline (e.g., before the recombinant vector of the present disclosure is administered to the subject). In some embodiments, a reference is the biomarker level and / or hepatosplenomegaly in a healthy subject or in a subject who does not have or has not been diagnosed with MPS II. In some embodiments, a reference is the biomarker level and / or hepatosplenomegaly in a subject or group of subjects with MPS II. In some embodiments, a reference is the biomarker level and / or hepatosplenomegaly in a healthy subject or in a subject who does not have or has not been diagnosed with MPS I. In some embodiments, a reference is the biomarker level and / or hepatosplenomegaly in a subject or group of subjects with MPS I. In some embodiments, a reference is the biomarker level and / or hepatosplenomegaly in a subject or group of subjects who is receiving or has received ERT treatment.

[0281] In some embodiments, ERT treatment is discontinued after administration of the recombinant vector of the present disclosure. For example, ERT treatment is stopped after the recombinant vector of the present disclosure is administered to a subject who has been treated with ERT and has anti-IDS Abs levels (e.g., anti-IDS Abs level as a result of ERT treatment). In some embodiments, a subject who has anti-IDS Abs levels has ERT treatment stopped as soon as or immediately after the recombinant vector of the present disclosure is administeredto the subject. In some embodiments, anti-IDS Abs levels is determined in a biological sample from a subject (e.g., serum sample). In some embodiments, anti-IDS Abs levels is determined in a biological sample obtained from a subject before the recombinant vector of the present disclosure is administered to the subject. In some embodiments, anti-IDS Abs levels is monitored before and / or after the recombinant vector of the present disclosure is administered to the subject. In some embodiments, if anti-IDS Abs levels is higher than a reference level, ERT treatment is stopped or discontinued. In some embodiments, a reference level is a predetermined level. In some embodiments, a reference level is the anti-IDS Abs level in a previous biological sample obtained from the subject. In some embodiments, a reference level is the anti-IDS Abs level from the subject at baseline (e.g., before the recombinant vector of the present disclosure is administered to the subject). In some embodiments, a reference level is the anti-IDS Abs level in a healthy subject or in a subject who does not have or has not been diagnosed with MPS II. In some embodiments, a reference level is the anti-IDS Abs level in a subject or group of subjects with MPS II. In some embodiments, a reference level is the anti-IDS Abs level in a subject or group of subjects who is receiving or has received ERT treatment. In some embodiments, if the anti-IDS Abs level is higher or increased by about or by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 250%, 500%, 750%, 1000%, or more than 1000% as compared to a reference level then ERT treatment is discontinued. In some embodiments, if the anti-IDS Abs level is higher or increased by about or by at least about 1-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85- fold, 90-fold, 95-fold, 100-fold, 150-fold, 250-fold, 500-fold, 750-fold, 1000-fold, or more than 1000-fold as compared to a reference level then ERT treatment is stopped or discontinued. In some embodiments, anti-IDS Abs level is measured at about, at least about, or every 1 day, 5 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 30 weeks, 35 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, 53 weeks, 54 weeks, 55 weeks, 56 weeks, 57 weeks, 58 weeks, 59 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15months, 18 months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years after ERT treatment is administered to the subject, after ERT treatment is stopped or discontinued, before a recombinant vector of the present disclosure is administered to the subject and / or after a recombinant vector of the present disclosure is administered to the subject

[0282] In some embodiments, efficacy of treatment or monitoring efficacy of treatment (e.g., with an rAAV9 encoding hIDUA) is based on the level of I0S6. In some embodiments, the level of I0S6 is determined from a biological sample from a subject. In some embodiments, the biological sample is plasma. In some embodiments, a decrease in the level of I0S6 after treatment with a recombinant vector of the disclosure (e.g., rAAV9 encoding hIDUA) in comparison with a reference, is indicative of efficacy of treatment. In some embodiments, the reference is a level of I0S6 in a biological sample from the subject obtained prior to administration of a recombinant AAV of the disclosure. In some embodiments, the reference is a predetermined value. In some embodiments, the reference is the level of I0S6 in a biological sample from another subject with MPS I or a population of subjects with MPS I. In some embodiments, the level of I0S6 is decreased after administration of a recombinant AAV of the disclosure as compared to before administration of the rAAV. In some embodiments, the subject was previously treated with ERT prior to rAAV administration and / or received ERT treatment after the rAAV administration. In some embodiments, the decrease in the level of I0S6 is a decrease of about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% as compared to the reference. In some embodiments, efficacy of MPS I treatment is an improvement in at least one subtest of the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III) as compared to a reference. In some embodiments, the at least one subtest is age equivalence score, cognitive developmental quotient (DQ), expressive language DQ, receptive language DQ, gross motor DQ, and / or fine motor DQ. In some embodiments, efficacy of MPS I treatment is an improvement in WASI and / or VABS score. In some embodiments, the rAAV9 encoding hIDUA is administered to the human subject in a solution comprising: (a) sodium chloride at a concentration of about 8.77 g / L, (b) magnesium chloride, at a concentration of about 0.244 g / L, (c) potassium chloride at a concentration of about 0.224 g / L, (d) calcium chloride at a concentration of about 0.206 g / L, (e) dextrose at aconcentration of about 0.793 g / L, (f) poloxamer 188 at a concentration of about 0.010 g / L,(g) sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and(h) sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L.5.5.2. Hepatosplenomegaly

[0283] In some embodiments, a subject diagnosed with MPS II has hepatosplenomegaly or is experiencing a symptom associated with hepatosplenomegaly. In some embodiments, hepatosplenomegaly is diagnosed by a physical examination. For example, a physician or medical care professional can observe the area around the abdomen for signs of organ enlargement. In some embodiments, a physician or medical care professional feels or palpates the abdomen area of the subject for sensitivity or enlargement of the liver and / or spleen. In some embodiments, a diagnostic test is performed to detect hepatosplenomegaly. For example, hepatosplenomegaly can be detected using a blood test (e.g., liver function test, blood count, and / or tests for clotting factors). In some embodiments, hepatosplenomegaly is detected using an imaging scan such as a computed tomography (CT) scan or ultrasound. In some embodiments, hepatosplenomegaly is detected via a biopsy.

[0284] In some embodiments, hepatosplenomegaly is measured before, during, and / or after treatment with a recombinant vector of the present disclosure. In some embodiments, hepatosplenomegaly is measured at different time points before, during, and / or after treatment with a recombinant vector of the present disclosure. In some embodiments, a decrease in hepatosplenomegaly is indicative that the treatment is effective. In some embodiments, assessing or monitoring hepatosplenomegaly is used to determine dosage amount, if dosage is to be increased, if dosage is to be decreased, and / or to determine dosage frequency.

[0285] In some embodiments, hepatosplenomegaly is determined before the subject starts treatment with a therapy or treatment of the present disclosure and / or after the subject start treatment with a therapy or treatment of the present disclosure. In some embodiments, hepatosplenomegaly is determined between dosage administrations e.g., between before the first dose (i.e., before treatment with the recombinant vector of the present disclosure commences) and after the first dose of the recombinant vector of the present disclosure is administered to the subject, between dose 0 and dose 2, between dose 0 and dose 3, between dose 1 and dose 2, between dose 1 and dose 3, and / or between dose 2 and dose 3). In some embodiments, hepatosplenomegaly is determined 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years after a dosage administration or after treatment commences (e.g., after a recombinant vector of the present disclosure is administered to the subject and / or after ERT treatment).

[0286] In some embodiments, hepatosplenomegaly (e.g., size of liver of spleen) is decreased by about or at least about 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, 200%, or more than 200% after a recombinant vector of the present disclosure is administered to the subject. In some embodiments, hepatosplenomegaly is decreased by about or at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 5-fold, 7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 97-fold, 100-fold, 200-fold, or more than 200-fold after a recombinant vector of the present disclosure is administered to the subject. In some embodiments, a decrease in hepatosplenomegaly corresponds to a decrease in liver and / or spleen dimension. In some embodiments, a decrease in hepatosplenomegaly corresponds to a decrease in liver and / or spleen mass or diameter. In some embodiments, a decrease in hepatosplenomegaly corresponds to a decrease in liver and / or spleen size. In some embodiments, hepatosplenomegaly decreases a recombinant vector of the present disclosure is administered to the subject and / or between dosage administrations. In some embodiments, hepatosplenomegaly decreases after a recombinant vector of the present disclosure is administered to the subject and / or a dosage administration (e.g., after the first dose, after the second dose, after the third dose, after the fourth dose, after the fifth dose, or after several doses). In some embodiments, hepatosplenomegaly decreases at about, after about, for at least about, or for up to about 1 day, 3 days, 5 days, 7 days, one week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 months, 50months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years, after a recombinant vector of the present disclosure is administered to the subject. In some embodiments, a decrease in hepatosplenomegaly is sustained for at least about or for about 1 day, 3 days, 5 days, 7 days, one week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years, after a recombinant vector of the present disclosure is administered to the subject. In some embodiments, the decrease in hepatosplenomegaly is greater in a subject undergoing a treatment of the present disclosure as compared to a decrease in hepatosplenomegaly if the subject was undergoing another treatment used for treating hepatosplenomegaly or MPS II. In some embodiments, the decrease in hepatosplenomegaly is greater in a subject who received treatment of a recombinant vector of the present disclosure as compared to a decrease in hepatosplenomegaly in a comparable subject having a comparable hepatosplenomegaly undergoing another treatment used for treating hepatosplenomegaly or MPS II. In some embodiments, a decrease in hepatosplenomegaly is greater by about or by at least about 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, 200%, or more than 200% (e.g., as compared to the decrease in hepatosplenomegaly if the subject was undergoing another treatment used for treating hepatosplenomegaly or MPS II).5.5.3. Tests for Neurocognitive and Neurodegeneration function

[0287] In certain embodiments, efficacy of treatment with the recombinant vector is monitored by measuring the level of cognitive function in the patient. Cognitive function may be measured by any method known to one of skill in the art. In certain embodiments, cognitive function is measured via a validated instrument for measuring intelligence quotient (IQ). In specific embodiments, IQ is measured by Wechsler Abbreviated Scale ofIntelligence, Second Edition (WASI-II). In some embodiments, neurocognition is determined by measuring intelligence quotient (IQ) as measured by Bayley’s Infantile Development Scale. In some embodiments, developmental quotient (DQ) is measured using Bayley Scales of Infant Development (BSID-III). In some embodiments, Bayley Scales of Infant and Toddler Development is used to measure motor, cognitive, language, social- emotional, and / or adaptive behavior development in babies, young children, or in a patient of the present disclosure. In some embodiments, efficacy of a treatment of MPS II according to the methods of the present disclosure (e.g., by administering an rAAV encoding hIDS to a subject) is determined by analyzing at least one subtest of BSID-III. In some embodiments, efficacy of a treatment of MPS I according to the methods of the present disclosure (e.g., by administering an rAAV encoding hIDUA to a subject) is determined by analyzing at least one subtest of BSID-III. In some embodiments, efficacy of a treatment of MPS II or MPS I according to the methods of the present disclosure (e.g., by administering an rAAV encoding hIDS or an AAV encoding hIDUA to a subject) is determined by analyzing age equivalence score, cognitive developmental quotient (DQ), expressive language DQ, receptive language DQ, gross motor DQ, and / or fine motor DQ. In some embodiments, the efficacy of MPS II or MPS I treatment is an improvement in at least one subtest of the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III) as compared to a reference. In some embodiments, the at least one subtest is age equivalence score, cognitive developmental quotient (DQ), expressive language DQ, receptive language DQ, gross motor DQ, and / or fine motor DQ. In some embodiments, the improvement is an improvement in DQ of about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100. In some embodiments, the age equivalence score is increased by about or at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, or more than 24 months as compared to a reference. In some embodiments, the reference is the score of the at least one subtest of the BSID-III obtained prior to administration of an rAAV of the present disclosure. In some embodiments, a reference is an average score of at least one subtest of BSID-III obtained from human subjects with MPS II of the same age as the human subject receiving an rAAV of the present disclosure. In some embodiments, a reference is an average score of at least one subtest of BSID-III obtained from human subjects with MPS I of the same age as the human subject receiving an rAAV of the present disclosure. In some embodiments, a level of a biomarker of the present disclosure is indicative of efficacy of an MPS II treatment. For example, in some embodiments, theI l llevel of D2S6 in a biological sample from a subject is inversely proportional to the efficacy of MPS II treatment. In some embodiments, a decrease in D2S6 level in a biological sample correlates with an improvement in at least one subtest of BSID-III. In some embodiments, a decrease in I0S6 level in a biological sample correlates with an improvement in at least one subtest of BSID-III. In some embodiments, a decrease in D2S6 level in a biological sample obtained from a subject after an rAAV encoding hIDS is administered to the subject (e.g., as compared to a reference or to the level of D2S6 prior to administration of the rAAV encoding hIDS), correlates with (or is indicative of) an improvement in the age equivalence score of a subject with MPS II. In some embodiments, a decrease in I0S6 level in a biological sample obtained from a subject after an rAAV encoding hIDUA is administered to the subject (e.g., as compared to a reference or to the level of I0S6 prior to administration of the rAAV encoding hIDUA), correlates with (or is indicative of) an improvement in the age equivalence score of a subject with MPS I. In some embodiments, a decrease in D2S6 level in a biological sample obtained from a subject after an rAAV encoding hIDS is administered to the subject (e.g., as compared to a reference or to the level of D2S6 prior to administration of the rAAV encoding hIDS), correlates with (or is indicative ...

Claims

WHAT IS CLAIMED IS:

1. A method for preventing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2- sulfatase (hIDS), wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear.

2. The method of claim 1, wherein the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 24 weeks, at least 48 weeks, at least about 1 year, at least about 52 weeks, at least about 1.5 years, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after the administration of the rAAV.

3. The method of claim 1 or 2, wherein the onset of hearing loss is indicated by an increase in a hearing threshold as measured by an auditory brain response (ABR) testing in the at least one ear after the administration of the rAAV as compared to a hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV.

4. The method of claim 3, wherein the hearing threshold is an estimate of behavioral threshold (eHL) derived from an ABR threshold (nHL).

5. The method of claim 3 or 4, wherein the method further comprises performing the ABR testing in the at least one ear prior to and after the administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV.

6. The method of any one of claims 1-5, wherein the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural as determined by a tympanometric testing.

7. The method of claim 6, wherein the method further comprises performing the tympanometric testing in the at least one ear prior to and after the administration of the rAAVto determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.

8. A method for stabilizing mucopolysaccharidosis II (MPS II)-associated hearing loss or preventing the further loss of hearing associated with MPS II in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents a further loss of the hearing in the at least one ear.

9. The method of claim 8, wherein the therapeutically effective amount of the rAAV stabilizes the hearing loss or prevents the further loss of the hearing in the at least one ear for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 24 weeks, at least 48 weeks, at least about 1 year, at least about 52 weeks, at least about 1.5 years, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after the administration of the rAAV.

10. The method of claim 9, wherein stabilizing the hearing loss or preventing the further loss of the hearing is indicated by no change or a small change in a hearing threshold as measured by an auditory brain response (ABR) testing in the at least one ear after the administration of the rAAV as compared to a hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV.

11. The method of claim 10, wherein the hearing threshold is an estimate of behavioral threshold (eHL) derived from an ABR threshold (nHL).

12. The method of claim 11, wherein stabilizing the hearing loss or preventing the further loss of the hearing is indicated by an about 10 dB or less change of the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV.

13. The method of any one of claims 10-12, wherein the method further comprises performing the ABR testing in the at least one ear prior to and after the administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV.

14. The method of any one of claims 8-13, wherein the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural as determined by a tympanometric testing.

15. The method of claim 14, wherein the method further comprises performing the tympanometric testing in the at least one ear prior to and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.

16. A method for improving mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2- sulfatase (hIDS), wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves the hearing in the at least one ear.

17. The method of claim 16, wherein the therapeutically effective amount of the rAAV improves the hearing in the at least one ear in about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 24 weeks, about 6 months, about 48 weeks, about 52 weeks, or about 1 year after the administration of the rAAV.

18. The method of claim 16 or 17, wherein improving the hearing is indicated by a decrease in a hearing threshold as measured by an auditory brain response (ABR) testing in the at least one ear after the administration of the rAAV as compared to a hearing threshold as measured by the ABR testing in the at least one ear prior to the administration of the rAAV.

19. The method of claim 18, wherein the therapeutically effective amount of the rAAV decreases the hearing threshold in the at least one ear for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the hearing threshold in the at least one ear prior to the administration of the rAAV.

20. The method of claim 18 or 19 , wherein the hearing threshold is an estimate of behavioral threshold (eHL) derived from an ABR threshold (nHL).

21. The method of claim 20, wherein improving the hearing is indicated by a more than about 10 dB decrease of the eHL in the at least one ear after the administration of the rAAV as compared to prior to the administration of the rAAV.

22. The method of any one of claims 18-21, wherein the method further comprises performing the ABR testing in the at least one ear prior to and after the administration of the rAAV to determine hearing thresholds in the at least one ear prior to and after the administration of the rAAV.

23. The method of any one of claims 8-22, wherein the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural as determined by a tympanometric testing.

24. The method of claim 23, wherein the method further comprises performing the tympanometric testing in the at least one ear prior to and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixed of conductive and sensorineural.

25. The method of any one of claims 1-24, wherein the at least one ear comprises the left ear or the right ear.

26. The method of any one of claims 1-25, wherein the at least one ear comprises the left ear and the right ear.

27. The method of any one of claims 1-26, wherein the method further comprises administering an additional treatment to the subject.

28. The method of claim 27, wherein the additional treatment is an enzyme replacement therapy comprising a recombinant idursulfase.

29. The method of claim 27, wherein the additional treatment is a second administration of the rAAV.

30. The method of claim 29, wherein the second administration of the rAAV is at a higher dose as compared to the first administration of the rAAV.

31. The method of any one of claims 1-30, wherein the rAAV is administered intravenously to the subject or to the cerebrospinal fluid (CSF) of the subject.

32. The method of any one of claims 1-31, wherein the rAAV is administered via intraci sternal (IC) administration.

33. The method of any one of claims 1-31, wherein the rAAV is administered via intracerebroventricular (ICV) administration.

34. The method of any one of claims 1-33, wherein the rAAV is administered at a volume that does not exceed 10% of the total cerebrospinal fluid volume of the subject.

35. The method of any one of claims 1-34, wherein the rAAV is administered intrathecally to the subject.

36. The method of any one of claims 1-35, wherein the rAAV is administered to the subject in a solution comprising:(a) sodium chloride at a concentration of about 8.77 g / L,(b) magnesium chloride, at a concentration of about 0.244 g / L,(c) potassium chloride at a concentration of about 0.224 g / L,(d) calcium chloride at a concentration of about 0.206 g / L,(e) dextrose at a concentration of about 0.793 g / L,(f) poloxamer 188 at a concentration of about 0.010 g / L,(g) sodium phosphate monobasic monohydrate at a concentration of about 0.0278 g / L, and(h) sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L.

37. The method of any one of claims 1-36, wherein the rAAV is administered at a dose of about 1.3 x io10GC / g brain mass, about 6.5 x io10GC / g brain mass, or about 2 x io11GC / g brain mass, wherein the brain mass is determined by MRI.

38. The method of any one of claims 1-30, wherein the rAAV is administered to the at least one ear.

39. The method of any one of claims 1-38, wherein the subject is 5 years old or older and less than 18 years old.

40. The method of any one of claims 1-38, wherein the subject is 4 months old or older and less than 5 years old.

41. The method of any one of claims 1-40, wherein the subject is receiving an enzyme replacement therapy (ERT) at the time of the administration of the rAAV.

42. The method of claim 41, wherein the subject is not responsive to the ERT.

43. The method of claim 41 or 42, wherein the ERT comprises a recombinant idursulfase.

44. The method of any one of claims 41-43, wherein the ERT is discontinued about 6 months, about 24 weeks, about 9 months, about 12 months, about 52 weeks, about 15 months, about 18 months, about 21 months, or about 24 months after the administration of the rAAV.

45. The method of any one of claims 1-44, wherein the rAAV is a recombinant adeno- associated virus serotype 9.

46. The method of claim 45, wherein the rAAV comprises a human IDS expression cassette, wherein the expression of the human IDS is driven by a hybrid of a cytomegalovirus (CMV) enhancer and a chicken beta actin promoter (CB7).

47. The method of claim 46, wherein the human IDS expression cassette comprises (i) an IDS transgene flanked by inverted terminal repeats (ITRs), (ii) the chicken beta actin intron, and (iii) a rabbit beta-globin polyadenylation (poly A) signal.

48. The method of claim 47, wherein the ITRs are AAV2 ITRs.

49. The method of any one of claims 46-48, wherein the human IDS expression cassette comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 45.

50. The method of any one of claims 1-49, further comprising administering an immune suppression therapy to the subject before or concurrently with the rAAV, and optionally continuing the immune suppression therapy thereafter.

51. The method of claim 50, wherein the immune suppression therapy comprises administering at least one corticosteroid, sirolimus, and / or tacrolimus.

52. The method of claim 51, wherein the at least one corticosteroid is methylprednisolone and / or prednisone.

53. The method of any one of claims 1-52, further comprising administering at least one antibiotics to the subject before or concurrently with the rAAV.

54. The method of claim 53, wherein the at least one antibiotics is trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atovaquone.

55. The method of any one of claims 1-54, further comprising administering at least one antifungal therapy to the subject before or concurrently with rAAV.

56. A therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for use in preventing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear.

57. A therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for use in stabilizing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof,wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents a further loss of the hearing in the at least one ear.

58. A therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for use in improving mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves the hearing in the at least one ear.

59. Use of a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) in preventing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents the onset of hearing loss in the at least one ear.

60. Use of a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) in improving mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves the hearing in the at least one ear.

61. Use of a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) in stabilizing mucopolysaccharidosis II (MPS II)-associated hearing loss in a subject in need thereof, wherein the therapeutically effective amount of the rAAV is administrable to the subject, wherein the subject has developed hearing loss in at least one ear prior to theadministration of the rAAV, and the therapeutically effective amount of the rAAV prevents a further loss of the hearing in the at least one ear.

62. A formulation suitable for administration to the ear comprising a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS).

63. The formulation of claim 62, wherein the rAAV comprises a human IDS expression cassette, wherein the expression of the human IDS is driven by a hybrid of a cytomegalovirus (CMV) enhancer and a chicken beta actin promoter (CB7).

64. The formulation of claim 63, wherein the human IDS expression cassette comprises (i) an IDS transgene flanked by inverted terminal repeats (ITRs), (ii) the chicken beta actin intron, and (iii) a rabbit beta-globin polyadenylation (poly A) signal.

65. The formulation of claim 64, wherein the ITRs are AAV2 ITRs.

66. The formulation of any one of claims 63-65, wherein the human IDS expression cassette comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 45.

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